Archive for the ‘Phosphorylase’ Category

Lesson 4 Cell Signaling And Motility: G Proteins, Signal Transduction: Curations and Articles of reference as supplemental information: #TUBiol3373

Curator: Stephen J. Williams, Ph.D.

Updated 7/15/2019

Below please find the link to the Powerpoint presentation for lesson #4 for #TUBiol3373.  The lesson first competes the discussion on G Protein Coupled Receptors, including how cells terminate cell signals.  Included are mechanisms of receptor desensitization.  Please NOTE that desensitization mechanisms like B arrestin decoupling of G proteins and receptor endocytosis occur after REPEATED and HIGH exposures to agonist.  Hydrolysis of GTP of the alpha subunit of G proteins, removal of agonist, and the action of phosphodiesterase on the second messenger (cAMP or cGMP) is what results in the downslope of the effect curve, the termination of the signal after agonist-receptor interaction.


Click below for PowerPoint of lesson 4

Powerpoint for lesson 4


Please Click below for the papers for your Group presentations

paper 1: Membrane interactions of G proteins and other related proteins

paper 2: Macaluso_et_al-2002-Journal_of_Cellular_Physiology

paper 3: Interactions of Ras proteins with the plasma membrane

paper 4: Futosi_et_al-2016-Immunological_Reviews


Please find related article on G proteins and Receptor Tyrosine Kinases on this Open Access Online Journal

G Protein–Coupled Receptor and S-Nitrosylation in Cardiac Ischemia and Acute Coronary Syndrome

Action of Hormones on the Circulation

Newer Treatments for Depression: Monoamine, Neurotrophic Factor & Pharmacokinetic Hypotheses

VEGF activation and signaling, lysine methylation, and activation of receptor tyrosine kinase


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New Studies toward Understanding Alzheimer Disease

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


There is no unifying concept of Alzheimer Disease beyond the Tau and beta amyloid roles.  Recently, Ingenbleek and Bernstein (journal AD) made the connection between the age related decline of liver synthesis of plasma transthyretin and the more dramatic decline of transthyretin at the blood brain barrier, and the relationship to inability to transfer vitamin A via retinol binding protein to the brain.  Related metabolic events are reported by several groups.


What else is New?


Amyloid-β peptide protects against microbial infection in mouse and worm models of Alzheimer’s disease.

Kumar DK, Choi SH, Washicosky KJ, Eimer WA, Tucker S, Ghofrani J, Lefkowitz A, McColl G, Goldstein LE, Tanzi RE, Moir RD.

Sci Transl Med. 2016 May 25;8(340):340ra72.  http://dx.doi.org:/10.1126/scitranslmed.aaf1059

They show that Aβ oligomerization, a behavior traditionally viewed as intrinsically pathological, may be necessary for the antimicrobial activities of the peptide. Collectively, our data are consistent with a model in which soluble Aβ oligomers first bind to microbial cell wall carbohydrates via a heparin-binding domain. Developing protofibrils inhibited pathogen adhesion to host cells. Propagating β-amyloid fibrils mediate agglutination and eventual entrapment of unatttached microbes….Salmonella Typhimurium bacterial infection of the brains of transgenic 5XFAD mice resulted in rapid seeding and accelerated β-amyloid deposition, which closely colocalized with the invading bacteria.

This is quite interesting in that infection drives the production of acute phase reactants resulting in decreased production of transthyretin.  Whether this also has ties to chronic disease in the elderly and risk of AD is not known.

Gain-of-function mutations in protein kinase Cα (PKCα) may promote synaptic defects in Alzheimer’s disease.

Alfonso SI, Callender JA, Hooli B, Antal CE, Mullin K, Sherman MA, Lesné SE, Leitges M, Newton AC, Tanzi RE, Malinow R.

Sci Signal. 2016 May 10;9(427):ra47.  http://dx.doi.org:/10.1126/scisignal.aaf6209.

Through whole-genome sequencing of 1345 individuals from 410 families with late-onset AD (LOAD), they identified three highly penetrant variants in PRKCA, the gene that encodes protein kinase Cα (PKCα), in five of the families. All three variants linked with LOAD displayed increased catalytic activity relative to wild-type PKCα as assessed in live-cell imaging experiments using a genetically encoded PKC activity reporter. Deleting PRKCA in mice or adding PKC antagonists to mouse hippocampal slices infected with a virus expressing the Aβ precursor CT100 revealed that PKCα was required for the reduced synaptic activity caused by Aβ. In PRKCA(-/-) neurons expressing CT100, introduction of PKCα, but not PKCα lacking a PDZ interaction moiety, rescued synaptic depression, suggesting that a scaffolding interaction bringing PKCα to the synapse is required for its mediation of the effects of Aβ. Thus, enhanced PKCα activity may contribute to AD, possibly by mediating the actions of Aβ on synapses.


Science Signaling Podcast for 10 May 2016: PKCα in Alzheimer’s disease.

Newton AC, Tanzi RE, VanHook AM.

Sci Signal. 2016 May 10;9(427):pc11. doi: 10.1126/scisignal.aaf9436.

Relevance of the COPI complex for Alzheimer’s disease progression in vivo.

Bettayeb K, Hooli BV, Parrado AR, Randolph L, Varotsis D, Aryal S, Gresack J,Tanzi RE, Greengard P, Flajolet M.

Proc Natl Acad Sci U S A. 2016 May 10;113(19):5418-23. http://dx.doi.org:/10.1073/pnas.1604176113

Inhibition of death-associated protein kinase 1 attenuates the phosphorylation and amyloidogenic processing of amyloid precursor protein.

Kim BM, You MH, Chen CH, Suh J, Tanzi RE, Ho Lee T.

Hum Mol Genet. 2016 Apr 19. pii: ddw114.

Extracellular deposition of amyloid-beta (Aβ) peptide, a metabolite of sequential cleavage of amyloid precursor protein (APP), is a critical step in the pathogenesis of Alzheimer’s disease (AD). While death-associated protein kinase 1 (DAPK1) is highly expressed in AD brains and its genetic variants are linked to AD risk, little is known about the impact of DAPK1 on APP metabolism and Aβ generation. This study demonstrated a novel effect of DAPK1 in the regulation of APP processing using cell culture and mouse models. DAPK1, but not its kinase deficient mutant (K42A), significantly increased human Aβ secretion in neuronal cell culture models. Moreover, knockdown of DAPK1 expression or inhibition of DAPK1 catalytic activity significantly decreased Aβ secretion. Furthermore, DAPK1, but not K42A, triggered Thr668 phosphorylation of APP, which may initiate and facilitate amyloidogenic APP processing leading to the generation of Aβ. In Tg2576 APPswe-overexpressing mice, knockout of DAPK1 shifted APP processing toward non-amyloidogenic pathway and decreased Aβ generation. Finally, in AD brains, elevated DAPK1 levels showed co-relation with the increase of APP phosphorylation. Combined together, these results suggest that DAPK1 promotes the phosphorylation and amyloidogenic processing of APP, and that may serve a potential therapeutic target for AD.

Recapitulating amyloid β and tau pathology in human neural cell culture models: clinical implications.

Choi SH, Kim YH, D’Avanzo C, Aronson J, Tanzi RE, Kim DY.

US Neurol. 2015 Fall;11(2):102-105.    Free PMC Article

The “amyloid β hypothesis” of Alzheimer’s disease (AD) has been the reigning hypothesis explaining pathogenic mechanisms of AD over the last two decades. However, this hypothesis has not been fully validated in animal models, and several major unresolved issues remain. Our 3D human neural cell culture model system provides a premise for a new generation of cellular AD models that can serve as a novel platform for studying pathogenic mechanisms and for high-throughput drug screening in a human brain-like environment.

The two key pathological hallmarks of AD are senile plaques (amyloid plaques) and neurofibrillary tangles (NFTs), which develop in brain regions responsible for memory and cognitive functions (i.e. cerebral cortex and limbic system) 3. Senile plaques are extracellular deposits of amyloid-β (Aβ) peptides, while NFTs are intracellular, filamentous aggregates of hyperphosphorylated tau protein 4.

The identification of Aβ as the main component of senile plaques by Drs. Glenner and Wong in 1984 5 resulted in the original formation of the “amyloid hypothesis.” According to this hypothesis, which was later renamed the “amyloid-β cascade hypothesis” by Drs. Hardy and Higgins 6, the accumulation of Aβ is the initial pathological trigger in the disease, subsequently leading to hyperphosphorylation of tau, causing NFTs, and ultimately, neuronal death and dementia 4,710. Although the details have been modified to reflect new findings, the core elements of this hypothesis remain unchanged: excess accumulation of the pathogenic forms of Aβ, by altered Aβ production and/or clearance, triggers the vicious pathogenic cascades that eventually lead to NFTs and neuronal death.

Over the last two decades, the Aβ hypothesis of AD has reigned, providing the foundation for numerous basic studies and clinical trials 4,7,10,11. According to this hypothesis, the accumulation of Aβ, either by altered Aβ production and/or clearance, is the initial pathological trigger in the disease. The excess accumulation of Aβ then elicits a pathogenic cascade including synaptic deficits, altered neuronal activity, inflammation, oxidative stress, neuronal injury, hyperphosphorylation of tau causing NFTs and ultimately, neuronal death and dementia 4,710.

One of the major unresolved issues of the Aβ hypothesis is to show a direct causal link between Aβ and NFTs 1214. Studies have demonstrated that treatments with various forms of soluble Aβ oligomers induced synaptic deficits and neuronal injury, as well as hyperphosphorylation of tau proteins, in mouse and rat neurons, which could lead to NFTs and neurodegeneration in vivo 1821. However, transgenic AD mouse models carrying single or multiple human familial AD (FAD) mutations in amyloid precursor protein (APP) and/or presenilin 1 (PS1) do not develop NFTs or robust neurodegeneration as observed in human patients, despite robust Aβ deposition 13,22,23. Double and triple transgenic mouse models, harboring both FAD and tau mutations linked with frontotemporal dementia (FTD), are the only rodent models to date displaying both amyloid plaques and NFTs. However, the NFT pathology in these models stems mainly from the overexpression of human tau as a result of the FTD, rather than the FAD mutations24,25.

Human neurons carrying FAD mutations are an optimal model to test whether elevated levels of pathogenic Aβ trigger pathogenic cascades including NFTs, since those cells truly share the same genetic background that induces FAD in humans. Indeed, Israel et al., observed elevated tau phosphorylation in neurons with an APP duplication FAD mutation 33. Blocking Aβ generation by β-secretase inhibitors significantly decreased tau phosphorylation in the same model, but γ-secretase inhibitor, another Aβ blocker, did not affect tau phosphorylation 33. Neurons with the APP V717I FAD mutation also showed an increase in levels of phospho tau and total tau levels 28. More importantly, Muratore and colleagues showed that treatments with Aβ-neutralizing antibodies in those cells significantly reduced the elevated total and phospho tau levels at the early stages of differentiation, suggesting that blocking pathogenic Aβ can reverse the abnormal tau accumulation in APP V717I neurons 28.

Recently, Moore et al. also reported that neurons harboring the APP V717I or the APP duplication FAD mutation showed increases in both total and phospho tau levels 27. Interestingly, altered tau levels were not detected in human neurons carrying PS1 FAD mutations, which significantly increased pathogenic Aβ42 species in the same cells 27. These data suggest that elevated tau levels in these models were not due to extracellular Aβ accumulation but may possibly represent a very early stage of tauopathy. It may also be due to developmental alterations induced by the APP FAD mutations.

As summarized, most human FAD neurons showed significant increases in pathogenic Aβ species, while only APP FAD neurons showed altered tau metabolism that may represent very early stages of tauopathy. However, all of these human FAD neurons failed to recapitulate robust extracellular amyloid plaques, NFTs, or any signs of neuronal death, as predicted in the amyloid hypothesis.

In our recent study, we moved one step closer to proving the amyloid hypothesis. By generating human neural stem cell lines carrying multiple mutations in APP together with PS1, we achieved high levels of pathogenic Aβ42 comparable to those in brains of AD patients 4446.

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Platform for AD drug screening in human neural progenitor cells with FAD mutations in a 3D culture system, which successfully reproduce human AD pathogenesis (amyloid plaques-driven tauopathy).

In addition to the impact on toxic Aβ species, our 3D culture model can test if these antibodies can block tau pathologies in 3D human neural cell culture systems 4446. Human cellular AD models can also be used to determine optimal doses of candidate AD drugs to block Aβ and/or tau pathology without affecting neuronal survival (Fig. 1).

While much progress has been made, many challenges still lie on the path to creating human neural cell culture models that comprehensively recapitulate pathogenic cascades of AD. A major difficulty lies in reconstituting the brain regions most affected in AD: the hippocampus and specific cortical layers. Recent progress in 3D culture technology, such as “cerebral organoids,” may also be helpful in rebuilding the brain structures that are affected by AD in a dish 52,53. These “cerebral organoids” were able to model various discrete brain regions including human cortical areas 52, which enabled them to reproduce microcephaly, a brain developmental disorder. Similarly, pathogenic cascades of AD may be recapitulated in cortex-like structures using this model. Adding neuroinflammatory components, such as microglial cells, which are critical in AD pathogenesis, will illuminate the validity of the amyloid β hypothesis. Reconstitution of robust neuronal death stemming from Aβ and tau pathologies will be the next major step in comprehensively recapitulating AD in a cellular model.


Family-based association analyses of imputed genotypes reveal genome-wide significant association of Alzheimer’s disease with OSBPL6, PTPRG, and PDCL3.

Herold C, Hooli BV, Mullin K, Liu T, Roehr JT, Mattheisen M, Parrado AR, Bertram L, Lange C, Tanzi RE.

Mol Psychiatry. 2016 Feb 2. http://dx.doi.org:/10.1038/mp.2015.218.

Relationship between ubiquilin-1 and BACE1 in human Alzheimer’s disease and APdE9 transgenic mouse brain and cell-based models.

Natunen T, Takalo M, Kemppainen S, Leskelä S, Marttinen M, Kurkinen KM, Pursiheimo JP, Sarajärvi T, Viswanathan J, Gabbouj S, Solje E, Tahvanainen E, Pirttimäki T, Kurki M, Paananen J, Rauramaa T, Miettinen P, Mäkinen P, Leinonen V, Soininen H, Airenne K, Tanzi RE, Tanila H, Haapasalo A, Hiltunen M.

Neurobiol Dis. 2016 Jan;85:187-205. http://dx.doi.org:/10.1016/j.nbd.2015.11.005.

Accumulation of β-amyloid (Aβ) and phosphorylated tau in the brain are central events underlying Alzheimer’s disease (AD) pathogenesis. Aβ is generated from amyloid precursor protein (APP) by β-site APP-cleaving enzyme 1 (BACE1) and γ-secretase-mediated cleavages. Ubiquilin-1, a ubiquitin-like protein, genetically associates with AD and affects APP trafficking, processing and degradation. Here, we have investigated ubiquilin-1 expression in human brain in relation to AD-related neurofibrillary pathology and the effects of ubiquilin-1 overexpression on BACE1, tau, neuroinflammation, and neuronal viability in vitro in co-cultures of mouse embryonic primary cortical neurons and microglial cells under acute neuroinflammation as well as neuronal cell lines, and in vivo in the brain of APdE9 transgenic mice at the early phase of the development of Aβ pathology. Ubiquilin-1 expression was decreased in human temporal cortex in relation to the early stages of AD-related neurofibrillary pathology (Braak stages 0-II vs. III-IV). There was a trend towards a positive correlation between ubiquilin-1 and BACE1 protein levels. Consistent with this, ubiquilin-1 overexpression in the neuron-microglia co-cultures with or without the induction of neuroinflammation resulted in a significant increase in endogenously expressed BACE1 levels. Sustained ubiquilin-1 overexpression in the brain of APdE9 mice resulted in a moderate, but insignificant increase in endogenous BACE1 levels and activity, coinciding with increased levels of soluble Aβ40 and Aβ42. BACE1 levels were also significantly increased in neuronal cells co-overexpressing ubiquilin-1 and BACE1. Ubiquilin-1 overexpression led to the stabilization of BACE1 protein levels, potentially through a mechanism involving decreased degradation in the lysosomal compartment. Ubiquilin-1 overexpression did not significantly affect the neuroinflammation response, but decreased neuronal viability in the neuron-microglia co-cultures under neuroinflammation. Taken together, these results suggest that ubiquilin-1 may mechanistically participate in AD molecular pathogenesis by affecting BACE1 and thereby APP processing and Aβ accumulation.

Correction to Cathepsin L Mediates the Degradation of Novel APP C-Terminal Fragments.

Wang H, Sang N, Zhang C, Raghupathi R, Tanzi RE, Saunders A.

Biochemistry. 2015 Sep 22;54(37):5781.  http://dx.doi.org:/10.1021/acs.biochem.5b00968. Epub 2015 Sep 8. No abstract available.

Massachusetts Alzheimer’s Disease Research Center: progress and challenges.

Hyman BT, Growdon JH, Albers MW, Buckner RL, Chhatwal J, Gomez-Isla MT, Haass C, Hudry E, Jack CR Jr, Johnson KA, Khachaturian ZS, Kim DY, Martin JB, Nitsch RM, Rosen BR, Selkoe DJ, Sperling RA, St George-Hyslop P, Tanzi RE, Yap L, Young AB, Phelps CH, McCaffrey PG.

Alzheimers Dement. 2015 Oct;11(10):1241-5. http://dx.doi.org:/10.1016/j.jalz.2015.06.1887. Epub 2015 Aug 19. No abstract available.

Alzheimer’s in 3D culture: challenges and perspectives.

D’Avanzo C, Aronson J, Kim YH, Choi SH, Tanzi RE, Kim DY.

Bioessays. 2015 Oct;37(10):1139-48. doi: 10.1002/bies.201500063. Epub 2015 Aug 7. Review.

Synaptotagmins interact with APP and promote Aβ generation.

Gautam V, D’Avanzo C, Berezovska O, Tanzi RE, Kovacs DM.

Mol Neurodegener. 2015 Jul 23;10:31. doi: 10.1186/s13024-015-0028-5.

Near-infrared fluorescence molecular imaging of amyloid beta species and monitoring therapy in animal models of Alzheimer’s disease.

Zhang X, Tian Y, Zhang C, Tian X, Ross AW, Moir RD, Sun H, Tanzi RE, Moore A, Ran C.

Proc Natl Acad Sci U S A. 2015 Aug 4;112(31):9734-9. doi: 10.1073/pnas.1505420112. Epub 2015 Jul 21.

A 3D human neural cell culture system for modeling Alzheimer’s disease.

Kim YH, Choi SH, D’Avanzo C, Hebisch M, Sliwinski C, Bylykbashi E, Washicosky KJ, Klee JB, Brüstle O, Tanzi RE, Kim DY.

Nat Protoc. 2015 Jul;10(7):985-1006. doi: 10.1038/nprot.2015.065. Epub 2015 Jun 11.

Cathepsin L Mediates the Degradation of Novel APP C-Terminal Fragments.

Wang H, Sang N, Zhang C, Raghupathi R, Tanzi RE, Saunders A.

Biochemistry. 2015 May 12;54(18):2806-16. doi: 10.1021/acs.biochem.5b00329. Epub 2015 Apr 28. Erratum in: Biochemistry. 2015 Sep 22;54(37):5781.

γ-Secretase modulators reduce endogenous amyloid β42 levels in human neural progenitor cells without altering neuronal differentiation.

D’Avanzo C, Sliwinski C, Wagner SL, Tanzi RE, Kim DY, Kovacs DM.

FASEB J. 2015 Aug;29(8):3335-41. doi: 10.1096/fj.15-271015. Epub 2015 Apr 22.

PLD3 gene variants and Alzheimer’s disease.

Hooli BV, Lill CM, Mullin K, Qiao D, Lange C, Bertram L, Tanzi RE.

Nature. 2015 Apr 2;520(7545):E7-8. doi: 10.1038/nature14040. No abstract available.

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Turning genetic information into working proteins

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series 2; 3.3

James E. Darnell Jr. (1930— )
Vincent Astor Professor Emeritus
2002 Albert Lasker Award for Special Achievement in Medical Science

Responsible for the various tasks required in turning genetic information into working proteins, ribonucleic acids are one of the most essential players in the life of a cell. First discovered in 1868, RNA today remains the subject of intense scientific scrutiny. Over the course of a career dedicated to understanding the intricate workings of gene transcription, Rockefeller University scientist James E. Darnell Jr. has revealed some of RNA’s most secretive and surprising mechanisms. For his half-century of illuminating research, Dr. Darnell received the 2002 Albert Lasker Award for Special Achievement in Medical Science.

In 1963, Dr. Darnell described a phenomenon he termed “RNA processing,” a step in the process of gene transcription, which had only recently been elucidated in bacterial systems. Working with mammalian cells — which differ from bacterial cells in that they contain a nucleus, where RNA is created — Dr. Darnell observed that very long strings of RNA disappear from the cell nucleus and that subsequently, shorter RNAs resembling the absent longer ones appear in the cytoplasm. Mammalian cells, he concluded, must distill their massive, immature nuclear RNA into shorter, mature forms that are individually coded for specific purposes by specific segments of the genome.

Dr. Darnell carried the principles of his finding — which he made in ribosomal RNA, part of the construction crew that builds cellular proteins — to other long nuclear RNA, including the longest one, which he named heterogeneous nuclear RNA (hnRNA). His hypothesis, that hnRNA is the precursor of the better known messenger RNA — which carries the genetic blueprint for protein building — soon bore fruit when he found a structural correlation between the two. Certain hnRNAs and nearly all messenger RNAs have a “tail” of adenine nucleotides at one end. Dr. Darnell followed this discovery with the observation that when an hnRNA string with an adenine tail disappears from the nucleus, a messenger RNA with the same tail then appears in the cytoplasm, suggesting a causal link between the two. When he found a second similarity — a cap at the end of the string opposite the adenine tail — he faced a conundrum. Scientific dogma had it that the order of nucleotides in any RNA mirrors that of DNA, whether the RNA is modeled from somewhere in the middle of the DNA or from one of the ends. The matching of a nuclear RNA to its cytoplasmic product by two end pieces glued together was surprising, but the concept was soon proven by colleagues at other institutions and called RNA splicing.

After a brief sojourn in Paris to work in François Jacob’s lab, Darnell worked at MIT, the Albert Einstein College of Medicine, and Rockefeller University on the relationship between mRNA and hnRNA. hnRNA was believed to be the precursor to mRNA, and despite making some key discoveries, Darnell admits that he could not free his imagination from the idea of colinearity and envision an hnRNA spliced to produce a smaller mRNA.

At this time, Darnell turned his attention to the question he had pondered since Paris: how were genes regulated in animal cells? This led to the discovery of the STAT and the Jak-STAT pathway of transcription control.

With the knowledge of RNA processing and splicing, Dr. Darnell next examined how cells begin the process of transcription and how they activate particular segments of DNA. Having moved to Rockefeller University in 1974, he found in the early 1980s that cells retain their specificity only in the context of their natural environment. Away from other liver cells, for example, a single liver cell stops producing liver-specific RNA, though it continues to make RNA for more generic cellular tasks. To pinpoint the signals responsible, which he believed must be coming from outside the cell, Dr. Darnell took a closer look at interferons (IFN), proteins that warn a cell when it’s time to raise its genetic defenses against harmful microbes.

Dr. Darnell’s laboratory studies how signals from the cell surface affect transcription of genes in the nucleus. Originally using interferon as a model cytokine, the Darnell group discovered that cell transcription was quickly changed by binding of cytokines to the cell surface. Introducing IFNβ into cell cultures, he watched as a particular type of mRNA accumulated in the cytoplasm, unaccompanied by any new protein synthesis. Analyzing the mRNA led him to the segment of DNA that had been activated, and the lack of new proteins told him that the cell contained its own, usually dormant, IFN-responsive transcription factor. By isolating a particular stretch of DNA from IFN-treated cells, he was able to call out of hiding the proteins that make up that factor, which, partly because they respond to signals very quickly, he called “STATs.” Dr. Darnell then traced the chemical relay that activates the STATs after IFN contact, called the Jak-Stat pathway.

The bound interferon led to the tyrosine phosphorylation of latent cytoplasmic proteins now called STATs (signal transducers and activators of transcription) that dimerize by reciprocal phosphotyrosine-SH2 interchange. They accumulate in the nucleus, bind DNA and drive transcription. This pathway has proved to be of wide importance, with seven STATs now known in mammals that take part in a wide variety of developmental and homeostatic events in all multicellular animals. Crystallographic analysis defined functional domains in the STATs, and current attention is focused on two areas: how the STATs complete their cycle of activation and inactivation, which requires regulated tyrosine dephosphorylation; and how persistent activation of STAT3 that occurs in a high proportion of many human cancers contributes to blocking apoptosis in cancer cells. Current efforts are devoted to inhibiting STAT3 with modified peptides that can enter cells.


Dr. Darnell received his M.D. in 1955 from the Washington University School of Medicine. His career has included poliovirus research with Harry Eagle at the National Institute of Allergy and Infectious Diseases, research with François Jacob at the Pasteur Institute in Paris and academic appointments at the Massachusetts Institute of Technology, the Albert Einstein College of Medicine and Columbia University. In 1974 Dr. Darnell joined Rockefeller as Vincent Astor Professor, and from 1990 to 1991 he was vice president for academic affairs.

A member of the National Academy of Sciences since 1973, he has received numerous awards, including the 2012 Albany Medical Center Prize in Medicine and Biomedical Research, the 2003 National Medal of Science, the 2002 Albert Lasker Award for Special Achievement in Medical Science, the 1997 Passano Award, the 1994 Paul Janssen Prize in Advanced Biotechnology and Medicine and the 1986 Gairdner Foundation International Award.

He is the coauthor with S.E. Luria of General Virology and the founding author with Harvey Lodish and David Baltimore of Molecular Cell Biology, now in its seventh edition. His book RNA, Life’s Indispensable Molecule was published in July 2011 by Cold Spring Harbor Laboratory Press. He is a member of the American Academy of Arts and Sciences and a foreign member of The Royal Society and The Royal Swedish Academy of Sciences.


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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD


Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at


This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:


Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 






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Angiogenesis Inhibitors [9.5]

Writer and Curator: Larry H Bernstein, MD, FCAP

This article has the following structure:

9.5.1 Motesanib (AMG 706)

9.5.2 Drugs that block cancer blood vessel growth (anti angiogenics)

9.5.3 Recent Advances in Anti-Angiogenic Therapy of Cancer

9.5.4 Angiogenesis inhibitors in cancer therapy: mechanistic perspective on classification and treatment rationales

9.5.5 LUCITANIB a VEGFR/FGFR dual kinase inhibitor in Phase 2 trials

9.5.1 Motesanib (AMG 706)



Motesanib (AMG 706) is an experimental drug candidate originally developed by Amgen[1] but is now being investigated by theTakeda Pharmaceutical Company. It is an orally administered small molecule belonging to angiokinase inhibitor class which acts as an antagonist of VEGF receptorsplatelet-derived growth factor receptors, and stem cell factor receptors.[2] It is used as the phosphate salt motesanib diphosphate.

Motesanib, also known as AMG-706, is an orally administered multikinase inhibitor that selectively targets VEGF receptors, platelet-derived growth factor receptors, and Kit receptors.






9.5.2 Drugs that block cancer blood vessel growth (anti angiogenics)


When it has reached 1 to 2mm across, a tumor needs to grow its own blood vessels in order to continue to get bigger. Some cancer cells make a protein called vascular endothelial growth factor (VEGF). The VEGF protein attaches to receptors on cells that line the walls of blood vessels within the tumour.

Drugs that block blood vessel growth factor

Some drugs block vascular endothelial growth factor (VEGF) from attaching to the receptors on the cells that line the blood vessels. This stops the blood vessels from growing.

A drug that blocks VEGF is bevacizumab (Avastin). It is also a monoclonal antibody.

Drugs that block signalling within the cell

Some drugs stop the VEGF receptors from sending growth signals into the blood vessel cells. These treatments are also called cancer growth blockers or tyrosine kinase inhibitors (TKIs).

Sunitinib (Sutent) is a type of TKI that blocks the growth signals inside blood vessel cells. It is used to treat kidney cancer and a rare type of stomach cancer called gastrointestinal stromal tumour (GIST).

Drugs that affect signals between cells

Some drugs act on the chemicals that cells use to signal to each other to grow. This can block the formation of blood vessels. Drugs that works in this way include thalidomide and lenalidomide (Revlimid).

Each drug has different side effects. You can look up the name of your drug in our cancer drug section to find out about the side effects you may have.

To find trials using anti angiogenesis treatment go to our clinical trials database and type ‘angiogenesis’ into the search box.


Tumors can cause their blood supply to form by giving off chemical signals that stimulate angiogenesis. Tumors can also stimulate nearby normal cells to produce angiogenesis signaling molecules. The resulting new blood vessels “feed” growing tumors with oxygen and nutrients, allowing the cancer cells to invade nearby tissue, to move throughout the body, and to form colonies of cancer cells, called metastases. Because tumors cannot grow beyond a certain size or spread without a blood supply, scientists are trying to find ways to block tumor angiogenesis.

Angiogenesis requires the binding of signaling molecules, such as vascular endothelial growth factor (VEGF), to receptors on the surface of normal endothelial cells. When VEGF and other endothelial growth factors bind to their receptors on endothelial cells, signals within these cells are initiated that promote the growth and survival of new blood vessels.

Angiogenesis inhibitors interfere with various steps in this process. For example, bevacizumab (Avastin®) is a monoclonal antibody that specifically recognizes and binds to VEGF (1). When VEGF is attached to bevacizumab, it is unable to activate the VEGF receptor. Other angiogenesis inhibitors, including sorafenib and sunitinib, bind to receptors on the surface of endothelial cells or to other proteins in the downstream signaling pathways, blocking their activities (2).

The U.S. Food and Drug Administration (FDA) has approved bevacizumab to be used alone forglioblastoma that has not improved with other treatments and to be used in combination with other drugs to treat metastatic colorectal cancer, some non-small cell lung cancers, and metastatic renal cell cancer. Bevacizumab was the first angiogenesis inhibitor that was shown to slow tumor growth and, more important, to extend the lives of patients with some cancers.

The FDA has approved other drugs that have antiangiogenic activity, including sorafenib (Nexavar®), sunitinib(Sutent®), pazopanib (Votrient®), and everolimus (Afinitor®). Sorafenib is approved for hepatocellular carcinoma and kidney cancer, sunitinib and everolimus for both kidney cancer and neuroendocrine tumors, and pazopanib for kidney cancer.

Angiogenesis inhibitors are unique cancer-fighting agents because they tend to inhibit the growth of blood vessels rather than tumor cells. In some cancers, angiogenesis inhibitors are most effective when combined with additional therapies, especially chemotherapy. It has been hypothesized that these drugs help normalize the blood vessels that supply the tumor, facilitating the delivery of other anticancer agents, but this possibility is still being investigated.

Angiogenesis inhibitor therapy does not necessarily kill tumors but instead may prevent tumors from growing. Therefore, this type of therapy may need to be administered over a long period.

Initially, it was thought that angiogenesis inhibitors would have mild side effects, but more recent studies have revealed the potential for complications that reflect the importance of angiogenesis in many normal body processes, such as wound healing, heart and kidney function, fetal development, and reproduction. Side effects of treatment with angiogenesis inhibitors can include problems with bleeding, clots in the arteries (with resultant stroke or heart attack), hypertension, and protein in the urine (35). Gastrointestinal perforation and fistulas also appear to be rare side effects of some angiogenesis inhibitors.

In addition to the angiogenesis inhibitors that have already been approved by the FDA, others that target VEGF or other angiogenesis pathways are currently being tested in clinical trials (research studies involving patients). If these angiogenesis inhibitors prove to be both safe and effective in treating human cancer, they may be approved by the FDA and made available for widespread use.

In addition, phase I and II clinical trials are testing the possibility of combining angiogenesis inhibitor therapy with other treatments that target blood vessels, such as tumor-vascular disrupting agents, which damage existing tumor blood vessels (6).

9.5.3 Recent Advances in Anti-Angiogenic Therapy of Cancer

Rajeev S. Samant and Lalita A. Shevde
Oncotarget. 2011 Mar; 2(3): 122–134.

More than forty anti-angiogenic drugs are being tested in clinical trials all over the world. This review discusses agents that have approved by the FDA and are currently in use for treating patients either as single-agents or in combination with other chemotherapeutic agents.

Tumor angiogenesis is generation of a network of blood vessels within the cancerous growth. This process can occur two ways: The more accepted model involves the release of signaling molecules by the tumor cells; these molecules activate the surrounding tissue to promote growth of new blood vessels. This stimulates vascular endothelial cells to divide rapidly [910]. The other model proposes the generation of new vasculature by vasculogenic mimicry. This model argues that the tumor cells trans-differentiate in endothelial-like cells and create structures from inside of the tumor tapping into a nearby blood vessel [4].

Escape of the tumor cell from the confines of the primary tumor to distant body parts is the pre-requisite for hematogenous metastasis. This escape route is provided by the tumor vasculature. Thus, it was envisioned that inhibition of angiogenesis will also lead to inhibition of metastasis. This phenomenon was demonstrated by very elegant mouse model studies using angiostatin [1112]. Angiostatin was also demonstrated to be secreted by some primary tumors leading to restricted growth of the metastasis leading to “dormancy” of the metastasis. Mice deficient in angiogenesis (Id1 & Id3 deficient) showed significantly less tumor take rates [13]. Independent studies showed absence of metastasis in angiogenesis deficient mice [1415]. Defective angiogenesis was attributed to impaired VEGF-dependent recruitment of precursor endothelial cells from the bone marrow to the newly developing tumor vasculature [16].

Metastasis of malignant tumors to regional lymph nodes is one of the early signs of cancer spread in patients, and it occurs at least as frequently as hematogenous metastasis [17]. Particularly, in cancers, such as breast cancer, lymphatic metastasis is a predominant route for tumor spread. The contribution of lymphatic system to the tumor growth is an area that is relatively less studied. However, lymphatic vessels are speculated to contribute to tumor growth and metastasis in a variety of ways. The VEGF, FGF2 and PDGF produced by vascular endothelial cells are proposed to be involved in the activation of lymphatic endothelial cells, which in turn produce matrix metalloproteases and urokinase plasminogen activator (uPA) that can promote malignant tumor growth. Thus, there exists a synergistic crosstalk between the tumor and the lymphatic vessels and blood vessels.

Angiogenesis is a complex and intricately regulated process. Like all other regulated biological phenomena, angiogenesis has activators or pro-angiogenic factors and inhibitors or anti-angiogenic factors [9].

The Activators

Tumor cells activate signaling pathways that promote uncontrolled proliferation and survival. These include the PI3K/AKT/mTOR pathway, Hedgehog pathway and, Wnt pathway [1824] that produce pro-angiogenic signaling intermediates [2526]. Among the several reported activators of angiogenesis present in cells two proteins appear to be the most important for sustaining tumor growth: vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). VEGF and bFGF are secreted by the tumor into the surrounding tissue. They bind to their cognate receptors on endothelial cells. This activates a signaling cascade that transmits a nuclear signal prompting target genes to activate endothelial cell growth. Activated endothelial cells also produce matrix metalloproteinases (MMPs). These MMPs break down the extracellular matrix and allow the migration of endothelial cells. The division and migration of the endothelial cells leads to formation of new blood vessels [2728].

The Inhibitors

If angiogenesis is so critical for the tumor growth, then agents that inhibit angiogenesis would have great therapeutic value. With the discovery of endostatin, the concept of anti-angiogenic therapy was launched and popularized by Dr. Folkman [29]. Angiogenesis inhibitors have been discovered from a variety of sources. Some are naturally present in the human body e.g. specific fragments of structural proteins such as collagen or plasminogen (angiostatin, endostatin, tumstatin) [30]. Others are natural products in green tea, soy beans, fungi, mushrooms, tree bark, shark tissues, snake venom etc. [31]. A plethora of synthetic compounds are also characterized to have anti-angiogenic properties [32].


Since angiogenesis is an event critical to primary tumor growth as well as metastasis, anti-angiogenic treatment of tumors is a highly promising therapeutic avenue [33]. Thus, for over last couple of decades, there has been a robust activity aimed towards the discovery of angiogenesis inhibitors [3435]. More than forty anti-angiogenic drugs are being tested in human cancer patients in clinical trials all over the world. From the several anti-angiogenic agents reported, we have focused this review on discussing those agents that have received FDA approval in the United States and are currently in use for treating patients either as a single-agent or in combination with other chemotherapeutic agents (Figure ​(Figure1).1). Based on functionality, the anti-angiogenic drugs can be sub-divided into three main groups:

angiogenesis inhibitors oncotarget-02-122-g001

angiogenesis inhibitors oncotarget-02-122-g001

Figure 1


Targets of FDA-approved angiogenesis inhibitors: Angiogenesis inhibitors impact both, the tumor as well as the endothelial cells resulting in the disruption of the effects of the microenvironment in promoting tumor growth and angiogenesis

Drugs that inhibit growth of endothelial cells

e.g. Endostatin and combretastatin A4, cause apoptosis of the endothelial cells [36]. Thalidomide is also a potent inhibitor of endothelial cell growth [37].

Drugs that block angiogenesis signaling

e.g. anti-VEGF antibodies (Avastin, FDA approved for colorectal cancer), Interferon-alpha (inhibits the production of bFGF and VEGF) [36].

Drugs that block extracellular matrix breakdown

e.g. inhibitors of MMPs [38].


Conventional chemotherapy is usually a systemic therapy that tries to capture a narrow therapeutic window offered by rapid proliferation of tumor cells compared to the normal cells. Chemotherapy has significant side effects such as hair loss, diarrhea, mouth ulcer, infection, and low blood counts. Anti-angiogenic therapy has several advantages over chemotherapy as it is mostly not directed towards directly killing cells but stopping the blood vessel formation, an event that is rare in tissues other than growing tumor. Hence it is well tolerated by the patients and has fewer side effects [29]. There are currently seven approved anti-cancer therapies in two primary categories:

  1. Monoclonal antibodies directed against specific pro-angiogenic growth factors and/or their receptors
  2. Small molecule tyrosine kinase inhibitors (TKIs) of multiple pro-angiogenic growth factor receptors.

Besides these, inhibitors of mTOR (mammalian target of rapamycin), proteasome inhibitors and thalidomide have also been reported to indirectly inhibit angiogenesis through mechanisms that are not completely understood.


Four monoclonal antibody therapies are approved to treat several tumor types:

Bevacizumab (Avastin®)

The first FDA approved angiogenesis inhibitor, Avastin is a humanized monoclonal antibody that binds biologically active forms of vascular endothelial growth factor (VEGF) and prevents its interaction with VEGF receptors (VEGFR-1 and VEGFR-2), thereby inhibiting endothelial cell proliferation and angiogenesis. Bevacizumab has been tested in phase I studies in combination with chemotherapy with a good safety profile [39]. This treatment is approved for metastatic colorectal cancer and non-small cell lung cancer [4043]. Bevacizumab has also evolved as a first line of treatment in combination with paclitaxel in breast cancer patients by virtue of its ability to double median progression-free survival (PFS) [44]. In combination with chemoendocrine therapy (including capecitabine and vinorelbine, and letrozole) bevacizumab treatment significantly decreased the percentage of viable circulating endothelial cells and prevented the chemotherapy-induced mobilization of circulating progenitors [45]. In combination with irinotecan, bevacizumab significantly increased PFS in glioma patients [4647]. VEGF has emerged as a compelling therapeutic target for leukemias. Inhibition of angiogenesis in hematological malignancies interdicts the angiogenesis within the bone marrow ecosystem comprised of multiple cell types, including fibroblasts, endothelial progenitor cells, endothelial cells, dendritic cells and, malignant cells, blocking the availability of nutrients to cancer cells and disrupting crosstalk between the various cell types to curtail the malignant phenotype [48].

Cetuximab (Erbitux®)

This is a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), preventing ligand binding and activation of the receptor resulting in internalization and degradation of the receptor culminating in inhibition of cell proliferation and angiogenesis. Cetuximab downregulated VEGF expression in a dose-dependent manner in a human colorectal carcinoma (CRC) cell line and in human CRC mouse xenografts [49]. The xenografts also showed a significant reduction in blood vessel counts following several rounds of cetuximab treatment [49], indicating that the tumor-promoting effects of EGFR overexpression may be mediated through VEGF stimulation and tumor angiogenesis. This treatment is approved for metastatic CRC and head and neck cancer [50] in patients who are refractory to irinotecan-based chemotherapy. In combination with irinotecan (an inhibitor of topoisomerase I), cetuximab is the first monoclonal antibody that has been approved by the FDA as second-line treatment for metastatic colorectal cancer [5152]. In Phase I and Phase III trials [5354] cetuximab significantly improved the effects of radiotherapy in patients with unresectable (cannot be removed by surgery) squamous cell carcinoma of the head and neck (SCCHN). Cetuximab has also been shown to sensitize cells to radiation and chemotherapy, potentially through blocking EGFR nuclear import and the associated activation of DNA protein kinase enzymes necessary for repairing radiation- and chemotherapy-induced DNA damage [55]. Compared to radiation alone, cetuximab plus radiation therapy can nearly double the median survival in patients with a certain kind of head and neck cancer that has not spread to other parts of the body [54] making cetuximab the only drug achieving interesting response rate in second line treatment of advanced SCCHN [56]. Cetuximab was also found to be tolerated well in combination with cisplatin, or carboplatin, and fluorouracil [5758].

Panitumumab (Vectibix™)

It is a fully humanized anti-EGFR monoclonal antibody that binds specifically to the human EGFR. Panitumumab is a recombinant human monoclonal antibody [59]; therefore, the risk of an infusion reaction is minimized. Vectibix® is indicated as a single agent for the treatment of EGFR-expressing, metastatic colorectal carcinoma with disease progression on or following fluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapy regimens [6062]. The effectiveness of Vectibix® as a single agent for the treatment of EGFR-expressing, metastatic CRC is based on progression-free survival [6364]. Panitumumab is used in patients who are not responding to regimens containing fluorouracil, oxaliplatin, and irinotecan [60]. Patients often receive panitumumab after receiving bevacizumab or cetuximab. Panitumumab can be given with FOLFOX (oxaliplatin, leucovorin, and fluorouracil) or FOLFIRI (irinotecan, leucovorin, and fluorouracil) regimens, or as a single agent. Currently no data are available that demonstrate an improvement in disease-related symptoms or increased survival with Vectibix® in colon cancer [65]. This drug is also being tested for aerodigestive track and head and neck cancer [6667].

Trastuzumab (Herceptin®)

Is a humanized monoclonal antibody that binds the extracellular domain of HER-2, which is overexpressed in 25-30% of invasive breast cancer tumors [68]. HER2-positive breast cancer is highly aggressive disease with high recurrence rate, poorer prognosis with decreased survival compared with HER2-negative breast cancer [69]. Herceptin® is designed to target and block the function of HER2 protein overexpression. This is the first humanized antibody is approved for Breast cancer [70]. Herceptin® is approved by the FDA to treat HER2 positive breast cancer that has metastasized after treatment with other anticancer drugs [71]. It is also approved to be used with other drugs to treat HER2-positive breast cancer that has spread to the lymph nodes to be used after surgery. The FDA first approved Herceptin in September 1998 [7173]. In November 2006, the FDA approved Herceptin as part of a treatment regimen containing doxorubicin, cyclophosphamide and paclitaxel, for the adjuvant treatment of patients with HER2-positive, node-positive breast cancer (http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/default.htm). In January 2008, the FDA approved Herceptin as a single agent for the adjuvant treatment of HER2-overexpressing node-negative (ER/PR-negative or with one high-risk feature) or node-positive breast cancer, following multi-modality anthracycline-based therapy (http://biopharminternational.findpharma.com/biopharm/News/FDA-Approves-Expanded-Adjuvant-Indications-for-Her/ArticleStandard/Article/detail/518867). Trastuzumab is also being studied in the treatment of other types of cancers such as pancreatic [74], endometrial [75], lung [76], cervical [77] and ovarian cancer [78]


Protein tyrosine kinases have emerged as crucial targets for therapeutic intervention in cancer especially because they play an important role in the modulation of growth factor signaling. As per ClinicalTrials.gov (www.clinicaltrials.gov), there are 43 ongoing studies on tyrosine kinase inhibitors in angiogenesis. Since discussing all of them is beyond the scope of this article, we have focused our discussion on the three TKIs that are currently approved as anti-cancer therapies:

Erlotinib (Tarceva®)

Erlotinib hydrochloride (originally coded as OSI-774) is an orally available, potent, reversible, and selective inhibitor of the EGFR (ErbB1) tyrosine kinase activity. Erlotinib hydrochloride has been approved by FDA for treatment of patients with locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen [7980]. Interesting recent studies have demonstrated that since Erlotinib and Bevacizumab act on two different pathways critical to tumor growth and dissemination, administering these drugs concomitantly may confer additional clinical benefits to cancer patients with advanced disease. This combination therapy may prove to be a viable second-line alternative to chemotherapy in patients with NSCLC [81]. Also, for patients with locally advanced, unresectable or metastatic pancreatic carcinoma, Erlotinib has received FDA approval for the treatment in combination with gemcitabine [8283]. Erlotinib is also being studied in the treatment of other types of cancers. For example combination of Erlotinib with Bevacizumab has been evaluated in metastatic breast cancer [84], hepatocellular carcinoma [85] and in metastatic renal cancer [86] as phase II trials. Outcomes for prostate, cervical and colorectal cancers treated with Erlotinib are cautiously optimistic [8789].

Sorafenib (Nexavar®)

Sorafenib is an orally active inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-β, and Raf-1 tyrosine kinase activity [90]. It has received the approval of FDA for the treatment of patients with unresectable hepatocellular carcinoma [91] and advanced renal cell carcinoma [92]. However, not all advanced hepatocellular carcinoma patients were able to tolerate sorafenib and some patients experienced tumor progression [91]. Sorafenib has shown improvements in PFS in patients with renal cell carcinoma [93]. It is one of the aggressively studied drugs. According to the NCI clinical trials search results, there are about 168 active clinical trials involving sorafenib in a variety of cancers.

Sunitinib (Sutent®)

Sunitinib targets activity of multiple tyrosine kinases such as VEGFR-1, VEGFR-2, VEGFR-3, PDGFR- β, and RET [94]. It is approved by FDA as Sunitinib malate for treating advanced (metastatic) renal cell carcinoma [95]. It is also approved by FDA for gastrointestinal stromal tumor (GIST) in patients whose disease has progressed or who are unable to tolerate treatment with imatinib (Gleevec), the current treatment for GIST patients [9596]. Sunitinib has shown early evidence of anti-tumor activity in Phase II trials in US, European and Asian patients with locally advanced, unresectable and metastatic hepatocellular carcinoma. A Phase III trial of sunitinib in hepatocellular carcinoma is ongoing [97]. According to the NCI clinical trials search results, Sunitinib is currently evaluated in about 150 active clinical trials. It is evaluated for ovarian [98], breast [99] and non small cell lung cancer [100] among others [101].

Inhibitors of mTOR

mTOR plays a part in the PI3 kinase/AKT pathway involved in tumor cell proliferation and angiogenesis [102]. Rapamycin and related mTOR inhibitors inhibit endothelial cell VEGF expression, as well as VEGF-induced endothelial cell proliferation [103]. Inhibitors of mTOR are an important class of anti-angiogenic agents. These include: deforolimus, everolimus, rapamycin (sirolimus), and temsirolimus [104105]. Temsirolimus (Toricel™) is a small molecule inhibitor of mTOR, approved for treating advanced renal cell carcinoma [106]. It is a type of rapamycin analog and a type of serine/threonine kinase inhibitor, it is also called CCI-779. In pre-clinical models combination therapy for treating breast cancer using anti-estrogen, ERA-923, and temsirolimus has been successfully tested [107]. It is found to be highly effective against human melanoma when tested in combination with cisplatin and DTIC (in independent studies) in a SCID mouse xenotranplantation model [108109]. There are over 41 active studies of Temsirolimus for a variety of solid tumors [110]. mTOR inhibition has also been strongly advocated in as a putative cancer therapeutic strategy for urologic malignancies [111]. In a pilot study (6 patients) with imatinib-resistant CML, rapamycin induced major and minor leukocyte responses, with an observed decrease in the mRNA levels of VEGFA in circulating leukaemic cells [112]. Combination treatments for breast cancer with aromatase inhibitor [113] and letrozol [114] are also being evaluated. Rapamycin treatment brought partial responses (>50% reduction in the absolute number of blood blasts) and stable disease in adult refractory/relapsed AML [115]. In a recent report, Deforolimus was studied in a Phase 2 trial in pretreated patients with various hematological malignancies, including ALL, AML, CLL, CML, MDS, agnogenic myeloid metaplasia, mantle cell lymphoma and T-cell leukemia/lymphoma [116]. Overall, 40% of deforolimus-treated patients experienced hematological improvement or stable disease.


Bortezomib (Velcade®)

Is a proteasome inhibitor that disrupts signaling of cancer cells, leading to cell death and tumor regression. It is the first compound in its class to be used in clinical practice. It has indirect anti-angiogenic properties [117]. While its exact mechanism is not understood, it induces the pro-apoptotic BH3-only family member NOXA in a p53 independent fashion triggering of a caspase cascade culminating in apoptosis in melanoma and myeloma cells [118]. It is FDA-approved for the treatment of myeloma that has relapsed after two prior treatments (or where resistance has developed following the last treatment). It was also found to induce high quality responses as third line salvage therapy with acceptable toxicity in a significant proportion of homogeneously pre-treated myeloma patients with progressive disease after autologous transplantation and thalidomide. [119]. In a Phase 3 trial involving 669 myeloma patients treated with at least one prior therapy, bortezomib increased median, improved overall survival, and increased response rate, compared with high-dose dexamethasone [120]. In combination with doxorubicin and gemcitabine, bortezomib was also found to be effective in heavily pretreated, advanced Cutaneous T cell Lymphomas (CTCL) [121]. Bortezomib was also reported to be active as a single agent for patients with relapsed/refractory CTCL and Peripheral T Cell Lymphoma (PTCL) with skin involvement [122]. On the contrary, the use of bortezomib was discouraged after a phase II study revealed that found in combination with dexamethasone, bortezomib is not active in heavily pre-treated patients with relapsed Hodgkin’s lymphoma [123124].

Thalidomide (Thalomid®)

Possesses immunomodulatory, anti-inflammatory, and anti-angiogenic properties, although the precise mechanisms of action are not fully understood. Thalidomide was the first angiogenesis inhibitor to demonstrate clinical efficacy in multiple myeloma [37125]. Specifically in myeloma, thalidomide down-regulated VEGF secretion from bone marrow endothelial cells obtained from patients with active disease. In a landmark Phase 2 clinical trial, 169 previously treated patients with refractory myeloma received thalidomide monotherapy [126]. Partial response, was achieved in 30% of patients, and 14% achieved a complete or nearly complete remission. The survival rate at 2 years was 48%. These results led to many subsequent clinical studies of thalidomide in myeloma, leading ultimately to FDA approval of the drug in 2006, for the treatment of newly diagnosed multiple myeloma, in combination with dexamethasone. In the pivotal Phase 3 trial, the response rate in patients receiving thalidomide plus dexamethasone was 63% compared to 41% with dexamethasone alone [127]. Long-term outcome measures, including time-to-progression (TTP) and PFS, were recently reported for a 470 patient randomized, placebo-controlled Phase 3 clinical trial of a similar protocol in newly diagnosed multiple myeloma, with comparable overall response rates [128]. Significant increases resulted in both median TTP and median PFS for the thalidomide plus dexamethasone group versus dexamethasone alone.

Thalidomide was found to be moderately tolerated and minimally effective in patients with histologically proven advanced hepatocellular carcinoma [129]. Thalidomide provided no survival benefit for patients with multiple, large, or midbrain metastases when combined with WBRT (whole-brain radiation therapy) [130]. On the contrary, thalidomide did not significantly add to the efficacy of the fludarabine, carboplatin, and topotecan (FCT) regimen in poor prognosis AML patients [131] and was also ineffective in improving prognosis or decreasing plasma VEGF levels in patients with persistent or recurrent leiomyosarcoma of the uterus [132].


While conventional anti-angiogenic therapy is based on Maximum Tolerated Doses (MTD), the cells involved in angiogenesis may regenerate during the three- to four-week interval between cycles of the chemotherapy. Taking advantage of the fact that endothelial cells are about 10–100 times more susceptible to chemotherapeutic agents than cancer cells, therapy based on daily, oral, low-dose chemotherapeutic drugs was designed. Metronomic chemotherapy refers to the close, rhythmic administration of low doses of cytotoxic drugs, with minimal or no drug-free breaks, over prolonged periods. Metronomic therapy appears promising mainly due to the fact that its anti-angiogenic and anti-tumorigenic effects are accompanied by low toxicity, limited side effects, no need for hospitalization and allowing for feasible combinations with selective inhibitors of angiogenesis. There are several foreseeable advantages and opportunities for metronomic chemotherapy: activity against the parenchymal and stromal components, pro-apoptotic activity, reduction of the likelihood of emergence of acquired resistance, feasibility of long term administration and acceptable systemic side effects [133]. In a pilot phase II study conducted by Correale et al [134] to investigate the toxicity and activity of the novel metronomic regimen of weekly cisplatin and oral etoposide in high-risk patients with NSCLC, the objective response rate was 45.2%, disease control was 58.1%, meantime to progression and survival were 9 and 13 months, respectively. Pharmacokinetic analysis showed that this regimen allowed a greater median monthly area under the curve of the drugs than conventional schedules. In a Phase I trial of metronomic dosing of docetaxel and thalidomide, of the 26 patients with advanced tumors enrolled, prolonged freedom from disease progression was observed in 44.4% of the evaluable patients [135].

Circulating endothelial progenitor cells (EPCs) also participate in tumor angiogenesis. In a study comparing the effects of metronomic chemotherapy over conventional dose-dense chemotherapy, it was found that the numbers of circulating EPCs and the plasma levels of VEGF increased sharply, doubling pre-therapeutic levels at day 21 after conventional chemotherapy, whereas under low-dose metronomic chemotherapy, the numbers of circulating EPCs decreased significantly and VEGF plasma concentrations remained unchanged. These observations provide evidence that conventional dose-dense chemotherapy leads to rebound EPC mobilization even when given with adjuvant intention, while low-dose metronomic scheduling of cytotoxic substances such as trofosfamide may sharply reduce EPC release into the circulation. [136].

Combined bevacizumab and metronomic oral cyclophosphamide was also discovered to be a safe and effective regimen for heavily pre-treated ovarian cancer patients [137]. Treatment with metronomic capecitabine and cyclophosphamide in combination with bevacizumab was shown to be effective in advanced breast cancer and additionally was minimally toxic [138]. Metronomic treatment with carboplatin and vincristine associated with fluvastatin and thalidomide significantly increased survival of pediatric brain stem tumor patients. Tumor volume showed a significant reduction accompanied by increased quality of life [139]. Thus, given the fact that the most evident effect of selective anti-angiogenic agents (i.e. bevacizumab) is the significant prolonging of the duration of response obtainable by chemotherapy alone, with minimal possible side effects of cytotoxic agents given in association metronomic chemotherapy should be considered both as novel up-front or maintenance treatment in patients with biologically poorly aggressive advanced cancer diseases [140].

Overall, metronomic chemotherapy was able to induce tumor stabilization and prolong the duration of clinical benefit, without much associated toxicity. Emerging evidence suggests that metronomic chemotherapy could also activate the host immune system and potentially induce tumor dormancy [141143].


While angiogenesis as a hallmark of tumor development and metastasis is now a validated target for cancer treatment, the overall benefits of anti-angiogenic drugs from the perspective of impacting survival have left much to desire, endorsing a need for developing more effective therapeutic regimens e.g., combining anti-angiogenic drugs with established chemotherapeutic drugs [144145]. There are now several agents that target the tumor vasculature through different pathways, either by inhibiting formation of the tumor neovasculature or by directly targeting the mature tumor vessels. The main body of evolving evidence suggests that their effects are compounded by their synergistic use with conventional chemotherapy rather than individual agents. Anti-angiogenic drugs such as bevacizumab can bring about a transient functional normalization of the tumor vasculature. This can have an additive effect when co-administered with chemo/radiotherapy. But long term inhibition of angiogenesis reduces tumor uptake of co-administered chemotherapeutic agents. This underscores the need for discovering new targets for anti-angiogenic therapy in order to effectively prohibit angiogenesis and circumvent mechanisms that contribute to resistance mechanisms that emerge with long term use of anti-angiogenic therapies. It also warrants a need to define reliable surrogate indicators of effectiveness of the anti-angiogenic therapy as well as dependable markers for identifying the patients who are most likely to benefit from the combination of anti-angiogenic therapy and conventional chemotherapy.

Several new frontiers are emerging. New advances in understanding endothelial cells, which constitute the tumor vasculature, towards developing antiangiogenic strategies are one of the important ones [146147]. Novel cellular targets such as integrins and microRNAs and novel treatment options such as possible use of pharmaconutrients to modulate angiogenic pathways need careful testing and evaluation [148151]. Finally, the administration of these drugs in a metronomic schedule is likely to improve the overall response to anti-angiogenic drugs making it feasible to administer them with conventionally toxic chemotherapeutic drugs, thus increasing the armamentarium of drug combinations that can be employed for treatment.

9.5.4 Angiogenesis inhibitors in cancer therapy: mechanistic perspective on classification and treatment rationales

El-Kenawi AE1, El-Remessy AB.
Br J Pharmacol. 2013 Oct; 170(4):712-29.

Angiogenesis, a process of new blood vessel formation, is a prerequisite for tumor growth to supply the proliferating tumor with oxygen and nutrients. The angiogenic process may contribute to tumour progression, invasion and metastasis, and is generally accepted as an indicator of tumor prognosis. Therefore, targeting tumor angiogenesis has become of high clinical relevance. The current review aimed to highlight mechanistic details of anti-angiogenic therapies and how they relate to classification and treatment rationales. Angiogenesis inhibitors are classified into either direct inhibitors that target endothelial cells in the growing vasculature or indirect inhibitors that prevent the expression or block the activity of angiogenesis inducers. The latter class extends to include targeted therapy against oncogenes, conventional chemotherapeutic agents and drugs targeting other cells of the tumor micro-environment. Angiogenesis inhibitors may be used as either monotherapy or in combination with other anticancer drugs. In this context, many preclinical and clinical studies revealed higher therapeutic effectiveness of the combined treatments compared with individual treatments. The proper understanding of synergistic treatment modalities of angiogenesis inhibitors as well as their wide range of cellular targets could provide effective tools for future therapies of many types of cancer.

Two major processes of blood vessel formation are implicated in the development of vascular system: vasculogenesis and angiogenesis. Vasculogenesis prevails in the embryo and refers to the formation ofde novo blood vessels by in situ differentiation of the mesoderm-derived angioblasts and endothelial precursors. Angiogenesis is the formation of new capillaries from pre-existing vessels and circulating endothelial precursors (Polverini, 2002; Chung et al., 2010; Ribatti and Djonov, 2012). Angiogenesis is a tightly controlled dynamic process that can occur physiologically in those tissues that undergo active remodeling in response to stress and hypoxia (Carmeliet, 2003; Folkman, 2007). However, it can be aberrantly activated during many pathological conditions such as cancer, diabetic retinopathy as well as numerous ischemic, inflammatory, infectious and immune disorders (Carmeliet, 2003; Ali and El-Remessy, 2009; Willis et al., 2011). Although the concept of proposing angiogenesis inhibitors as anticancer drugs received considerable skepticism when first presented by Dr. Folkman in the early 1970s (Folkman, 1971), active research in the field and subsequent clinical trials eventually resulted in US Food and Drug Administration (FDA) approval of bevacizumab for colorectal cancer in 2004 (Cohen et al., 2007). Since then, several angiogenic inhibitors have been identified. This review will provide an overview of the key mechanisms involved in tumor angiogenesis, classification of angiogenesis inhibitors as well as treatment rationales from the mechanistic point of view.

Sustained angiogenesis as a hallmark of cancer

Proliferating tumours tend to activate an angiogenic phenotype to fulfil their increased demand of oxygen and nutrients (Hanahan and Folkman, 1996; Carmeliet, 2005). Additionally, paracrine release of anti-apoptotic factors from activated endothelial cells in the newly formed vasculature supplies tumour cells with a survival privilege (Folkman, 2003). Consequently, in order to progress, tumors tend to activate an event called ‘angiogenic switch’ by shifting the balance of endogenous angiogenesis inducers and inhibitors towards a pro-angiogenic outcome. As a result, dormant lesion progresses into outgrowing vascularized tumor and eventually into a malignant phenotype (Hanahan and Folkman, 1996; Baeriswyl and Christofori, 2009). Hypoxia drives such imbalance through up-regulation of the transcription factor hypoxia inducible factor-1α (HIF-1α), which in turn increases the expression of many angiogenesis inducers as well as suppresses the expression of endogenous angiogenesis inhibitors (Pugh and Ratcliffe, 2003). In spite of that, accumulating evidence indicates that angiogenic cascade can be also driven by alternative HIF-1-independent pathways (Mizukami et al., 2007; Arany et al., 2008; Lee, 2013).

As summarized in Table 1, the angiogenesis inducers are a wide range of mediators that include many growth factors, a plethora of cytokines, bioactive lipids, matrix-degrading enzymes and a number of small molecules (Folkman, 1995; Folkman, 2003; Lopez-Lopez et al., 2004; Bouis et al., 2006; El-Remessy et al., 2007; Bid et al., 2011; MacLauchlan et al., 2011; Murakami, 2011; Fagiani and Christofori, 2013; Qin et al., 2013). Pro-angiogenic growth factors mostly activate a series of surface receptors in a series of paracrine and autocrine loops with the VEGF-A signaling representing the critical rate-limiting step, physiologically and pathologically. VEGF-A (traditionally known as VEGF) is the most potent VEGF isoform that acts mainly on VEGF receptor 2 (VEGFR2) to mediate vascular permeability, endothelial proliferation, migration and survival (Takahashi and Shibuya, 2005; Bouis et al., 2006). In spite of the well-established master roles of VEGF signaling in literature, those processes are probably accomplished through a highly regulated interplay between VEGF and other pro-angiogenic factors. In this context, basic fibroblast growth factor (bFGF) activation of the endothelium is required for maintenance of VEGFR2 expression and the ability to respond to VEGF stimulation (Murakami et al., 2011). Similarly, sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid that can directly contribute to tumor angiogenesis (reviewed in Sabbadini, 2011), is needed for VEGF-induced blood vessel formation, indicating the cooperation between S1P and VEGF in tumor angiogenesis (Visentin et al., 2006). As a net result, the pro-angiogenic interplay of those ligands and others dominates over the activities of two dozen endogenous angiogenesis inhibitors that can be either matrix-derived inhibitors or non–matrix-derived inhibitors (Nyberg et al., 2005).

Table 1. Pro-angiogenic mediators implicated in tumor angiogenesis

Category Examples References
Growth factors VEGFs Bouis et al., 2006
FGFs Ibid
TGFs Ibid
PDGFs Ibid
Insulin-like growth factors Lopez-Lopez et al., 2004; Bid et al., 2011
ANGs Fagiani and Christofori, 2013
Cytokines IL-8 Strieter et al., 2004
CSF-1 Lin et al., 2006
Bioactive lipids PGE2 Wang and Dubois, 2010
S1P Murakami, 2011
Matrix-degrading enzymes MMPs Bourboulia and Stetler-Stevenson, 2010
Heparanases Vlodavsky and Friedmann, 2001
Small mediators NO MacLauchlan et al., 2011
Peroxynitrite El-Remessy et al., 2007
Serotonin Qin et al., 2013
Histamine Qin et al., 2013

The multistep angiogenic process starts with vasodilation and increased permeability of existing vessels in response to tumor cell-secreted VEGF. This is accompanied by loosening of pericytes covering mediated by angiopoietin-2 (ANG2), a ligand of tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (TIE2) receptor (Bergers and Benjamin, 2003; Jain, 2003; Fagiani and Christofori, 2013). Meanwhile, many secreted matrix-degrading enzymes, such as MMPs and heparanases, function in concert to dissolve the basement membrane and to remodel the extracellular matrix (ECM) as well as to liberate more pro-angiogenic growth factors (bFGF and VEGF) from matrix heparan sulfate proteoglycans (HSPGs) respectively (Houck et al., 1992; Whitelock et al., 1996; Vlodavsky and Friedmann, 2001; Tang et al., 2005; van Hinsbergh and Koolwijk, 2008). The overall chemotactic angiogenic stimuli guide endothelial cells to migrate, to align into tube-like structures and to eventually form new blood vessels. However, such blood vessels are characterized by being disorganized, chaotic, hemorrhagic and poorly functioning (Bergers and Benjamin, 2003).

The angiogenic phenotype in tumor micro-environment can further be sustained and extravagated by the recruitment of other types of stromal cells. Stromal cells such as fibroblasts, mesenchymal stem cells and various bone marrow-derived myeloid cells including macrophages, TIE2-expressing monocytes, neutrophils and mast cells contribute to tumor angiogenesis through their production of growth factors, cytokines and proteases (Murdoch et al., 2008; Joyce and Pollard, 2009; Cirri and Chiarugi, 2011). For example, in response to cancer cell-derived TGF-β, PDGF or bFGF, fibroblasts are transformed to an activated phenotype with a higher proliferative activity and myofibroblastic characteristics (Kalluri and Zeisberg, 2006; Cirri and Chiarugi, 2011). Such carcinoma-associated fibroblasts (CAFs) were shown to promote angiogenesis and metastasis by secreting large amounts of MMP-2 and MMP-9 as well as by expressing many cytokines and chemokines that resulted in immune cell infiltration (Gerber et al., 2009; Giannoni et al., 2010). Furthermore, it has been shown that PDGF-C produced by CAFs is able to elicit VEGF production from tumor cells, thereby sustaining the angiogenic shift (Crawford et al., 2009). Similarly, tumor-associated macrophages (TAMs), one of the bone marrow myeloid-derived cells, are induced to develop into polarized type II (alternatively activated or M2 macrophages), upon exposure to tumor hypoxia and tumor cell-derived cytokines (Leek et al., 2002; Rogers and Holen, 2011). M2 macrophages tend to produce many pro-angiogenic growth factors, cytokines and matrix-degrading enzymes such as VEGF, PDGF, bFGF, TNF-α, COX-2, MMP-9, MMP-7 and MMP-12 (Lewis and Pollard, 2006).

From another perspective, angiogenesis may be dispensable for progression of some malignancies. For example, some tumours may co-opt pre-existent vessels as an alternative way to obtain blood supply. Vessel co-option was first described in the brain, one of the most densely vascularized organs, in which tumours may develop in earlier stages without the activation of angiogenic response (Holashet al., 1999; Leenders et al., 2002; Bergers and Benjamin, 2003; Hillen and Griffioen, 2007). In another example, hypovascularized tumors such as pancreatic ductal adenocarcinoma may involve certain adaptation to flourish in the absence of prominent angiogenesis (Bergers and Hanahan, 2008). Obviously, in both cases, tumors may be intrinsically indifferent to angiogenesis inhibitors. However, in most other cases, therapy directed towards the vasculature of solid tumors is being considered as an important direction in cancer treatment.

Classification of angiogenesis inhibitors

Growth of newly formed vessels in tumor micro-environment can be inhibited directly by targeting endothelial cells in the growing vasculature or indirectly by targeting either tumor cells or the other tumor-associated stromal cells. Therefore, angiogenesis inhibitors can be classified into direct and indirect inhibitors (Kerbel and Folkman, 2002; Folkman, 2007).

Direct endogenous inhibitors of angiogenesis

Direct endogenous inhibitors of angiogenesis, such as angiostatin, endostatin, arrestin, canstatin, tumstatin and others, are fragments released on proteolysis of distinct ECM molecules. Endogenous inhibitors prevent vascular endothelial cells from proliferating, migrating in response to a spectrum of angiogenesis inducers, including VEGF, bFGF, IL-8 and PDGF (Kerbel and Folkman, 2002; Abdollahi et al., 2004; Mundel and Kalluri, 2007; Ribatti, 2009). This direct anti-angiogenic effect may be mediated by interference with endothelial integrins along with several intracellular signaling pathways (Mundel and Kalluri, 2007). For example, the ability of tumstatin-derived active peptide to inhibit angiogenesis and tumour growth is associated with the expression of the adhesion receptor, αvβ3 integrin, on tumor endothelial cells (Eikesdal et al., 2008). Through binding αvβ3 integrin, full tumstatin was found to inhibit endothelial cell activation of focal adhesion kinase, PI3K, Akt, mammalian target of rapamycin (mTOR) and others (Maeshima et al., 2002). Direct targeting of those signaling pathways by endogenous inhibitors was thought to be the least likely to induce acquired drug resistance because they target endothelial cells with assumed genetic stability rather than unstable mutating tumour cells (Kerbel and Folkman, 2002). However, endostatin has not yet led to any documented benefit to patients in randomized phase III trials, or even modest activity in phase II trials (Ellis and Hicklin, 2008).

Indirect inhibitors of angiogenesis

Indirect inhibitors of angiogenesis classically prevent the expression or block the activity of pro-angiogenic proteins (Folkman, 2007). For example, Iressa, an EGF receptor (EGFR) TK inhibitor (TKI), blocks tumour expression of many pro-angiogenic factors; bevacizumab, a monoclonal antibody, neutralizes VEGF after its secretion from tumour cells whereas sunitinib, a multiple receptor TKI, blocks the endothelial cell receptors (VEGFR1, VEGFR2 and VEGFR3), preventing their response to the secreted VEGF (Folkman, 2007; Roskoski, 2007). In addition, this class extends to include conventional chemotherapeutic agents, targeted therapy against oncogenes and drugs targeting other cells of the tumor micro-environment (Kerbel et al., 2000; Ferrara and Kerbel, 2005).

Conventional chemotherapeutic agents

Conventional chemotherapeutic agents have been shown to have anti-angiogenic properties in addition to the ability to induce direct cancer cell death. Such chemotherapeutic agents can affect the endothelial cell population in the tumour bed during treatment cycles because they have significantly higher proliferation rates than resting endothelium outside a tumor, making them more susceptible to cytotoxic effect (Kerbel et al., 2000; Folkman, 2003). However, the cyclic treatment rationale of cytotoxic drugs allows the potential damage to the tumour vasculature to be repaired during the long breaks. Thus, continuous low doses of chemotherapeutic agents were suggested as a way to reduce side effects and drug resistance (Drevs et al., 2004). This modality is termed metronomic therapy, and clinically, it refers to the daily administration of 5–10% of the phase II-recommended dose of the chemotherapeutic agent (Penel et al., 2012). The extended use of such low doses of cytotoxic agents elicits an anti-angiogenic activity through induction of endothelial cell apoptosis and decreasing the level of circulating endothelial precursors (Hamano et al., 2004; Shahrzad et al., 2008). In clinical investigations, metronomic dosing of cyclophosphamide and others showed promising efficacy in patients with advanced, multiple metastasized and/or multiple pretreated solid tumours (Lord et al., 2007; Fontana et al., 2010; Nelius et al., 2011; Gebbia et al., 2012; Briasoulis et al., 2013; Navid et al., 2013).

VEGF-targeted therapy

VEGF-targeted therapy includes neutralizing antibodies to VEGF (e.g. bevacizumab) or VEGFRs (e.g. ramucirumab), soluble VEGFR/VEGFR hybrids (e.g. VEGF-Trap) and TKIs with selectivity for VEGFRs (e.g. sunitinib and sorafenib; Baka et al., 2006; Ellis and Hicklin, 2008; Hsu and Wakelee, 2009). Bevacizumab, a humanized monoclonal antibody against all isoforms of VEGF-A, has been approved for the treatment of colorectal, lung, glioblastoma and renal cell carcinoma (Hsu and Wakelee, 2009). Many other clinical trials with promising efficacy were also conducted in other cancers such as head and neck cancer, hepatocellular carcinoma, ovarian cancer, metastatic melanoma and gastric cancer (Argiris et al., 2011; 2013; Burger et al., 2011; Ohtsu et al., 2011; Fang et al., 2012; Minor, 2012; Schuster et al., 2012; Van Cutsem et al., 2012). However, for metastatic breast cancer, bevacizumab had been initially granted an accelerated FDA approval, which was later withdrawn due to lack of improvement evidence in disease-related symptoms or overall survival (Burstein, 2011; Montero et al., 2012). Similarly, clinical trials showed that the addition of bevacizumab to the treatment regimens of advanced pancreatic cancer did not extend overall survival (Chiu and Yau, 2012). The neutralization of VEGF-A can also be achieved by soluble receptor construct (VEGF-Trap) that monomerically ‘traps’ the different isoforms of VEGF-A, in addition to VEGF-B and placental growth factor (Rudge et al., 2007). VEGF-Trap showed clinical benefit in a phase III trial of oxaliplatin pretreated metastatic patients with colorectal cancer, and is currently being investigated in a prostate cancer phase III trial (Gaya and Tse, 2012). TKIs are small molecules with different chemical structures that have the ability to interact physically with the highly conserved kinase domain shared by different VEGFRs as well as PDGF receptors (PDGFRs), FGF receptors (FGFRs), EGFR, Raf kinases and c-Kit (a receptor of the pluripotent cell growth factor, stem cell factor). Such interaction directly inhibits tyrosine phosphorylation and the subsequent many downstream pro-angiogenic signaling networks (Baka et al., 2006; Ivy et al., 2009). Those multi-targeted TKIs demonstrated efficacy against various solid malignancies in different clinical trials, some of which have lead eventually to FDA approval of sunitinib and sorafenib. Sunitinib, known to inhibit several receptor TKs (RTKs) including VEGFR1–3, PDGFR-α, PDGFR-β, c-Kit, colony-stimulating factor-1 receptor (CSF-1R) and Flt-3, was approved for the treatment of renal cell carcinoma and gastrointestinal stromal cell tumours. Sorafenib that acts also by inhibiting VEGFR1–3 and PDGFR-β in addition to the serine–threonine kinases Raf-1, B-Raf, was approved for hepatocellular carcinoma in addition to renal cell carcinoma (Llovet et al., 2008; Ivy et al., 2009; Huang et al., 2010).

FGF-targeted therapies

FGF-targeted therapies were recently reconsidered as promising anti-angiogenic and anti-tumor agents after a long period of little attention for drug development, partly due to redundancy (Bono et al., 2013). The FGFR superfamily with its 18 ligands and four receptors has been involved in endothelial cell migration, proliferation and differentiation (Presta et al., 2005). Therapeutic targeting of FGF/FGFR signalling was accomplished by either monoclonal antibodies that inhibit FGFs binding, small molecules that inhibit FGFR TK activity or allosteric modulators that bind the extracellular FGFR domain. Monoclonal antibodies against bFGF displayed potent anti-tumor and anti-angiogenic effects in different preclinical cancer models, which warrant further clinical evaluation (Zhao et al., 2010; Wang et al., 2012). Pan inhibitors of the FGFR TKs such as AZD4547 (blocks the activity of FGFR1–3) and ponatinib (blocks all the FGFR isoforms) elicited potent anti-tumor activities in preclinical investigations so they are currently being evaluated in clinical trials. Those inhibitors displayed the greatest potency in FGFR-driven cancer models, which may be attributed to the interference with the oncogenic functions of either amplified or constitutively active FGFR (Dutt et al., 2011; Zhao et al., 2011; Gavine et al., 2012; Gozgit et al., 2012). Accordingly, further studies are needed to evaluate the relative contribution of angiogenic versus oncogenic inhibitory mechanisms towards the overall anti-tumor activity. The allosteric antagonist of the FGFR, SSR128129E, showed a strong anti-angiogenic activity in addition to tumour growth and metastasis inhibitory effects in animal models of arthritis and cancer respectively. Because allosteric modulators leave a residual level of baseline signalling, they have the ability to fine-tune target biological responses. As a result, allosteric multi-FGFR inhibitors may have an improved benefit/risk ratio that is not attainable with the other TKIs (Bonoet al., 2013; Herbert et al., 2013). However, preclinical findings suggest that long-term clinical outcomes may improve with blockade of additional pro-angiogenic RTKs that may also reduce the risk of drug resistance (Hilberg et al., 2008). For example, dual inhibition of VEGFRs and FGFRs using brivanib produced enduring tumour stasis and angiogenic blockade following the failure of VEGF-targeted therapies (Allen et al., 2011). Furthermore, triple inhibition of FGFRs, VEGFRs and PDGFR(s) using dovitinib (TKI258) or nintedanib (BIBF 1120) displayed broad-spectrum anti-tumour activities in several tumour xenograft models as well as promising data in clinical trials. Combined inhibition of FGFR/VEGFR/PDGFR targets not only tumour cells, but also endothelial cells, pericytes and smooth muscle cells, resulting in an effective inhibition of tumour growth, angiogenesis and metastasis even in advanced tumour stages (Hilberg et al., 2008; Ledermann et al., 2011; Taeger et al., 2011; Chenet al., 2012; Angevin et al., 2013).

Oncogene-targeted therapy

Oncogenes, genes that cause the transformation of normal cells into cancerous cells, are thought to up-regulate many pro-angiogenic proteins. Therefore, anticancer drugs that were developed for their capacity to block an oncogene also have an indirect anti-angiogenic activity (Kerbel et al., 2000; Bergers and Benjamin, 2003; Folkman, 2003). For example, dasatinib and other inhibitors of sarcoma (Src), an aberrantly activated non-RTK associated with many human malignancies, showed potent anti-angiogenic effects through the down-regulation of VEGF and IL-8 (Summy et al., 2005; Han et al., 2006; Haura et al., 2010). Another example is to target the oncogenic Ras using farnesyl transferase (FT) inhibitors, which inhibit post-translational farnesylation of Ras that governs the latter’s activity (Awada et al., 2002). FT inhibitors were found to inhibit tumor VEGF expression and block FTase-dependent Ras activation, which is critically involved in VEGF-elicited angiogenic signal transduction and angiogenesis (Han et al., 2005; Izbicka et al., 2005; Kim et al., 2010). In addition to classical oncogenes inhibition, interference with other tumor-deregulated signaling pathways would offer another approach in targeting angiogenesis. For example, inhibitors of heat shock protein 90 (HSP90), a chaperone molecule known to protect oncoproteins from misfolding and degradation in the protein-rich intracellular environment, were found to prevent VEGF production and to disrupt multiple pro-angiogenic signalling pathways in numerous cancer cells. They were also shown to inhibit tumour growth and vascularity of different human tumor xenografts (Sanderson et al., 2006; Langet al., 2007; Eccles et al., 2008; Trepel et al., 2010; Moser et al., 2012). Proteasome inhibitors, such as bortezomib (PS-341) or MG-132, were also shown to reduce tumour growth and vascularity of squamous cell carcinoma and pancreatic cancer xenograft probably through inhibition of NF–κB-dependent release of pro-angiogenic gene products, VEGF and IL-8 (Sunwoo et al., 2001; Nawrocki et al., 2002; Matsuo et al., 2009). Similarly, inhibition of B-cell lymphoma 2 (Bcl-2), a prosurvival protein that regulates apoptosis by preventing the mitochondrial release of pro-apoptogenic factors, was shown to prevent NF-κB-mediated release of the pro-angiogenic factors IL-8 and CXC chemokine ligand 1 (CXCL1) as well as VEGF in tumor-associated endothelial cells and pancreatic cell lines respectively (Karl et al., 2005; Wang et al., 2008). Moreover, (−)-gossypol, a natural BH3 mimetic that inhibits BH3 domain of Bcl-2 as well as related prosurvival proteins (Bcl-xL and Mcl-1), was shown to remarkably decrease microvessel density in human prostate tumour PC-3 xenografts through decrease of VEGF and IL-8 release as well as blocking multiple steps in VEGF-activated biological events (Karaca et al., 2008; Pang et al., 2011).

Matrix degrading and remodelling-targeted therapy

Matrix degrading and remodelling are activated by tumors to modify local micro-environment, which in turn promote their angiogenic potential (Bergers et al., 2000; Vlodavsky and Friedmann, 2001). Up-regulation of expression and activity of several endogenous MMPs including MMP-2, MMP-9 as well as MMP-3 and MMP-7 have been identified in invasive tumors (for a review, see Bourboulia and Stetler-Stevenson, 2010). Consequently, inhibitors of MMPs were extensively pursued as a therapeutic strategy for treating cancer. Unfortunately, MMPs intervention strategies had met with limited clinical success because of severe toxicities and associated metastasis-promoting effect (Coussens et al., 2002; Devy et al., 2009). Furthermore, the paradoxical roles of tissue inhibitors of metalloproteinases (TIMPs) may contribute to such failure depending on the net balance of TIMPs and MMPs in tumour stroma (Jiang et al., 2002). As a result, efforts were directed at therapies exploiting endogenous MMP inhibitors, TIMPs or monoclonal antibodies against individual MMPs (Martens et al., 2007; Jarvelainen et al., 2009). For example, DX-2400, a highly selective fully human MMP-14 inhibitory antibody, was found to block pro-MMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions (Devy et al., 2009). Alternatively, in order to reduce toxicity and enhance drug delivery, polymeric nanoparticulate delivery systems could be used to target individual components of ECM. For example, targeted delivery of antisense inhibitors of laminin-8, a vascular basement membrane component, by conjugation to the natural drug carrier β-poly(L-malic acid) significantly reduced tumour microvessel density and increased animal survival in an experimental model of glioblastoma (Fujita et al., 2006). Similarly, a nano delivery system that incorporate peptides against proteolytically processed type IV collagen significantly accumulated in tumors and blocked angiogenesis in experimental models (Mueller et al., 2009). However, the highly sulfated oligosaccharides, Heparan (HS) mimetics highly sulfated oligosaccharides, were shown to have a heparanase-inhibiting effect sequestering, in turn, many heparan sulfate proteoglycan (HSPG)-binding factors (Johnstone et al., 2010; Dredge et al., 2011). In preclinical studies, HS mimetics have effectively targeted multiple HSPG-dependent functions and have resulted in decreased in vivo tumor growth, tumor invasion, tumor metastasis and angiogenesis (Johnstone et al., 2010; Dredge et al., 2011; Zhou et al., 2011). Clinically, the heparanase inhibitor PI-88 showed preliminary efficacy as an adjunct therapy for post-operative hepatocellular carcinoma (Liu et al., 2009).

Tumour-associated stromal cell-targeted therapy

Tumour-associated stromal cells crosstalk is a perquisite for the formation of a tumour vasculature, an essential step for tumour progression (Lorusso and Ruegg, 2008). Interference with those crosstalk circuits through intervention of cellular adhesion (highlighted in next paragraph) or tumor-induced recruitment of different stromal cells may be considered as an indirect way of anti-angiogenic therapy (Ferrara and Kerbel, 2005). The latter can be supported by studies in which inhibition of macrophage infiltration, for example, by either genetic ablation of the macrophage CSF-1 or liposomal clodronate-induced macrophage depletion, was shown to delay the angiogenic switch and malignant transition (Giraudo et al., 2004; Lin et al., 2006). Furthermore, CSF-1R kinase inhibitors were found to reduce tumor-associated vascularity in two different tumor mouse models (Kubota et al., 2009; Mantheyet al., 2009). In addition, clodronate and other related bisphosphonates, originally used to treat skeletal complications in patients with tumour-induced osteolysis, were shown to exert potent anti-tumour and anti-angiogenic effects in many other studies (Fournier et al., 2002; Santini et al., 2003; Stathopoulos et al., 2008). Zoledronic acid, a third-generation bisphosphonate, was also found to reduce a number of tumour-associated macrophages and shift their phenotype from M2 to M1, resulting in a reduction in TAM-associated production of VEGF in murine models of spontaneous mammary carcinogenesis and mesothelioma (Coscia et al., 2010; Veltman et al., 2010). Clinically, repeated low-dose therapy with zoledronic acid, which maintains active drug plasma concentration, was able to induce an early remarkable and long-lasting decrease of VEGF levels in patients with cancer (Santini et al., 2007). In another example, inhibition of mobilization of neutrophils, from bone marrow and their infiltration into tumour, using neutralizing anti–prokineticin-2, an antibody against a secreted protein known also as BV8, was shown to impair the initial angiogenic switch in a multistage pancreatic beta cell tumorigenesis model (Shojaei et al., 2008). Furthermore, the neutralizing anti-BV8 was found to prevent myeloid cell-dependent tumour angiogenesis in several xenograft models (Shojaei et al., 2007). Cancer-associated fibroblasts (CAF) can also be targeted with thapsigargin analogue coupled with peptides specific for fibroblast activation protein (FAP), a CAF membrane-bound protease whose catalytic site has access to the peritumoural fluid of the tumor micro-environment. This extracellular activation results in the death of CAFs as well as pericytes and endothelial cells within milieu of different human tumor xenografts (Brennen et al., 2012).

Cell adhesion molecules (CAMs)-targeted therapy

CAMs are cell surface proteins known to be involved in binding with other counter-receptors on adjacent cells or surrounding ECM macromolecules (Aplin et al., 1998). Many CAMs, such as αv-integrins, E-selectin, N-cadherin and VE-cadherin, have been implicated in tumour angiogenesis (Bischoff, 1997; Tei et al., 2002; Nakashima et al., 2003; Weis and Cheresh, 2011). For example, αv-integrins are expressed on surface of endothelial cells and can determine whether cells can adhere to and survive in a particular micro-environment. A number of matrix-derived fragments have the ability to act as endogenous angiogenesis inhibitors through binding to integrins on endothelial cells, disrupting physical connections and suppressing signalling events associated with cell survival, migration and proliferation (Nyberg et al., 2005). Consequently, integrins antagonism using peptidomimetics (e.g. cilengitide), monoclonal antibodies (e.g. volociximab) or oral small-molecule compounds have been investigated in a wide range of malignancies (Huveneers et al., 2007). Cilengitide is a cyclized pentapeptide peptidomimetic designed to compete for the arginine-glycine-aspartic acid (RGD) peptide sequence, thereby blocking the ligation of the αvβ3 and αvβ5 integrins to matrix proteins (Hariharan et al., 2007). Cilengitide is mainly under clinical development for glioblastoma; however, clinical trials of other malignancies such as head and neck cancer as well as lung cancer were also initiated (Reardon and Cheresh, 2011; Vermorken et al., 2012; Manegold et al., 2013). Alternatively, cyclic peptides containing RGD motif could guide nanoparticulate delivery system, which incorporates anti-angiogenic cytotoxic agents such as doxorubicin, paclitaxel or combretastatin A4, to accumulate specifically in tumor vasculature with no overt systemic toxicity (Murphy et al., 2008; Ruoslahti et al., 2010; Wang et al., 2011). Volociximab, a chimeric humanized monoclonal antibody that selectively inhibits the αvβ1 integrin interaction with fibronectin, has been evaluated also in clinical trials for solid tumours such as renal cell carcinoma, recurrent ovarian cancer, advanced non–small-cell lung cancer and metastatic pancreatic cancer (Figlin et al., 2006; Evans et al., 2007; Jarvelainen et al., 2009; Vergote et al., 2009; Besse et al., 2013). Cadherins constitute a superfamily of molecules that mediate calcium-dependent cell–cell adhesions. The intracellular domains of cadherins directly bind to β-catenin and link with cytoskeletal components, providing the molecular basis for stable cell–cell adhesion (Zhang et al., 2010). Targeting cadherin signalling may also represent another way for tumor angiogenesis intervention. For example, ADH-1, a cyclic pentapeptide containing the cell adhesion recognition site (His-Ala-Val) required for N-cadherin adhesion, was shown to possess anti-angiogenic and anti-tumour activity (Blaschuk et al., 2005; Blaschuk, 2012). Similarly, monoclonal antibody directed against specific region of VE-cadherin was able to inhibit tumor angiogenesis and growth with no side effects on normal vasculature (Corada et al., 2002; May et al., 2005).

Inflammatory angiogenesis-targeted therapy

Targeting inflammatory angiogenesis, responsible for a substantial part of tumour vascularization initiated by infiltrating leukocytes, may be considered as another indirect anti-angiogenic strategy (Albini et al., 2005). Moreover, as mentioned before, tumour-infiltrating leukocytes contribute into malignant progression through production of many pro-inflammatory cytokines, chemokines and enzymes that can mostly induce angiogenic cascade (Balkwill et al., 2005). Such vital roles have been supported by the early observation that nonsteroidal anti-inflammatory drugs can inhibit tumour angiogenesis and, in turn, tumor progression (Albini et al., 2005). For example, ibuprofen was found to decrease tumor growth and metastatic potential in mice models through modulation of angiogenesis (Yao et al., 2005). Moreover, selective inhibitors of COX-2, an inducible enzyme that catalyses the production of prostanoids from arachidonic acid, were also shown to inhibit angiogenesis (Tsujii et al., 1998; Wei et al., 2004). The anti-angiogenic effect of COX-2 inhibitors may be contributed, in part, by decreasing the COX-2 metabolic product PGE2, the predominant PG in solid tumors known to stimulate cancer cells to produce pro-angiogenic factors such as VEGF and bFGF as well as many other factors belonging to CXC chemokines family (Strieter et al., 2004; Wang et al., 2006; Wang and Dubois, 2010). Members of the CXC chemokine family are heparin-binding proteins that possess disparate regulative roles in angiogenesis. For example, the ELR+ CXC chemokines, characterized by highly conserved three amino acid motifs (Glu-Leu-Arg; ‘ELR’ motif), are potent promoters of angiogenesis, whereas the IFN-inducible (ELR−) CXC chemokines are inhibitors of angiogenesis (Strieter et al., 2004). The use of repertaxin, originally designed to target the ELR+ CXC chemokine receptors CXCR1 and CXCR2 on neutrophils to prevent their migration to sites of inflammation, was found to inhibit tumor angiogenesis, thereby suppressing tumour progression in a genetic model of pancreatic ductal adenocarcinoma (Ijichi et al., 2011). It would be beneficial to explore other small-molecule CXCR2 antagonists that have already been developed for the treatment of inflammatory diseases in different preclinical models of cancer, especially inflammation-associated cancers (refer to Chapman et al., 2009 for a list of newly developed CXCR2 antagonists used in the treatment of inflammatory diseases of the lung).

Mechanisms of enhanced therapeutic efficacy

  • Dual targeting of tumor vasculature
  • Targeting different cell types of tumor micro-environment
  • Normalization of tumor vasculature
  • Chemosensitization of tumor cells
  • Interference with the repair of cytotoxic drug-induced damage and resistance mechanisms

Consequences of anti-angiogenic therapy with other anticancer therapy

  • Contrary to initial expectations, treatment with angiogenesis inhibitors was associated with unexpected toxicities. The toxicity profiles of those inhibitors reflect the systemic disturbance of growth factor signalling pathways that mediate their anti-angiogenic activity (Elice and Rodeghiero, 20102012). In this context, disturbance of the tight endothelial cell-platelet interaction that maintains vascular integrity results in bleeding complications, gastrointestinal perforations, and disturbed wound and ulcer healing (Verheul and Pinedo, 2007). In general, the incidence of those adverse effects increases when anti-angiogenic agent is combined with chemotherapy. For example, bleeding complications have been observed in patients with colorectal cancer treated with chemotherapy in combination with bevacizumab (Kabbinavar et al., 2003; Giantonio et al., 2006). In non–small-cell lung cancer, some patients treated with bevacizumab in combination with carboplatin and paclitaxel experienced severe or fatal pulmonary haemorrhage (Johnson et al., 2004). Furthermore, a higher incidence of gastrointestinal perforation was observed in patients with colorectal cancer given bevacizumab in combination with chemotherapy compared with chemotherapy alone (Hurwitz et al., 2004). Similarly, thrombotic events have been observed in patients treated with angiogenesis inhibitors, especially when these agents are given in combination with chemotherapy (Verheul and Pinedo, 2007). Treatment of patients with cancer with angiogenesis inhibitors is frequently associated with hypertension, which may require the addition of regular anti-hypertensive agent (Izzedine et al., 2009).

Summary and future directions

  • Angiogenesis is a critical process that occurs pathologically in many malignancies due to changing balance of endogenous angiogenesis inducers and inhibitors, leading to the activation of nearby endothelial cells to form new vasculature. Consequently, angiogenesis can be targeted to restrict initiation, growth and progression of most of angiogenesis-dependent malignancies. Numerous angiogenic inhibitors have been identified, some of which are currently being investigated in clinical trials and some others were even approved for cancer therapies. These angiogenesis inhibitors were classified based on their target into two main classes: direct and indirect inhibitors. Indirect angiogenesis inhibitors can be further subclassified based on their interference mechanisms with the angiogenic cascade. A list of major categories and molecular targets for angiogenesis inhibitors is shown in Table 2.
  • Most angiogenesis inhibitors conferred clinical benefits mainly when combined with other chemotherapeutic/targeted therapies rather than being used as monotherapy. Unfortunately, many anti-angiogenic agents were shown to be associated with overt systemic toxicity as well as resistance emergence and disease recurrence. Drug resistance in anti-angiogenic therapy may result from a plethora of pro-angiogenic factors released by inappropriately functioning host cells in the tumor micro-environment as a compensatory mechanism. Therefore, the strategy of targeting endothelial cells alone may not be enough as explained in the previous texts, requiring the proposal of different rationales in which other cellular compartments of tumor micro-environment are targeted to attain proper anti-angiogenic and anti-tumor response. That highlights the importance of considering tumor micro-environment as a dynamic system, as depicted in Figure 1 in which interference with any of its components may be an approach to interfere with cancer hallmarks, including angiogenesis.

9.5.5 LUCITANIB a VEGFR/FGFR dual kinase inhibitor in Phase 2 trials

Dr.  Anthony Melvin Crasto

source: http://medcheminternational.blogspot.com/2015/01/lucitanib-vegfrfgfr-dual-kinase.html

6-(7-((l-aminocyclopropyl)methoxy)-6-methoxyquinolin-4-yloxy)- N-methyl- 1 -naphthamide
1058137-23-7 (E-3810 free base); 1058137-84-0  (E-3810 HCl salt)
E-3810, E-3810 amine, UNII-PP449XA4BH, E3810, Lucitanib [INN], AL3810
Molecular Formula:C26H25N3O4
Molecular Weight:443.4944 g/mol
Spiro Substituted Compounds As Angiogenesis Inhibitors [US8163923] 2008-09-18 2012-04-24
A 4-(3-methoxypropoxy)-3-methylpyridinyl derivative of timoprazole that is used in the therapy of STOMACH ULCERS and ZOLLINGER-ELLISON SYNDROME. The drug inhibits H(+)-K(+)-EXCHANGING ATPASE which is found in GASTRIC PARIETAL CELLS.
For in advanced solid tumors.
Lucitanib (E-3810): Lucitanib, also known as E-3810,  is a novel dual inhibitor targeting human vascular endothelial growth factor receptors (VEGFRs) and fibroblast growth factor receptors (FGFRs) with antiangiogenic activity. VEGFR/FGFR dual kinase inhibitor E-3810 inhibits VEGFR-1, -2, -3 and FGFR-1, -2 kinases in the nM range, which may result in the inhibition of tumor angiogenesis and tumor cell proliferation, and the induction of tumor cell death. Both VEGFRs and FGFRs belong to the family of receptor tyrosine kinases that may be upregulated in various tumor cell type
Lucitanib (E-3810) Structure


Lucitanib is an oral, potent inhibitor of the tyrosine kinase activity of fibroblast growth factor receptors 1 through 3 (FGFR1-3), vascular endothelial growth factor receptors 1 through 3 (VEGFR1-3) and platelet-derived growth factor receptors alpha and beta (PDGFR α-ß). We own exclusive development and commercial rights to lucitanib on a global basis, excluding China. Lucitanib rights to markets outside of the U.S. and Japan have been sublicensed to Les Laboratoires Servier (Servier). We are collaborating with Servier on the global clinical development of lucitanib.

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Wnt/β-catenin Signaling

Writer and Curator: Larry H. Bernstein, MD, FCAP 


7.10 Wnt/β-catenin signaling

7.10.1 Wnt signaling and hepatocarcinogenesis. The hepatoblastoma model

7.10.2 The Wnt.β-catenin pathway in ovarian cancer : a review.

7.10.3 Wnt Signaling in the Niche Enforces Hematopoietic Stem Cell Quiescence and Is Necessary to Preserve Self-Renewal In Vivo

7.10.4 Wnt.β-Catenin Signaling in Development and Disease

7.10.5 Wnt.β-Catenin Signaling. Components, Mechanisms, and Diseases

7.10.6 Wnt.β-Catenin Signaling. Turning the Switch

7.10.7 Wnt–β-catenin signaling

7.10.8 Extracellular modulators of Wnt signaling

7.10.9 FOXO3a modulates WNT.β-catenin signaling and suppresses epithelial-to-mesenchymal transition in prostate cancer cells

7.10.1 Wnt signalinbg pathway in liver cancer Wnt signaling and hepatocarcinogenesis. The hepatoblastoma model

Armengol C1Cairo SFabre MBuendia MA.
Int J Biochem Cell Biol. 2011 Feb; 43(2):265-70.

The Wnt/β-catenin pathway plays a key role in liver development, regeneration and tumorigenesis. Among human cancers tightly linked to abnormal Wnt/β-catenin signaling, hepatoblastoma (HB) presents with the highest rate (50-90%) of β-catenin mutations. HB is the most common malignant tumor of the liver in childhood. This embryonic tumor differs from hepatocellular carcinoma by the absence of viral etiology and underlying liver disease, and by distinctive morphological patterns evoking hepatoblasts, the bipotent precursors of hepatocytes and cholangiocytes. Recent studies of the molecular pathogenesis of hepatoblastoma have led to identify two major tumor subclasses resembling early and late phases of prenatal liver development and presenting distinctive chromosomal alterations. It has been shown that the molecular signature of Wnt/β-catenin signaling in hepatoblastoma is mainly imposed by liver context, but differs according to developmental stage. Finally, the differentiation stage of tumor cells strongly influences their invasive and metastatic properties, therefore affecting clinical behavior. Targeting the Wnt/β-Catenin Signaling Pathway in Liver Cancer Stem Cells and Hepatocellular Carcinoma Cell Lines with FH535

Roberto Gedaly ,Roberto Galuppo, Michael F. Daily, Malay Shah, Erin Maynard, et al.
PLoS ONE 2014; 9(6): e99272.     http://dx.doi.org:/10.1371/journal.pone.0099272

Activation of the Wnt/β-catenin pathway has been observed in at least 1/3 of hepatocellular carcinomas (HCC), and a significant number of these have mutations in the β-catenin gene. Therefore, effective inhibition of this pathway could provide a novel method to treat HCC. The purposed of this study was to determine whether FH535, which was previously shown to block the β-catenin pathway, could inhibit β-catenin activation of target genes and inhibit proliferation of Liver Cancer Stem Cells (LCSC) and HCC cell lines. Using β-catenin responsive reporter genes, our data indicates that FH535 can inhibit target gene activation by endogenous and exogenously expressed β-catenin, including the constitutively active form of β-catenin that contains a Serine37Alanine mutation. Our data also indicate that proliferation of LCSC and HCC lines is inhibited by FH535 in a dose-dependent manner, and that this correlates with a decrease in the percentage of cells in S phase. Finally, we also show that expression of two well-characterized targets of β-catenin, Cyclin D1 and Survivin, is reduced by FH535. Taken together, this data indicates that FH535 has potential therapeutic value in treatment of liver cancer. Importantly, these results suggest that this therapy may be effective at several levels by targeting both HCC and LCSC.

Hepatocellular carcinoma (HCC), the most common liver cancer, is the fifth most common cancer and the third highest cause of cancer-related mortality worldwide [1][2]. The alarming rise in HCC incidence in Europe and North America in recent years is related mainly to hepatitis C virus infection, although other factors such as excessive alcohol consumption and obesity also contribute to this increase [3]. The etiology of HCC is complex and involves numerous genetic and epigenetic alterations and the disruption of various signaling pathways including the Wnt/β-catenin, Ras/Raf/MAPK, PI3K/AKT/mTOR, HGF/c-MET, IGF, VEGF and PDGF pathways. Among these, the Wnt/β-catenin pathway is considered among the most difficult to inhibit [4]. Currently, few chemical agents targeting the Wnt/β-catenin pathway are available or under investigation [5].

Activation of the canonical Wnt/β-catenin pathway involves the binding of Wnt proteins to cell surface Frizzled receptors and LRP5/6 co-receptors. In the absence of Wnt proteins, much of the cellular β-catenin is bound to E-cadherin on the cell membrane. Cytosolic β-catenin is constitutively phosphorylated at specific serine residues by an enzymatic complex that includes adenomatous polyposis coli (APC), Axin, and the kinases glycogen synthase kinase-3β (GSK-3β) and casein kinase I, marking it for ubiquitin-mediated proteolysis. Under these conditions, the TCF/LEF transcription factors are bound to their cognate DNA recognition elements along with members of the Groucho family of co-repressors, insuring the transcriptional silencing of β-catenin target genes. Engagement of Wnt proteins with the Frizzled receptor activates the Dishevelled protein, resulting in the dissociation of the cytosolic destructive complex and inhibition of GSK-3β. This leads to the stabilization and accumulation of cytoplasmic β-catenin, which then enters the nucleus, binds TCF/LEF proteins and leads to the subsequent dissociation of groucho co-repressors, recruitment of the coactivator p300 and activation of β-catenin target genes [6][9]. Many of the β-catenin targets, including Cyclin D1, c-myc and Survivin, promote cell cycle progression and inhibit apoptosis [10][12]. Consistent with this data, activation of the Wnt/β-catenin pathway is seen in a variety of cancers, including HCC. Aberrant activation of the Wnt/β-catenin pathway has been observed in at least 1/3 of HCC, and roughly 20% of HCCs have mutations in the β-catenin gene. More than 50% of HCC tumors display nuclear accumulation of β-catenin indicating that other factors may be involved such as aberrant methylation of the tumor suppressors APC and E-cadherin, inactivation of casein kinase and GSK-3β, or increased secretion of Wnt ligants [4][5].

There has been increasing interest in the role of liver cancer stem cells (LCSC) in tumorigenesis, tumor progression, invasion and metastases. The cancer stem cell theory suggests that a tumor is comprised of a heterogeneous population of cells that form a distinct cellular hierarchy. Recent studies have provided convincing evidence that these cells do exist in solid tumors of many types including, brain, breast, colorectal, liver, pancreas and prostate cancers. In 2006, two different groups isolated a CD133+ subpopulation from HCC cell lines and described higher proliferative and tumorigenic potential, consistent with stem cell properties. CD44 was also found as an important marker used in combination with other stem cell markers to better define the surface phenotype of LCSC. It has been demonstrated that CD133+ and CD90+ cells co-expressing CD44+ are more aggressive than those expressing CD133 or CD90 alone [13][14].

The chemical agents used to target Wnt-/β-catenin pathway are at the membrane, cytosol and transcription factor levels [5]. The small molecular agent FH535 is a dual inhibitor of peroxisome proliferator-activated receptor (PPAR) and β-catenin/TCF/LEF. FH535 has been shown to inhibit proliferation of HCC and hepatoblastoma cell lines and its specificity on inhibition of β-catenin/TCF/LEF activity was illustrated in hepatoblastoma cell line HepG2 [15].

The aim of this study was to determine if FH535 can inhibit the activation of β-catenin-regulated genes by endogenous and ectopically expressed β-catenin in the HCC cell lines Huh7, Hep3B and PLC and liver cancer stem cells (LCSC). The specificity of FH535 on inhibition of β-catenin via TCF/LEF activation was assayed in dual luciferase reporter transfected in LCSC and in HCC cells. Proliferation, cell cycle, and other targeted genes and proteins were assayed.

FH535 inhibits transcriptional activation mediated by wild-type and constitutively active β-catenin

FH535 has been shown to block signaling through endogenous β-catenin in several cell lines, including the hepatoblastoma cell line HepG2 [15]. To further explore this regulation and to test whether FH535 could block ectopic β-catenin, co-transfections with β-catenin expression vectors and the TCF4-dependent luciferase reporter vector TOPFlash were performed in the human HCC cell lines Huh7 and Hep3B (Fig. 1). In both cell lines, co-transfected wild-type β-catenin expression vector increased luciferase activity from TOPFlash nearly 15-fold compared to cells co-transfected with the empty vector (E.V.) control. This β-catenin-dependent increase was inhibited by FH535 in a dose-dependent manner. β-catenin is often mutated in various cancers, including HCC. One natural mutation changes the serine at position 37; this altered form of β-catenin is resistant to degradation by the APC complex and thus has higher stability. To test whether this form of activated form of β-catenin could also be blocked by FH535, an expression vector for βCatS37A, in which the serine at position 37 has been changed to an alanine, was co-transfected with TOPFlash. As expected, βCatS37A-mediated transactivation of TOPFlash was significantly higher than transactivation by wild-type β-catenin. However, in both cell lines, βCatS37A-mediated transactivation was significantly inhibited by FH535. As controls, cells were also co-transfected with FOPFlash, which is identical to TOPFlash except that the TCF4 sites have been mutated and therefore no longer responsive to β-catenin; FOPFlash was not activated by wild-type β-catenin or βCatS37A as shown in Figure 1.


Figure 1. FH535 inhibits β-catenin dependent transcriptional activation in HCC cell lines.

Huh7 (Panel A) and Hep3B (Panel B) HCC cells were transfected with the luciferase reporter genes TOPFlash (left panels), which contains three TCF binding sites, or E3-pGL3 (right panels), which contains the AFP enhancer element E3 that has a highly conserved TCF site. Cells were additionally co-transfected with an expression vector that contained no insert (empty vector control, E.V.), wild-type β-catenin (β-catenin), or a constitutively active form of β-catenin (βcatS37A). Renilla luciferase was used to control for variations in transfection efficiency. Six hours after the addition of DNA, cells were treated with DMSO alone (no treatment) or increasing amounts of FH535. After 48 hours, luciferase levels were determined; firefly luciferase was normalized to renilla. In both cell lines, FH535 inhibited β-catenin-dependent activation of target genes. *P<0.05. The experiment was done twice with similar results.


TOPFlash contains three consensus TCF4 binding motifs that confer responsiveness to β-catenin. To test whether FH535 could also block β-catenin-mediated transactivation of a TCF4 motif in the context of a natural regulatory region, co-transfections were performed with E3-pGL3. E3 is a ~340 bp fragment that contains alpha-fetoprotein (AFP) enhancer element E3, one of three enhancers that control hepatic expression of the mouse AFP gene. E3 contains binding sites for multiple factors, including Foxa/HNF6, C/EBP, orphan nuclear receptors, and TCF4 [26][27]. We recently showed that this enhancer is regulated by β-catenin in cells and transgenic mice [21]. E3-pGL3 was transactivated by β-catenin and to a greater extent by βCatS37A (Fig. 1). However, this transactivation by both wild-type and S37A forms of β-catenin was blocked by FH535 in a dose-dependent manner.

3.2 FH535 inhibits β-catenin-mediated transcriptional activation in LCSC

Previous studies have shown that β-catenin signaling is elevated in EpCAM positive cells with LCSC properties [28]. We previously described that CD133+, CD44+, CD24+ LCSC aggressively form tumors when small numbers of these cells are injected into nude mice [29]. To test the ability of FH535 to inhibit β-catenin in these LCSCs, transient transfections were performed with TOPFlash. As controls, TOPFlash was also transfected into the HCC cell lines Huh7 and PLC (Fig. 2). In all three populations, untreated cells exhibited low luciferase levels. When treated with the GSK-3β inhibitor LiCl, which leads to endogenous β-catenin activation[30], TOPFlash activity increased dramatically. FH535 effectively blocked LiCl-mediated activation of TOPFlash in a dose-dependent manner. Interestingly, this inhibition was more robust in LCSC than in either HCC cell line. As a control, transfections were also performed with FOPFlash, which is no longer responsive to β-catenin. As expected, luciferase activity in FOPFlash-transfected cells was neither increased by LiCl nor inhibited by FH535.


Figure 2. FH535 inhibits TOPFlash activation in LCSC and HCC cell lines.

LCSC (left panel), Huh7 (middle panel) and HPLC (right panel) cells were co-transfected with TOPFlash or FOPFlash luciferase reporter genes along with renilla luciferase. After 6 hours, cells were left untreated (no treatment) or treated with LiCl alone or LiCl with increasing amounts of FH535. LiCl is a known activator of β-catenin. After an additional 36 hours, cells were harvested and luciferase levels were determined; firefly luciferase was normalized to renilla. TOPFlash activity was highly induced in all three cell populations; this activation was inhibited by FH535. The negative control FOPFlash showed minimal response to LiCl or FH535. TOPFlash inhibition by FH535 was more robust in LCSC than in either HCC cell line. * P<0.003, # P<0.001. The experiment was done twice with similar results.


3.3 FH535 inhibits proliferation of LCSC and HCC cell lines

Numerous studies have demonstrated that β-catenin plays an important role in proliferation during normal development and in cellular transformation in many tissues, including the liver. Liver development is impaired in the absence of β-catenin, and mutations that activate the β-catenin pathway are found in about 1/3 of HCC [4][5]. Furthermore, the growth of adult liver progenitor stem cells (oval cells) can be inhibited by blocking the β-catenin pathway. Since our data indicated that FH535 can block β-catenin-mediated transcriptional activation, we also tested whether proliferation of LCSC and HCC cell lines was affected by this compound. LCSC were cultured in the presence of 10% or 1% serum and with between 5 µM and 30 µM FH535 for 72 hours, and cell proliferation was monitored by 3H-thymidine incorporation (Figs. 3A and 3B, respectively). Proliferation decreased with increasing amounts of FH535, with a more dramatic reduction observed in cells grown in the presence of lower serum; the concentration of FH535 to cause a 50% inhibition of cell grown (IC50) was 13.8 µM for cells grown in 10% serum and 5.1 µM for cells grown in 1% serum. This inhibition was more potent than that seen with XAV939 (IC50 = 55 µM), which inhibits tankyrase, thus stabilizing axin and promoting β-catenin degradation (Fig. 3C) [31]. FH535 also blocked proliferation of HCC cells at concentrations that were similar to that seen with LCSC (IC50 of 10.9 µM, 9.25 µM and 6.6 µM for Huh7, PLC and Hep3B, respectively; Fig.3D). To confirm that FH535 indeed inhibited cell proliferation and did not lead to increased cell death, FH535 and 3H-thymidine were added simultaneously to Huh7 cells, which were then cultured for 18 h. In this scenario, we observed a significant inhibition of proliferation at 2.5, 5, 10 and 15 µM of FH535 treatment as compared to control (p<0.05, n = 6), with FH535 at 15 µM causing a 41% inhibition (Figure S3). This data indicates that FH535 is inhibiting cell proliferation rather than increasing cell death.


Figure 3. FH535 inhibits proliferation of LCSC and HCC cell lines.

Cells were seeded in 96-well plates in 0.2 ml of media as described below for 72 hours, followed by the addition of 3H-thymidine at 1 µCi/well for 4 hours. Incorporation of 3H-thymidine was determined by scintillation counting. In panels A, B and D, the final concentration of DMSO in each well was 0.05%; in panel C, the final DMSO concentration in each well was 0.1%. (A) LCSCs were plated at 1000 cells/well in DMEM with 10% FBS along with DMSO alone or with increasing amounts of FH535. (B). LCSCs were plated at 5000 cells/well in DMEM with 1% FBS with DMSO alone or with increasing concentrations of FH535. (C). LCSCs were plated in DMEM with 10% FBS at 1000 cells/well with DMSO alone or increasing concentrations of XAV939. (D). Huh7, Hep3B and PLC cells were plated in DMEM with 10% FBS at 1000, 2500, and 5000 cells/well, respectively, with DMSO alone or increasing concentrations of FH535. Pvalues are for all the three cell lines treated with FH535 are compared to controls. The experiment was done twice with similar results.


3.4 FH535 induces cell cycle arrest in the HCC cell line Huh7 and in LCSC

The ability of FH535 to inhibit cell proliferation prompted us to investigate the cell cycle distribution following treatment. Huh7 cells were synchronized by growth in 0.1% FBS for 24 hours and then cultured in the presence of 10% FBS and with no FH535 or FH535 at 7.5 µM and 15 µM. After 24 hours, cells were harvested and DNA content was analyzed by propidium iodide staining. In the presence of FH535, there was a statistically significant increase in the number of cells in G0/G1 and a corresponding decreased in the percentage of cells in S phase compared to cells grown in the absence of FH535 (Fig. 4A). The number of cells in G2 was not significantly altered by FH535. In addition, there was no sub-G1 peak detected by flow cytometry, indicating that FH535 was not promoting apoptosis at the concentrations being use (see Figure S4). We also did cell cycle analysis in LCSC after FH535 treatment and found FH535 at 15 µM significantly caused G1 phase arrest in LCSC (P = 0.012). FH535 also significantly reduced G2/M phase in the LCSC after 24 h of 7.5 µM and 15 µM FH535 treatment (P = 0.038 and P<0.001 respectively), no significant S phase inhibition was observed in LCSC (p = 0.446) (Fig. 4B.). Our data are similar to previously published results and reflects β-catenin regulation of cell cycle is different in different cell types [32][33]. Cell cycle regulators (cyclins, CDKs and regulators) can vary in different cell types, which could lead to different responses after FH535 treatment. This may worth exploring in our future study.


Figure 4. FH535 alters cell cycle progression in Huh7 and LCSC cells.

A. Huh7 cells were cultured in DMEM +10%FBS for 24 h. The cells were washed with serum free DMEM 3 times, then cultured in DMEM +0.1% FBS for 24 h for cell synchronization. Cells were then cultured in DMEM+10% FBS along with different concentrations of FH535 for 24 h. The cells were harvested and stained with propidium iodide (PI) and analyzed by flow cytometry according to the GenScript protocol (Piscataway, NJ, USA). Treatment with FH535 increased the percentage of cells in G1 and decreased the percentage of cells in S phase. The experiment was done twice with similar results. B. LCSC cells were cultured in CelProgen complete LCSC culture medium for 24 h. Cells were then washed with serum free CelProgen medium 3 times and cultured in CelProgen Medium +0.1% FBS for 24 h for synchronization of the cells. The cells were then returned to CelProgen Complete Medium +10% FBS with different concentrations of FH535 for 24 h. Cell cycle was assayed as per Huh7 described above.


3.5 Expression of β-catenin target genes cyclin D1 and Survivin is inhibited by FH535

β-catenin controls cell proliferation and survival by regulating the expression of numerous targets genes. Two well-established targets are the genes encoding Survivin (Birc5) and Cyclin D1 (CcnD1). Survivin is an anti-apoptotic protein that also regulates progression through mitosis [34]; Cyclin D1 controls proliferation by activating the G1 kinases cdk4 and cdk6 [35]. Survivin and Cyclin D1 transcription are regulated through TCF elements in their promoter regions [36]. To test whether FH535 inhibits expression of these two β-catenin target genes, real-time RT-PCR was performed with LCSC and HCC cells that were treated with increasing amounts of FH535. Cyclin D1 and Survivin mRNA levels were reduced by FH535 in all three cell populations in a dose-dependent manner (Fig. 5). To confirm that this reduction in mRNA levels also led to lower protein levels, western analysis was performed using whole cell extracts from Huh7 cells. Both Cyclin D1 and Survivin protein levels were reduced in a dose-dependent manner, with the greatest reduction seen in the presence of 10 µM FH535 (Fig. 6.). Densitometric analysis indicated that FH535 at 5 and 10 µM inhibited Cyclin D1 28% and 64% respectively; FH535 at 5 and 10 µM inhibited surviving 24% and 48% respectively (Fig. 6).


Figure 5. FH535 reduces cyclin D1 and survivin mRNA levels in LCSC and in HCC cell lines.

LCSCs, Huh7 and Hep3B cells were treated with DMSO alone or increasing concentrations of FH535 for 38-time PCR for expression of Cyclin D1 (Panel A) or Survivin (Panel B). In both cases, mRNA levels were plotted relative to β2-microglobulin. The experiment was done twice with similar results.



Figure 6. FH535 reduces cyclin D1 and Survivin protein levels in Huh7 cells.

Huh7 cells were treated with DMSO alone or increasing amounts of FH535 for 38-PAGE, and transferred for Western analysis with antibodies against Cyclin D1, Survivin, and β-actin. The top of shows the western blot image; the bottom graph shows densitometric analysis of the western data. This densitometric analysis indicated that FH535 at 5 and 10 µM inhibited Cyclin D1 protein levels 28% and 64% respectively; FH535 at 5 and 10 µM inhibited Survivin protein levels 24% and 48% respectively. The experiment was done twice with similar results.



In recent years, numerous signaling pathways have been implicated in hepatic carcinogenesis. The β-catenin pathway is essential in stem cells for self-renewal and maintenance of stem cell properties. Disruption of this balance results in both genetic and epigenetic changes, found in many cancers, including colon cancer and HCC [4]. In this study, we used FH535 as an inhibitor of the β-catenin pathway. This compound has been used previously to inhibit β-catenin expression in cells from colon and lung as well as in cells from hepatoblastoma and HCC [15]. In this report, the authors concluded that FH535 was toxic to a number of cell lines, including Huh7. However, their assays could not distinguish between toxicity and reduced cell proliferation. Our data indicates that FH535 does indeed inhibit cell proliferation; we did not directly measure toxicity.

FH535 inhibition of LCSC proliferation is of interest due to its potential therapeutic effect in chemo-resistant HCC. Our group and others have focused on strategies to inhibit the proliferation of LCSC and differences in resistance patterns with non-liver cancer stem cell lines in vitro and in vivo.

Despite numerous efforts, the etiology of HCC tumorigenesis, whether transformed cells originate from mature hepatocytes or stem/progenitor cells remains unclear. Stem cells are defined by their potential for self-renewal and by their ability to proliferate and differentiate into diverse cell types [37]. In recent years, studies have provided convincing evidence that these cells do exist in solid tumors of many types including, brain, breast, colorectal, liver, pancreas and prostate cancers [27]. In this study we have used LCSC that are 64.4%, 83.2%, 96.4% and 96.9% positive, respectively, for CD133, CD44, CD24 and Aldehyde A1 as determined by flow cytometry. These cells have been previously profiled not only by checking the LCSC markers but also by evaluating their tumorigenic potential using low cell numbers (using 2000 LCSCs instead of 100,000 HCC cells to generate tumors) and studying resistance to several drugs. We previously found that these LCSC have intermediate to high resistance to drugs compare to non- liver cancer stem cell lines using different inhibitors.

In this study, we found that FH535, LCSC inhibition of proliferation was affected by FBS concentration in the culture medium, suggesting that the PPAR pathway may be involved in LCSC proliferation as found in the human cancer cell line HCT116 [15]. This could be explained by a variety of fatty acids and their derivatives present in the FBS that are natural agonists to PPAR. It is possible the PPAR agonists suppress the inhibitory effects of FH535 in cell culture. Indeed, in HCT116 cells, FH535 inhibition of β-catein/TCF-dependent luciferase reporter genes was five times stronger in serum-free medium than in media containing 10% FBS. The ability of FH535 to inhibit tumor growth was dramatically increased when 10% FBS was replaced with 10% BSA [15]. Lysophosphatidic acid was found to be an effective PPAR agonist that could reverse FH535 induced inhibition of HCT116 growth [15]. However, the potential function of PPAR in LCSC is beyond the scope of this study and needs further investigation. Recently, FH535 was found to be the most potent drug among several other Wnt/β-catenin inhibitors on human biliary tract cancer cells cultured in serum-free medium [38]. Our study found that FH535 is much more potent than XAV939 in 10%FBS DMEM. This may be related to the PPAR inhibition potential of FH535. Our study found that FH535 inhibited HCC cell lines Huh7, Hep3B and PLC proliferation, indicating that Wnt/β-catenin signaling plays an important role not only in LCSC but also in HCC.

FH535 inhibition of LCSC and HCC proliferation was illustrated by its ability to inhibit β-catenin/TCF/LEF-dependent luciferase reporter activity. To our knowledge, this is the first report on the ability of FH535 to inhibit β-catenin/TCF/LEF activity in LCSC and in HCC cell lines. Previously, Handeli and Simon reported that FH535 inhibits β-catenin/TCF/LEF activity in the HepG2 cell line, which was mistakenly labeled as HCC by these authors [15]. For over thirty years this cell line was considered HCC by numerous investigators. Lopez et al., who initially isolated these cells, recently concluded that HepG2 cells should in fact be considered a hepatoblastoma cell line [39]. Further studies will be needed to investigate how FH535 inhibition of β-catenin influences LCSCs and HCCs. As shown here, cyclin D1 and Survivin expression are inhibited by FH535. Survivin is an anti-apoptotic protein that also regulates progression through mitosis [26], whereas Cyclin D1 controls proliferation by activating the G1 kinases [35]. Real-time RT-PCR and Western analysis confirmed that the expression of these target genes was evident at the mRNA and protein level. Our preliminary data indicate that FH535 treatment does not alter CD133, CD13 and EPCAM expression in LCSC and HCC cell lines (data not shown). Further analysis of these and other stem cell markers are warranted.

In conclusion, our data show that FH535 is a potent inhibitor of the Wnt/β-catenin pathway in LCSCs and HCC cell lines. Whether its ability to inhibit PPAR also affects the growth of LCSCs and HCC cells will require further investigation. Further studies will also be needed to investigate the in vivo efficacy and toxicity of FH535 on HCC xenografts in an animal model. The role of combination therapy using FH535 with other anti-HCC drugs and the possibility of finding cross-talk of Wnt/β-catenin pathway with other signaling pathways should be investigated. Wnt signaling in hepatocellular carcinoma: analysis of mutation and expression of beta-catenin, T-cell factor-4 and glycogen synthase kinase 3-beta genes.

Hepatocellular carcinoma (HCC) is a common killer cancer in the world. Recently, abnormal activation of the Wnt pathway has been found to be involved in the carcinogenesis of several human cancers including HCC. The goal of the present study was to investigate the mechanism of inappropriate activation of the Wnt pathway in hepatocarcinogenesis. We analyzed the alterations of three key components of the Wnt pathway: beta-catenin, glycogen synthase kinase (GSK)-3beta and T-cell factor (Tcf)-4 in 34 HCC and paracancerous normal liver by immunohistochemistry, polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP), direct sequencing, and quantitative real-time reverse transcription (RT)-PCR. We found that 61.8% (21/34) of all HCC examined showed an abnormal beta-catenin protein accumulation in the cytoplasm or nuclei. The RT-PCR-SSCP and direct sequencing showed that beta-catenin exon 3 mutations existed in 44.1% (15/34) of the HCC. No mutations of GSK-3beta or Tcf-4 were detected in HCC. Moreover, messenger RNA of beta-catenin and Tcf-4, but not GSK-3beta, was found to be overexpressed in HCC. On analyzing the relationship between alterations of beta-catenin or Tcf-4 and C-myc or Cyclin D1 expression, we found that mutations of beta-catenin, as well as overexpression of beta-catenin or the Tcf-4 gene were independently correlated with C-myc gene overexpression in HCC. Our present findings strongly suggest that mutations of beta-catenin, as well as overexpression of beta-catenin and the Tcf-4 gene, independently activate the Wnt pathway in HCC, with the target gene most likely to be C-myc. Wnt signaling and cancer
Genes & Dev. 2000. 14:1837-1851

The regulation of cell growth and survival can be subverted by a variety of genetic defects that alter transcriptional programs normally responsible for controlling cell number. High throughput analysis of these gene expression patterns should ultimately lead to the identification of minimal expression profiles that will serve as common denominators in assigning a cancer to a given category. In the course of defining the common denominators, though, we should not be too surprised to find that cancers within a single category may nevertheless exhibit seemingly disparate genetic defects. The wnt pathway has already provided an outstanding example of this. We now know of three regulatory genes in this pathway that are mutated in primary human cancers and several others that promote experimental cancers in rodents (Fig. 1). In all of these cases the common denominator is the activation of gene transcription by β-catenin. The resulting gene expression profile should provide us with a signature common to those cancers carrying defects in the wnt pathway. In this review, the wnt pathway will be covered from the perspective of cancer, with emphasis placed on molecular defects known to promote neoplastic transformation in humans and in animal models.

Figure 1.

Oncogenes and tumor suppressors in the wnt signaling pathway. Lines ending with arrows or bars indicate activating or inhibitory effects, respectively. Green and red indicate proto-oncogenic and tumor suppressive activity, respectively, in human cancer or transgenic animals. Definition of the genes and the basis for their activities are described in the text.

The wnt signaling mechanism

The model illustrated in Figure 2 is a proposed mechanism for wnt signaling and is based on the following literature. Signaling is initiated by the secreted wnt proteins, which bind to a class of seven-pass transmembrane receptors encoded by the frizzled genes (Bhanot et al. 1996; Yang-Snyder et al. 1996; He et al. 1997). Activation of the receptor leads to the phosphorylation of the dishevelled protein which, through its association with axin, prevents glycogen synthase kinase 3β (GSK3β) from phosphorylating critical substrates (Itoh et al. 1998; Kishida et al. 1999; Lee et al. 1999; Peters et al. 1999; Smalley et al. 1999). In vertebrates, the inactivation of GSK3β might result from its interaction with Frat-1 (Thomas et al. 1999; Yost et al. 1998; Li et al. 1999a; Salic et al. 2000). The GSK3β substrates include the negative regulators axin and APC, as well as β-catenin itself (Rubinfeld et al. 1996; Yost et al. 1996; Yamamoto et al. 1999). Unphosphorylated β-catenin escapes recognition by β-TRCP, a component of an E3 ubiquitin ligase, and translocates to the nucleus where it engages transcription factors such as TCF and LEF (Behrens et al. 1996; Molenaar et al. 1996;Hart et al. 1999). Additional components in the pathway include casein kinases I and II, both of which have been proposed to phosphorylate dishevelled (Sakanaka et al. 1999; Willert et al. 1997; Peters et al. 1999). The serine/threonine phosphatase PP2A associates with axin and APC, although its functional role in the pathway remains obscure (Hsu et al. 1999; Seeling et al. 1999). Also obscure is the manner by which the wnt receptors communicate with dishevelled.

Figure 2.

Proposed mechanism for the transmission of wnt signals. In the absence of wnt –wnt) GSK3β phosphorylates APC and axin, increasing their binding affinities for β-catenin, which too is phosphorylated by GSK3β, marking it for destruction. In the presence of wnt (+wnt) FRAT prevents GSK3β from phosphorylating its substrates, and β-catenin is stabilized. Casein kinase1ε (CK1ε) binds to and phosphorylates dishevelled (dvl) modulating the FRAT1/GSK3β interaction. RGS, PDZ, and DIX are protein interaction domains.

 Receptors, ligands, and related proteins

The proto-oncogenic effects of wnt were discovered over 18 years ago inciting intense investigation into the role of wnt genes in human cancer (Nusse and Varmus 1982). The subsequent discovery of wingless, the fly homolog of wnt-1, paved the way for assembling a signaling pathway subsequently found to contain cancer causing genes (Cabrera et al. 1987; Rijsewijk et al. 1987). Although wnt was the prototypical oncogene in this pathway, no formal proof for its involvement in human cancer has ever been documented. There have been numerous reports on the overexpression, and sometimes underexpression, of wnt genes in human cancers, but mRNA expression levels are merely correlative. More compelling evidence, such as amplification, rearrangement, or mutation of genes encoding wnt ligands or receptors has not been forthcoming. In lieu of these sorts of findings, we are left to speculate on the consequences of epigenetic events implicating these genes in human cancer. In doing so we can use animal and cell culture models to guide our interpretation.

The wnt ligands, of which there are at least 16 members in vertebrates, are secreted glycoproteins that can be loosely categorized according to their ability to promote neoplastic transformation (for review, seeWodarz and Nusse 1998). For example, the activation of wnt-1, wnt-3, or wnt-10b by retroviral insertion in the mammary gland will promote tumor formation in mice (Lee et al. 1995; Nusse and Varmus 1982; Roelink et al. 1990). Oncogenic potential can also be assessed in cultured mammalian cells, such as C57MG and CH310T1/2, where expression of the proto-oncogenic wnts results in morphological transformation (Bradbury et al. 1994; Wong et al. 1994). These cells are transformed by wnt-1, wnt-2, wnt3a but not by wnt-4, wnt-5a, and wnt-6. The transforming wnt genes also promote the accumulation of β-catenin in some cultured mammalian cells (Shimizu et al. 1997). Some aspects of the wnt cancer pathway are also recapitulated inXenopusdevelopment, where injection of transforming wnts into early embryos results in duplication of the dorsal axis (Wodarz and Nusse 1998). A caveat here is that the lack of specific receptors for certain wnts might also explain their inactivity in some of these assays (He et al. 1997). Nevertheless, identifying those wnts capable of neoplastic transformation will aid the interpretation of epigenetic evidence implicating wnts in cancer. For example, expression of thewnt-16 gene is activated by the E2A–Pbx1 fusion product in acute lymphoblastoid leukemia (McWhirter et al. 1999), but the oncogenic potential of wnt-16 is unknown.

As might be expected from the plethora of wnt genes, there are also numerous wnt receptors. At least 11 vertebrate frizzled genes have been identified, but how they differ in function and ligand specificity is far from clear. The analysis of mere binding specificity may not be sufficient to sort out the appropriate combinations of functional receptor-ligand interactions. Wnt-3a and wnt-5a both bind to Human frizzled 1 (Hfz1), yet only wnt-3a mediates TCF-dependent transcription (Gazit et al. 1999). This suggests that the activation of TCF/LEF-dependent transcription is a good correlate to neoplastic transformation. Implementation of this assay, along with a second assay involving the translocation of PKC to the cell membrane, resulted in the categorization of murine wnt receptors into two exclusive groups (Sheldahl et al. 1999). Human FzE3 fell into the TCF/LEF activation group, consistent with previous work showing that its overexpression resulted in nuclear localization of β-catenin (Tanaka et al. 1998). This receptor was also expressed in numerous human esophageal cancers, but not in matched normal tissue (Tanaka et al. 1998).

In addition to the frizzled receptors, there exists a family of secreted proteins bearing homology to the extracellular cysteine-rich domain of frizzled. The so-called secreted frizzled-related proteins (sFRP) bind to the wnt ligands, thereby exerting antagonistic activity when overexpressed in wnt signaling assays (Leyns et al. 1997; Wang et al. 1997). The vertebrate sFRPs, like the frizzled proteins, exhibit functional specificity with respect to the various wnts. InXenopus assays, the prototypical frizzled related protein frzb, now known as sFRP-3, inhibited wnt-1 and wnt-8, but not wnt-5a (Leyns et al. 1997; Lin et al. 1997; Wang et al. 1997). Assays in mammalian cells showed that FrzA, now termed sFRP-1, inhibited wnt-1-induced accumulation of β-catenin (Dennis et al. 1999;Melkonyan et al. 1997). Again, binding specificity may not relate to functional specificity, as wnt-5a associated with sFRP-3 but was unable to inhibit its activity (Lin et al. 1997). Even the significance of specific functional interactions might be suspect based on recent titration experiments with purified soluble sFRP-1. At low concentrations sFRP-1 enhanced signaling activity by soluble wingless protein, whereas at higher concentrations it was inhibitory (Uren et al. 2000). The authors proposed high and low states of binding affinity that involved the carboxy-terminal heparin binding domain and the amino-terminal cysteine-rich domain of sFRP-1, respectively. Binding to the cysteine-rich domain might confer inhibition while binding to the carboxy-terminal region could facilitate presentation of active ligand to receptor. The potential for some sFRPs to activate wnt signaling is consistent with a previous study in which sFRP-2, then known as SARP-1, increased the intracellular concentration of β-catenin and conferred anti-apoptotic properties to cultured MCF-7 cells (Melkonyan et al. 1997). Functional studies are further complicated by the binding of a sFRP to the putative human receptor frizzled-6, underscoring additional possible modes of regulation (Bafico et al. 1999). The sFRPs have not been directly linked to cancer, but one could speculate that the anti-apoptotic activity observed with the SARP-1 could contribute to tumor progression. Alternatively, the identification of sFRP-2 as a target of the hedgehog signaling pathway might be relevant to human basal cell cancers (Lee et al. 2000). Additional structurally distinct secreted inhibitors of wnt signaling include the recently discovered dickopft-1 and wif-1 proteins (Fedi et al. 1999; Glinka et al. 1998;Hsieh et al. 1999).


The serine/threonine kinase GSK3β binds to and phosphorylates several proteins in the wnt pathway and is instrumental to the down regulation of β-catenin (Dominguez et al. 1995; He et al. 1995; Hedgepeth et al. 1999b; Ikeda et al. 1998;Itoh et al. 1998;Li et al. 1999a; Nakamura et al. 1998b; Rubinfeld et al. 1996;Yamamoto et al. 1999; Yost et al. 1996). As a negative regulator of wnt signaling, GSK3β would qualify as a potential tumor suppressor. However, mutations or deletions in the gene coding for GSK3β were not been detect ed in a survey of colorectal tumors (Sparks et al. 1998). Perhaps GSK3β can compensate for the loss of GSK3β and the biallelic inactivation of both these genes is unlikely in tumor progression. Alternatively, the utilization of GSK3β by pathways independent of wnt could make its overall ablation incompatible with cell viability. Nevertheless, inactivation of GSK3β can still be achieved by a means other than genetic ablation and can occur in a manner that uniquely affects wnt signaling. This mode of inactivation involves the association of GSK3β with Frat-1. Frat-1 was identified by insertional mutagenesis in a screen for genes that enhanced the progression of transplanted T-cell lymphomas in mice (Jonkers et al. 1997). Subsequent transgenic expression of Frat-1 alone did not induce spontaneous lymphomas, but greatly enhanced lymphomagenesis initiated either by leukemia virus M-MuLV or expression of the Pim1 oncogene (Jonkers et al. 1999). A connection to GSK3β was realized by the discovery of the Frat-1 Xenopushomolog GBP, a GSK3β binding protein inhibitory to wnt signaling when expressed in Xenopus embryos (Yost et al. 1998). Frat-1 is also antagonistic to wnt signaling in mammalian cells, presumably because it competes with axin for binding to GSK3β (Li et al. 1999a; Thomas et al. 1999). GBP also inhibited the phosphorylation and degradation of β-catenin in vitro when added to Xenopusextracts (Salic et al. 2000). Although Frat-1 contributes to cancer progression in a transgenic mouse model, its contribution to human cancer has not been documented.


The genetic analysis of dishevelled in developmental systems has defined it as a positive mediator of wnt signaling positioned downstream of the receptor and upstream of β-catenin (Noordermeer et al. 1994). Overexpression or constitutive activation of dishevelled would be expected to promote neoplastic transformation, but its involvement in human cancers has not been reported. This might reflect the dual function of dishevelled, one that transduces wnt signals for the stabilization of β-catenin and a second that relays signals for the activation of jun kinases (Li et al. 1999b; Moriguchi et al. 1999). Although these two functions are housed in physically separable regions of the protein, dysregulation of one function, without impacting the other, could place severe constraints on selection for potential oncogenic mutations. A possible connection of dishevelled to cancer is through casein kinase II, which binds to and phosphorylates dishevelled and also promotes the formation of lymphomas when expressed in transgenic mice (Seldin and Leder 1995; Song et al. 2000; Willert et al. 1997).


Mutations in the β-catenin gene (CTNNb1) affecting the amino-terminal region of the protein make it refractory to regulation by APC (Morin et al. 1997; Rubinfeld et al. 1997). These mutations affect specific serine and threonine residues, and amino acids adjacent to them, that are essential for the targeted degradation of β-catenin (for review, see Polakis 1999). The mutations abrogate the phosphorylation dependent interaction of β-catenin with β-TRCP, a component of an E3 ubiquitin ligase that makes direct contact with amino terminal sequence in β-catenin (Hart et al. 1999). This regulatory sequence in β-catenin is mutated in a wide variety of human cancers as well as in chemically and genetically induced animal tumors. Importantly, β-catenin mutations in tumors are exclusive to those that inactivate APC. This is particularly apparent in colorectal cancer where the vast majority of these tumors contain APC mutations and the overall frequency of β-catenin mutations is quite low (Samowitz et al. 1999; Sparks et al. 1998;Kitaeva et al. 1997) (Table 1). When colorectal tumors lacking APC mutations were analyzed separately, the likelihood of finding a CTNNb1 mutation was greatly increased (Iwao et al. 1998; Sparks et al. 1998). The exclusivity of CTNNb1 and APC mutations in colorectal cancer was also evident from the analysis of replication error-positive tumors identified by microsatellite instability. Both the hereditary and sporadic forms of replication error-positive colorectal cancers had a relatively high frequency of β-catenin mutations, whereas APC mutations were relatively rare (Mirabelli-Primdahl et al. 1999; Miyaki et al. 1999) (Table 1). Interestingly, this correlation between microsatellite instability andCTNNb1 mutations was not apparent in endometrial cancers (Mirabelli-Primdahl et al. 1999).

Table 1. 
Beta-catenin mutations in human cancers
Aggressive fibromatosis, otherwise known as desmoid tumor, is a locally invasive fibrocytic growth that occurs with increased incidence in patients with familial adenomatous polyposis coli (FAP). FAP individuals carry APC mutations in their germline and present with multiple intestinal adenomas at an early age. Desmoids also occur sporadically and, with the exception of colorectal cancer, represent a rare example of biallelic inactivation of APC in individuals without a pre-existing germline mutation in APC (Alman et al. 1997). Not surprisingly, mutations inCTNNb1 have also been detected in sporadic desmoid tumors (Shitoh et al. 1999;Tejpar et al. 1999). The β-catenin mutations were found in over half of the 42 desmoids analyzed, while inactivating mutations in APC were detected in nine and, again, there was no overlap between APC and β-catenin mutations (Tejpar et al. 1999). The β-catenin mutations were all of the missense variety and were confined to codons 41 and 45. Some of the desmoids lacked mutations in either β-catenin or APC, but all displayed increased expression of β-catenin, implying that yet unidentified defects in β-catenin regulation exist in some of these tumors.

There appears to be a low probability of accruing biallelic inactivating mutations in APC in most sporadic cancers, despite increased cancer incidence at numerous extracolonic sites in FAP patients. This suggests that the stabilization of β-catenin can promote cancer in many tissue types, but the biallelic inactivation of APC is an unlikely means to this end. Components in the wnt pathway other than APC, such as β-catenin, might make easier targets for oncogenic mutations. Indeed, several mutations in CTNNb1 were recently identified in gastric cancers, which occur with increased incidence in FAP patients (Park et al. 1999). In this study, 27% of intestinal type gastric cancers harbored mutations in β-catenin. Hepatoblastoma also occurs with increased incidence in FAP individuals (Hughes and Michels 1992;Giardiello et al. 1996; Cetta et al. 1997), but biallelic inactivation of APC is uncommon in the sporadic forms of these tumors. In three separate studies, mutations in β-catenin were identified at high frequency in hepatoblastoma, while no APC mutations were found (Koch et al. 1999; Jeng et al. 2000; Wei et al. 2000). Hepatoblastoma is also associated with Beckwidth–Wiedemann syndrome (BWS), however, a direct link between wnt signaling and the genetic defects underlying BWS are unlikely as a tumor from one of these patients also contained a somatic mutation in β-catenin (Wei et al. 2000). By contrast, a subset of patients with Turcot’s syndrome harbor germline mutations in APC and are at increased risk of medulloblastoma (Hamilton et al. 1995; Lasser et al. 1994). Although inactivating mutations in APC have not been detected in the sporadic forms of medulloblastoma, CTNNb1mutations were found in a small percentage (Zurawel et al. 1998).

Hepatocellular carcinoma (HCC) has become one of the most common tumors harboring mutations in the wnt pathway. Based on five separate studies, the frequency of CTNNb1 mutations in hepatocellular carcinoma (HCC) was ∼20% overall and perhaps higher still for HCCs associated with hepatitis C virus (de La Coste et al. 1998; Miyoshi et al. 1998;Huang et al. 1999; Legoix et al. 1999; Van Nhieu et al. 1999) (Table1). Preliminary data indicated a poorer prognosis associated with nuclear accumulation of β-catenin in HCC and histological data indicated enhanced nuclear staining in the invasive and intravascular compartments of the tumors (Huang et al. 1999; Van Nhieu et al. 1999). In one of these studies an inverse correlation between β-catenin mutations and loss of heterozygosity in the genome was noted (Legoix et al. 1999). This suggests that chromosomal instability and mutations inCTNNb1 represent alternative modes of tumor progression in HCC.

It is noteworthy that c-myc and cyclin D genes are amplified in a subset of HCCs and both these genes are downstream targets of β-catenin (He et al. 1998; Nishida et al. 1994; Peng et al. 1993;Shtutman et al. 1999; Tetsu and McCormick 1999). It would be of interest to determine whether any overlap exists between their amplification and CTNNb1mutations in HCC. Animal models of HCC have provided some clues toward understanding the relationship between these genes in cancer. HCCs induced by transgenic expression of SV40 T antigen in murine liver did not contain mutations in CTNNb1 (Umeda 2000). As T antigen activates cyclin D kinase by sequestration of Rb, the activation of the cyclin D gene by mutant β-catenin may no longer be required. By contrast, activating mutations inCTNNb1 were identified in half of the HCCs generated by transgenic expression of c-myc in murine liver (de La Coste et al. 1998). This animal model suggests that β-catenin mutations occur as a second “hit” in HCC tumor progression in cooperation with a distinct cancer pathway initiated by c-myc. That CTNNb1mutations can occur subsequent to other oncogenic defects is also evident from their occurrence in Wilm’s tumor. Mutations in β-catenin were detected in 15% of these pediatric kidney cancers and in two of these cases they were concomitant with mutations in the Wilm’s tumor gene WT1 (Koesters et al. 1999). One of these cases was associated with Denys-Drash syndrome, a familial disorder attributable to germline mutations in WT1.

It makes sense that extracolonic tumors associated with FAP, such as desmoids, medulloblastoma, and HCC, would contain CTNNb1mutations in their sporadic forms. Thyroid cancers also occur with increased incidence in FAP and, not surprisingly, a high frequency ofCTNNb1 mutations was recently reported for anaplastic thyroid cancers (Cetta et al. 2000; Garcia-Rostan et al. 1999). Although many of these mutations affected amino acids known to influence the regulation of β-catenin, many of them affected residues for which the consequence of their mutation is unknown (Garcia-Rostan et al. 1999). In particular, the substitution K49R was detected nine times. This mutation was frequently detected in the context of independentCTNNb1 mutations in the same thyroid tumor, and up to four independent CTNNb1 mutations were found in some tumors. The occurrence of multiple independent CTNNb1 mutations was also noted in some HCCs and might reflect the multifocal origin of some cancers (Huang et al. 1999; Legoix et al. 1999; Van Nhieu et al. 1999). In one HCC study, examination of different tumor areas from the same patient revealed distinct CTTNb1 mutations in two independent cases (Huang et al. 1999).

Some cancers, such as endometrial ovarian tumors, do not occur with increased incidence in patients with FAP, yet they contain activating mutations in CTNNb1(Palacios and Gamallo 1998; Gamallo et al. 1999; Wright et al. 1999). Perhaps inactivation of the remaining wild-type APC allele in FAP individuals is unlikely in this tissue, or the expression of an alternative APC gene compensates for its loss. The CTNNb1 mutations associated with ovarian cancer appeared to be confined to the endometrioid subtype. In this tissue, cancers with activated β-catenin signaling were reported to be less aggressive than their nonactivated counterparts. In one report, a more favorable prognosis was associated with cancers exhibiting enhanced nuclear staining of β-catenin and another indicated higher frequency ofCTNNb1 mutations in lower grade tumors (Palacios and Gamallo 1998; Wright et al. 1999). A similar inverse correlation between tumor grade and occurrence ofCTNNb1 mutations was also reported for uterine endometrial cancers (Fukuchi et al. 1998). The overlap between mutations in CTNNb1 and other gene defects in ovarian cancers has not been explored in detail, although one study noted coexisting mutations in the PTEN tumor suppressor andCTNNb1 in endometrioid tumors (Wright et al. 1999).

Additional types of cancers with CTNNb1 mutations, albeit at low frequency, include melanoma and prostate. Although only one of sixty-five melanomas contained detectable mutations, nuclear localization of the protein was seen in one-third (Rimm et al. 1999). Thus, additional mechanisms for β-catenin activation likely occur in these tumors. Possibly the highest percentage ofCTNNb1mutations occurs in a common skin tumor known as pilomatricomas (Chan et al. 1999). That these tumors might contain CTNNb1 mutations was surmised from the genesis of similar tumors in transgenic mice expressing mutant β-catenin in the skin (Gat et al. 1998). The tumors appeared to originate from the hair follicle, which is consistent with the lack of hair in mice homozygous for mutations in LEF, a transcription factor responsive to β-catenin (van Genderen et al. 1994).


Axin was originally identified as an inhibitor of wnt signaling inXenopus embryos and was subsequently shown to bind directly to APC, β-catenin, GSK3β and dishevelled (for review, see Peifer and Polakis 2000). A plethora of in vitro and in vivo studies inXenopus, Drosophila, and cultured mammalian cells has demonstrated that axin is central to the down regulation of β-catenin (Zeng et al. 1997; Behrens et al. 1998; Hart et al. 1998;Ikeda et al. 1998; Nakamura et al. 1998a; Sakanaka et al. 1998; Fagotto et al. 1999; Hedgepeth et al. 1999a; Li et al. 1999a; Willert et al. 1999a; Farr et al. 2000). It is not entirely clear how axin functions, but it has been proposed to facilitate the phosphorylation of β-catenin and APC by GSK3β (Hart et al. 1998; Ikeda et al. 1998). Thus axin would be viewed as a tumor suppressor based on its ability to downregulate signaling, and this has now been verified by documentation of its biallelic inactivation in human hepatocellular cancers and cell lines (Satoh et al. 2000). Importantly, these mutations were identified in those HCCs that lacked activating mutations inCTNNb1. All of the mutations were predicted to truncate the axin protein in a manner that eliminated the β-catenin binding sites. Axin, which should now be regarded as a tumor suppressor, constitutes the third genetic defect in the wnt pathway that contributes to human cancer. There also exists a close homolog of axin termed conductin, which exhibits of all the binding and regulatory functions of axin (Behrens et al. 1998). That this apparent redundancy did not suppress axin mutations in HCC suggests conductin is either not functionally equivalent to axin or not expressed at levels sufficient to compensate for its loss in HCCs.


The dependence upon serine/threonine kinases for the regulation of β-catenin implies that phosphatases are also involved. Indeed, the rapid dephosphorylation of the axin protein is a consequence of wnt signaling and has been proposed to both destabilize axin and reduce its affinity for β-catenin (Willert et al. 1999b;Yamamoto et al. 1999). Although axin binds directly to the PP2A catalytic subunit, the phosphatase affecting axin in response to wnt signaling has not been identified (Hsu et al. 1999). If PP2A is this phosphatase, it would be viewed as proto-oncogenic because it downregulates the tumor suppressor axin. On the contrary, expression of the PP2A regulatory subunit B56 in human colon cancer cells results in the downregulation of β-catenin, consistent with a tumor suppressive function in the wnt pathway (Seeling et al. 1999). Moreover, the beta isoform of the PP2A A subunit is deleted in some human colon tumors, again implying tumor suppression (Wang et al. 1998). Also, disruption of twins, aDrosophila gene coding for a PP2A subunit, complemented the overexpression and underexpression of the β-catenin homolog armadillo, in a manner consistent with negative regulation of wnt signaling (Greaves et al. 1999). By all accounts, PP2A plays a role in wnt signaling, but its potential role as proto-oncogene or tumor suppressor might be dependent upon the precise nature of the defect.


Genetic analysis of FAP families led to the identification of theAPC gene, and subsequent studies demonstrating an interaction with β-catenin placed it tentatively in the wnt pathway (Groden et al. 1991; Kinzler et al. 1991; Munemitsu et al. 1995; Rubinfeld et al. 1993; Su et al. 1993). Experiments in Drosophilaultimately revealed that genetic ablation of APC indeed resulted in upregulation of β-catenin signaling (Ahmed et al. 1998). In some systems, such as Xenopus andCaenorhabditis elegans, a positive role for APC in the wnt pathway has been proposed, but the former study suffers from potential overexpression artifacts and the latter from a lack of relatedness to the vertebrate APC protein (Rocheleau et al. 1997; Vleminckx et al. 1997). In any case, APC is a tumor suppressor in human cancers and its mutation relates strongly to the regulation of β-catenin. The spectrum of APC mutations, which typically truncate the protein, suggest selection against β-catenin regulatory domains, albeit not necessarily against β-catenin binding (for review, see Polakis 1999). The selective pressure might be directed against the presence of Axin binding sites, of which there are three, dispersed across the central region of the APC protein (Behrens et al. 1998). The presence of axin binding sites are critical to APC in the regulation of β-catenin levels and signaling in cultured cells (Kawahara et al. 2000). Moreover, mice lacking wild-type APC but expressing a truncated mutant APC retaining a single axin binding site are viable and do not develop intestinal neoplasia (Smits et al. 1999). This has not been the case for mice with more extensive truncations in APC (Oshima et al. 1995a; Su et al. 1992). Also, milder forms of colorectal polyposis, as well as familial infiltrative fibromatosis (desmoid tumors), have been associated with germline mutations in the 3′ region of the APC open reading frame. These mutations predict truncated proteins that retain only one or two of the three axin binding sites in APC (Walon et al. 1997; Kartheuser et al. 1999; Scott et al. 1996;van der Luijt et al. 1996). A recent study has also demonstrated that the expression of just the central region of APC, which contains all of the axin and β-catenin binding sites, was sufficient to elicit cellular growth suppression (Shih et al. 2000). This effect is consistent with previous work showing that a like fragment of APC was sufficient to downregulate β-catenin levels in cancer cells (Munemitsu et al. 1995).

Although both copies of the APC gene are typically inactivated in colorectal cancers, it remains possible that a mutant truncated APC could contribute to cancer progression. This was tested by transgenic expression of two different APC mutants in a wild-type intestinal background (Oshima et al. 1995b). This did not result in cancer-prone mice, despite high levels of expression of mutant proteins and, therefore, argues against a dominant negative effect by these particular mutants. However, it does not rule out an additive contribution to tumor progression by mutant APC protein in a background lacking wildtype APC. In fact, genetic evidence argues in favor of selection for a somewhat specific mutant APC protein. The mutation cluster region (MCR) in APC, roughly defined by codons 1250–1500, is not only consistent with selection against specific sequence, but also retention of an APC molecule that extends into the MCR (Fig.3.). A correlation between the presence of a germline mutation in the MCR and the severity of polyposis has been noted (Ficari et al. 2000; Nagase et al. 1992; Wu et al. 1998). The enhanced severity of polyposis suggests there should also be selective pressure for somatic mutations in the MCR, which indeed appears to be the case (Miyoshi et al. 1992). Selective pressure for an MCR mutant has also been proposed based on the occurrence of somatic mutations in the MCR relative to the position of the germline mutation in FAP (Lamlum et al. 1999). Tumors from FAP patients with a germline MCR mutation exhibited frequent inactivation of the remaining APC allele by LOH, while those without a germline MCR mutation had frequent somatic mutations in the MCR (Fig. 3). Therefore, a germline mutation in the MCR could relieve the constraint for a subsequent somatic MCR mutation, thereby increasing the likelihood of polyposis. This implies that a truncated MCR APC mutant has an interfering or gain of function property that enhances tumor progression beyond simple loss of APC function. An interfering function would probably not involve interaction with wild-type APC, as recently suggested, because the MCR mutant is still selected for in the absence of a wild-type APC gene copy (Dihlmann et al. 1999). Finally, some of the germline mutations in APC do not disrupt the open reading frame yet correlate with increased risk of colorectal cancer (Frayling et al. 1998; Gryfe et al. 1999; Laken et al. 1997). These mutations have been proposed to increase the occurrence of subsequent truncating mutations by enhancing the mutational susceptibility of the affected nucleotide tract.

Figure 3.

Mutations in APC. A compilation of germline and somatic mutations in APC illustrates selection for mutations in the mutation cluster region (MCR). MCR mutations result in truncated proteins retaining β-catenin binding but not regulatory activity. Somatic MCR mutations are more frequently selected for in FAP patients with germline mutations outside of the MCR.

Transcription factors

Prior to discussing the potential role for LEF/TCF transcription factors in cancer, it is important to outline the mechanism by which they have been proposed to operate. Although LEF/TCFs bind directly to DNA through their HMG domains, they are incapable of independently activating gene transcription (Eastman and Grosschedl 1999; Roose and Clevers 1999). This has best been illustrated for LEF, which through its binding to the cofactor ALY, makes indirect contacts with a second transcription factor AML (Bruhn et al. 1997). The TCFs do not contain the ALY binding site, but like LEF they cannot activate test genes comprised of multimerized TCF/LEF binding sites and a minimal promotor sequence. However, these reporter genes are activated on coexpression of TCF with β-catenin, suggesting that β-catenin supplies additional cofactors required for transcriptional activation (Molenaar et al. 1996). This activity was localized to the carboxy-terminal region of the Drosophila β-catenin armadillo, which when fused directly to TCF resulted in β-catenin independent transcriptional activation (van de Wetering et al. 1997).

The simple interpretation is that the TCF/LEF-β-catenin complex comprises a bipartite positive acting transcription factor in the wnt pathway. This interpretation agrees well with developmental studies in which the manipulation of LEF/TCF function results in phenotypes consistent with the genetic manipulation of wnt/β-catenin signaling (Behrens et al. 1996; Brunner et al. 1997; Huber et al. 1996; van de Wetering et al. 1997). For example, a zygotic homozygous null mutation inDrosophila LEF results in a loss of naked cuticle in the larval epidermis, a phenotype typical of loss of function wingless mutations (Brunner et al. 1997). Moreover, the formation of excess naked cuticle by ectopic expression of armadillo in wild-type embryos does not occur in the LEF null mutants. Exactly how β-catenin contributes to transcriptional activation is unclear, but might involve additional proteins that bridge the TCF/β-catenin complex to the basal transcriptional machinery. The bridging function might be fulfilled by Pontin 52, a TATA-binding protein that was reported to bind to β-catenin (Bauer et al. 1998). Also, a mutant form of β-catenin incapable of binding LEF squelched LEF-dependent reporter gene activation, presumably by titration of an essential cofactor (Prieve and Waterman 1999). Finally, the carboxy-terminal region of armadillo binds to the Zinc finger protein teashirt, a homeotic gene essential for a subset of wingless signaling outputs in Drosophila (Gallet et al. 1999).

The simple model of positive transcriptional activation by the TCF-β-catenin complex is not in accord with all experiments. Mutation of the TCF/LEF binding sites in the promotors of the wingless responsive gene ultrabithorax and the Wnt-responsive Xenopus gene Siamois enhanced their activities under conditions where the wingless/β-catenin signal input was weak (Brannon et al. 1999; Riese et al. 1997). The mammalian cyclin D gene was recently identified as a wnt target and, again, mutation of the corresponding TCF binding sites enhanced its basal activity (Tetsu and McCormick 1999). These results suggest TCF represses transcription of its target genes in unstimulated cells and the binding of β-catenin promotes derepression. Derepression cannot fully account for signaling activity, however, as mutations in the TCF binding sites compromise target gene activation under conditions of active wnt signaling (Brannon et al. 1999; Riese et al. 1997). Repression of gene expression by TCF is consistent with its direct physical interaction with at least three different gene products, the Groucho/TLE and CtBP corepressors, and the CREB binding protein CBP (Brannon et al. 1999;Cavallo et al. 1998; Levanon et al. 1998; Roose et al. 1998; Waltzer and Bienz 1998).

The groucho/TLE proteins bind to the central region of TCF/LEF at a site distinct from that of β-catenin binding and inhibit gene activation of TCF target genes (Levanon et al. 1998; Roose et al. 1998). By contrast, CtBP binds to two independent sites in the carboxy-terminal region of Xtcf-3, which when mutated abrogated the repressor function of this region of Xtcf-3 (Brannon et al. 1999). The binding sites for CtBP are not present in LEF, which might explain the ability of LEF, but not Xtcf-3, to induce axis duplication in Xenopus embryos. Finally, the Drosophila CREB binding protein CBP was reported to bind to the HMG domain of dTCF (Waltzer and Bienz 1998). Loss-of-function CBP mutants displayed some features of wingless over expression and also suppressed phenotypes resulting from loss of the β-catenin homolog armadillo. The genetics imply suppression of wingless by CBP, which is somewhat paradoxical when considering the role of CBP acetyltransferase activity in chromatin remodeling and gene activation. However, it was shown that CBP acetylates a lysine proximal to the armadillo binding site in TCF, thereby reducing its affinity for armadillo. Repression of β-catenin/TCF signaling by CBP does not occur in all settings, though, as two recent studies demonstrated activation ofXenopus TCF target genes by CBP (Hecht et al. 2000;Takemaru and Moon 2000). CBP directly associated with carboxy-terminal sequence in β-catenin and overexpression of E1A, which also directly binds CBP, reduced β-catenin dependent transactivation.

Does the activation of TCF/LEF target genes by β-catenin cause cancer? Good evidence to this effect was provided by the expression of a chimeric protein consisting of the LEF DNA binding sequence fused to the transcriptional activation domain of either VP16 or the estrogen receptor (Aoki et al. 1999). Expression of these constructs in chicken embryo fibroblasts resulted in their neoplastic transformation. The proliferative potential of TCF was also apparent from the phenotype resulting from homozygous disruption of TCF-4 in the germline of mice. These animals were incapable of maintaining a proliferative stem cell compartment in the small intestine and died shortly after birth (Korinek et al. 1998). Whether the TCF/LEF genes are directly activated by mutations in cancer is unclear, but mutations in TCF-4 have been detected in a subset of colorectal tumors (Duval et al. 1999). The mutations all occur as single base deletions in an (A)9 nucleotide repeat within the 3′ coding region of the gene. These deletions generate frame shifts predicted to effect the proportion of the long and short forms of TCF that normally result from alternative mRNA splicing. The mutations were also found in cancer cell lines, all of which possessed mutations in either APC or β-catenin. This indicates that the TCF mutations do not substitute for APC/β-catenin mutations but could act in an additive manner.

An additional mechanism by which TCFs could contribute to cancer was gleaned from the phenotype of mice homozygous for mutations in TCF-1 (Roose et al. 1999). Fifteen percent of these animals developed adenomatous intestinal polyps by one year of age, implicating TCF-1 as a tumor suppressor. The major isoforms of TCF-1 do not contain a β-catenin binding site and could therefore act in a dominant negative manner in wnt signaling. Crossing TCF-1 null mice with cancer-prone ApcMin/+ mice resulted in offspring with ten times the number of intestinal polyps relative to ApcMin/+ littermates. This experimental model suggests that the genetic ablation of TCF-1 could modify, or even independently contribute to cancer progression in humans. Additional potential mechanisms for cancer would include the inactivation of corepressors such as CtBP and TLE/groucho.

Cross talk

Defects leading to activation of the wnt pathway could also occur in signaling systems that are seemingly unrelated to wnt signaling. One potential mode of cross talk includes the kinase TAK1, which can substitute for MAPK kinase kinase in the yeast pheromone pathway. TAK1 (TGF-β activatedkinase) is activated by TGF-β in mammalian cells and has also been implicated in interleukin-1 activation of NFκB (Ninomiya-Tsuji et al. 1999; Yamaguchi et al. 1995). In c. elegans, the TAK1 homolog MOM-4 negatively regulates the TCF homolog POP-1 by activating another kinase LIT-1, which then phosphorylates POP-1 (Meneghini et al. 1999;Shin et al. 1999). LIT-1 is thought to gain access to POP-1 through its direct binding to the β-catenin homolog WRM-1 (Shin et al. 1999). Parallel interactions have been demonstrated for the mammalian counterparts of these proteins where the phosphorylation of TCF, by the LIT-1 homolog NLK, reduces its DNA binding affinity (Ishitani et al. 1999). Thus a MAPK-like signaling system might downregulate the wnt-1 pathway. A second opportunity for cross talk with wnt signaling was realized by a physical interaction between the β-catenin-TCF complex and SMAD4, a mediator of TGF-β signaling (Nishita et al. 2000). This interaction was proposed to be synergistic with respect to the activation of theXenopus wnt target gene twin. How this relates to cancer is somewhat puzzling when considering that TGF-β signaling is typically compromised by genetic and epigenetic defects during tumor progression.

An additional mode of cross regulation was recently revealed by the discovery that retinoids inhibit β-catenin dependent gene transcription (Easwaran et al. 1999). β-catenin associated with a retinoic acid receptor (RAR) and cooperated with retinoids to enhance activation of a retinoic acid responsive promotor. Moreover, the identification of RAR-γ as a target of wnt signaling inXenopus also points to an interaction between these signaling systems (McGrew et al. 1999). Signaling by β-catenin was also reported to be repressed by expression of sox3 and sox17 transcription factors, which associated directly with β-catenin (Zorn et al. 1999). Although inhibition of β-catenin signaling was clearly demonstrated, it is also possible that β-catenin drives gene activation independent of LEF/TCF, through its association with the sox proteins. Finally, the activation of the WISP genes by β-catenin is highly dependent upon the presence of a CREB binding site in the WISP promotor (Xu et al. 2000). This implies that cAMP-dependent protein kinase A feeds into wnt signaling and might cooperate with the activation of some wnt target genes. The binding of CBP to β-catenin is particularly relevant with respect to this proposal (Hecht et al. 2000; Takemaru and Moon 2000).


It is apparent that wnt signaling causes cancer and that tumor promotion by this pathway can proceed through a number of different genetic defects. Additional mechanisms by which defects in the regulation of wnt signaling contribute to tumor progression probably remain undiscovered. The manifestation of cancer by aberrant wnt signaling most likely results from inappropriate gene activation mediated by stabilized β-catenin. Target genes need not contain TCF/LEF binding sites in their promotors, though, as new potential modes of gene activation by β-catenin are becoming apparent. Several target genes of β-catenin signaling have now been identified and some of their functions are consistent with control of cellular growth, differentiation, and survival. For an excellent summary of wnt target genes, and a wealth of information on wnt signaling in general, I refer the reader to the Wnt Home Page posted by the Nusse lab (http://www.stanford.edu/rnusse/wntwindow.html).

7.10.2 The Wnt.β-catenin pathway in ovarian cancer : a review.

Arend RC1Londoño-Joshi AIStraughn JM JrBuchsbaum DJ.
Gynecol Oncol. 2013 Dec; 131(3):772-9.

Objective: Ovarian cancer is the deadliest gynecologic malignancy and the fifth leading cause of death from cancer in women in the U.S. Since overall survival remains poor, there is a need for new therapeutic paradigms. This paper will review the Wnt/β-catenin pathway as it relates to epithelial ovarian cancer, specifically its role in chemoresistance and its potential role as a target for chemosensitization. Methods: A PubMed search was performed for articles published pertaining to Wnt/β-catenin pathway specific to ovarian cancer. Wnt/β-catenin signaling pathways play an active role in cancer stem cells (CSCs) and carcinogenesis of all ovarian cancer subtypes. Studies also have shown that ovarian CSCs are involved in chemoresistance, metastasis, and tumor recurrence. Results: Wnt/β-catenin target genes regulate cell proliferation and apoptosis, thereby mediating cancer initiation and progression. The Wnt/β-catenin pathway is one of the major signaling pathways thought to be involved in epithelial-to-mesenchymal transition (EMT). Alterations affecting Wnt pathway proteins on the cell membrane, in the cytoplasm, and in the nucleus have been shown to play important roles in the tumorigenesis of ovarian cancer. Conclusions: Wnt signaling is activated in epithelial ovarian cancer. Given the role of the Wnt/β-catenin pathway in carcinogenesis, more pre-clinical studies are warranted to further investigate other Wnt inhibitors in ovarian cancer. The Wnt pathway should also be investigated as a potential target in the development of new drugs for ovarian cancer as a single agent and in combination with chemotherapy or other targeted agents.

Ovarian cancer is the deadliest gynecologic malignancy and the fifth leading cause of death from cancer in women in the U.S. In 2013, there will be an estimated 22,240 newly diagnosed cases of ovarian cancer and an estimated 14,030 deaths in the United States [1].A major contributor to the high mortality rate is the fact that 70% of women with ovarian cancer initially present with metastases throughout the peritoneal cavity. Over the last two decades, advances in chemotherapy have improved the overall survival in patients with advanced stage ovarian cancer [2]. These advances include the introduction of taxane/platinum-based chemotherapy, intraperitoneal delivery of chemotherapy,dose-dense chemotherapy, and the availability of novel agents such as bevacizumab [3,4].Since overall survival remains poor, there is a need for new therapeutic paradigms. Further research is needed to understand how molecular pathways contribute to the development of metastasis, recurrence, and resistance of ovarian cancer to chemotherapeutic agents. Studies have shown that ovarian cancer stem cells (CSCs) are also involved in chemoresistance, metastasis, and tumor recurrence [5]. CSCs area subpopulation of cancer cells that possess characteristics associated with normal stem cells and are able to generate tumors through the stem cell processes of self-renewal and differentiation.These cells are proposed to persist in tumors as a distinct population that cause recurrence and metastasis by giving rise to new tumors. Recently, chemoresistance has been reported to be associated with acquiring epithelial to mesenchymal transition (EMT) in ovarian cancer cells [6].CancercellsundergoingEMT are unique in that they have stem-like properties that enable cancer cell dissemination and metastasis formation [7]. Major signaling pathways involved in EMT include TGF-β, Wnt/ β-catenin, Notch, Hedgehog, and others [8]. Endometrioid ovarian carcinomas often harbor mutations in the β-catenin gene, but mutations in the Wnt/β-catenin pathway are rare in serous, clear cell, and mucinous ovarian carcinomas [9]. There is emerging data that suggests that despite not having mutations, the Wnt/β-catenin pathway plays a role in carcinogenesis of all ovarian cancer subtypes [10–12]. It has been suggested that the Wnt/β-catenin target genes can be divided into two groups: a “stemness/proliferation group” that is active early in tumor progression and an “EMT/ dissemination group” that is expressed in late stage tumors. The Wnt/ β-catenin pathway has been shown to be a therapeutic molecular target for CSCs[13].Wnt/β-catenin target genes regulate cell proliferation and apoptosis,thereby mediating cancer initiation and progression [14,15]. Given the role of the Wnt/β-catenin pathway in carcinogenesis, we will review the Wnt/β-catenin pathway as it relates to epithelial ovarian cancer, specifically its role in chemoresistance and its potential role as a target for chemosensitization.

Historical perspective of Wnt signaling in the ovary

In the late 1990s, the importance of the Wnt pathways in the embryonic development of the ovary was established. Wnt-4, a Wnt ligand, demonstrated a critical role in embryonic ovarian development [16]. Wnt-7a was shown to affect sex-specific differentiation of the reproductive tract [17]. In 2002, Ricken et al. reported that components of the Wnt signaling pathways are expressed in the immature rat ovary, and that their expression is localized to specific ovarian compartments [18]. This study reported the expression of three different Wnt transcripts (Wnt-2b, Wnt-5a, Wnt-11) that were common to five ovarian cancer cell lines derived from histologically varied human ovarian carcinomas.These results raised the possibility that aberrant Wnt expression may be involved in ovarian tumorigenesis in humans. Prior to this study, alterations in Wnt expression had been described in a variety of female human tumors, including breast and endometrial cancer, but this was the first study to suggest its involvement in ovarian cancer. When β-catenin gene mutations were initially discovered in ovarian cancer, they were thought to be limited to the endometrioid subtype [19]. A study by Wu et al. carried out a comprehensive molecular analysis of 45 tumor specimens of primary ovarian endometrioid adenocarcinomas and two ovarian endometrioid adenocarcinomaderived cell lines. They found Wnt/β-catenin pathway defects in both the cell lines and in nearly half of the primary ovarian endometrioid adenocarcinomas analyzed. β-catenin deregulation was most often attributable to a mutation of the β-catenin gene (CTNNB1) itself, although less frequently β-catenin deregulation may have resulted from inactive mutations in the APC, AXIN1, orAXIN2 genes [20]. Depending on the study, a wide range (3–59%) of serous ovarian cancers have also been reported to stain positive for cytoplasmic and nuclear β-catenin by immunohistochemistry even in the absence of mutations in APC, Axin or β-catenin, which are more common in the endometrioid subtype [21–23]. More recent data have shown that although gene mutations in the Wnt/β-catenin pathway are relatively uncommon in ovarian cancer in general, especially in serous ovarian cancer,components of the pathway are still important in the molecular events that lead to ovarian cancer development [12]. There are three main Wnt signaling pathways: the canonical Wnt/β-catenin pathway, the non-canonical planar cell polarity pathway, and the non-canonical Wnt–Ca2+ pathway. These pathways belong to one of two categories: canonical or non-canonical. The difference between these two categories is the presence or absence of β-catenin. The canonical Wnt/β-catenin pathway involves this protein and the non-canonical pathway operates independently of it.

Components of the Wnt signaling pathway

Non-canonical Wnt signaling pathways

Wnt proteins, which serve as ligands for the Wnt pathway, consist of 19 cysteine-rich glycoproteins. They bind to the Frizzled (Fzd) transmembrane receptor, one of the main receptors of the Wnt pathways [24]. When Wnt binds to Fzd, it can activate one of the three distinct intracellular signaling pathways. While the canonical Wnt/β-catenin signaling pathway leads to the accumulation and stabilization of cytosolic, unphosphorylated (“free”) β-catenin, the non-canonical pathways promote an increase in intracellular calcium or mediate cell polarity. In all three pathways, a Wnt ligand binds to Fzd receptor and promotes recruitment of Dishevelled (Dsh) protein (Figs. 1 and 2). In the planar cell polarity non-canonical pathway, this complex binds to the Dsh-associated activator of morphogenesis (Daam1). This cascade of events leads to the activation of Rac and RhoA GTPases which mediate cell polarity (Fig. 1). In the Wnt-Ca2+ noncanonical pathway, the Wnt/Fzd/Dsh complex binds with a G protein (Ror 1/2) as shown in Fig. 1, which leads to activation of calmodulindependent kinase II, protein kinase C and the phosphatase calcineurin. This binding promotes the increase in intracellular calcium levels which then mediates other signaling pathways. The Wnt pathways are critical to embryonic development of a variety of organs including the ovaries. Activation of Wnt signaling occurs via the canonical Wnt/β-catenin pathway and the non-canonical cell polarity pathway and the Wnt/ Ca2+ pathway; however, as it relates to oncology research the Wnt/β-catenin canonical pathwayis the mostrelevant [25].

Canonical (Wnt/β-catenin) signaling pathway

In the canonicalWnt/β-catenin pathway, the pathway is “off” when either there is no Wnt ligand, no receptor, or the receptor is being blocked (Fig. 2A). Dikkopf family (DKK1–4) binds directly to one of the transmembrane receptors (Fzd, LRP5/6) and blocks Wnt from binding. Wnt-inhibitory factor (WIF-1) and the family of secreted Fzd receptor proteins (SFRP1-5) bind to Wnt itself and prevent them from binding to the receptors. If the Wnt ligand does not bind to the receptors, β-catenin is degraded by a destruction complex that is comprised of Axin, adenomatous polyposis coli (APC), and glycogen synthase kinase 3β (GSK3β). β-Catenin is phosphorylated by the kinases casein kinase 1 (CK1) and GSK3β, followed by ubiquitination and proteasomal degradation by the 26S proteasome. Low cytoplasmic levels of β-catenin allow for the recruitment of the corepressor Groucho to lymphoid enhanced factor–T-cell factor (TCF/LEF) transcription factors,which blocks the target genes from being activated and ensures transcriptional repression (Fig. 2A). Activation of the canonical Wnt pathway involves the stabilization of β-catenin through the binding of Wn tligands to cell surface receptors including Fzd family receptors and low-density lipoprotein receptor (LDLR)-related proteins: LRP5 and LRP6. When the Wnt pathway is “on”, cytosolic β-catenin is stabilized (Fig. 2B). LRP6/LRP5 is phosphorylated by the kinases CK1 and GSK3β. Dsh molecules are recruited to the plasma membrane to interact with Fzd. Interaction of Axin with phosphorylated LRP6/LRP5 and Dsh leads to inactivation of the destruction complex and degradation of β-catenin is inhibited. βCatenin accumulates in the cytoplasm and enters the nucleus and activates Wnt target genes by binding to the transcriptional factors of the TCF/LEF family by displacing Groucho and interacting with coactivators such as B-cell lymphoma 9/Legles (BCL9/LGS) and Pygopus (Pygo) to promote transcription of target genes [26]. TCF/LEF, BCL9/ LGS, and Pygo all bind with β-catenin in the nucleus to form a transcriptional activation complex (Fig. 2B). β-Catenin promotes transcription of genes related to proliferation and survival. Some of the key downstream proteins and genes that are activated with the binding of β-catenin to the transcriptional factors of the canonical pathway include c-MYC (MYC), Cyclin D1 (CCND1), Survivin (BIRC5), Axin2 (AXIN2), and matrix metalloproteinases (MMPs). There have been over 100 target genes identified as regulated by the Wnt pathway and 23 of them have been shown to be overexpressed in ovarian cancer [27].

Regulation of the Wnt pathway

The remainder of the review will focus on the canonical Wnt/ β-catenin pathway, because the Wnt/β-catenin pathway has been the most well described in the literature as it relates to cancer research and specifically ovarian cancer. It is regulated at multiple levels: gene mutations, extracellular inhibitors, and intranuclear transcription cofactors. These all contribute to the diverse mechanisms that are involved in the Wnt pathway.When there is no Wnt ligand, a destruction complex regulates β-catenin levels. Specifically, CK1 and unphosphorylated GSK3β phosphorylate β-catenin and target the protein for ubiquitination and proteasomal degradation. Phosphorylation of GSK3β by protein kinases (A, B, and C), Akt/PI3K, and MAPK inhibits its ability to phosphorylate and target β-catenin for degradation. The majority of ovarian cancers have an activation of PI3K (phosphoinositide 3-kinase) by gene amplification, which can potentially phosphorylate GSK3β, impeding the phosphorylation of β-catenin and resulting in cellular differentiation, division, and survival [28,29].

Alterations of the Wnt pathway in ovarian cancer

Membranous factors

The first event in the activation of the Wnt pathway is the binding of a Wnt ligand to Fzd and LRP6/LRP5. Two subtypes of the Fzd receptor are increased in epithelial ovarian cancer, Fzd1 and Fzd5. A higher number of malignant ovarian specimens stained positive for both receptors than normal ovary and the Fzd5-positive tumors had a worse 6-year survival than those that were Fzd5-negative [30]. During metastatic spread of epithelial ovarian cancer, there is adhesion of cancer cells to submesothelial interstitial collagens. When β1 integrin mediated anchoring to the mesothelium and submesothelial matrix occurs, it facilitates the formation of metastatic tumor sites on other peritoneal organs. The engagement of collagen-binding β1 integrins have been shown to upregulate LRP6, WNT5A, MMP9, PTGS2 (COX2), PLAUR (uPAR), VIM (vimentin), SNAII (Snail) at the mRNA level [31]. This suggests tha tmetastatic spread of ovarian cancer is likely facilitated by the upregulation of LRP6 and targeting LRP6 may be an effective strategy for treating ovarian cancer.

There are several proteins that act as antagonists to the Wnt pathway. These proteins include: the Dikkopf family (DKK1–4), Wnt inhibitory factor (WIF-1) and the family of secreted Fzd receptor proteins (SFRP1-5)(Fig.2A). SFRPs bind directly to the Wnt ligand or Fzd receptor and inhibit Wnt from binding to Fzd and activating the pathway. Loss of SFRP4 expression correlates with a more aggressive ovarian cancer phenotype and the level of SFRP4 is directly related to prognosis [32]. Investigators have studied the re-expression of SFRP4 in epithelial ovarian cancer cell lines, and found that re-expression inhibited the Wnt/ β-catenin signaling pathway, thereby inhibiting cell migration and EMT. These proteins provide important potential therapeutic targets by either re-expression, if their expression is lost,or potentially upregulated.

Cytoplasmic and nuclear factors

Endometrioid ovarian carcinomas often have mutations in the βcatenin gene. Table 1 summarizes the studies that show β-catenin mutations in human ovarian cancer, from 16% to 54% in endometrioid cancers and 14% in mucinous cancers. Despite no reported mutations in the CTNNB1 gene in serous and clear cell cancers, nuclear β-catenin has been observed in serous and clear cell ovarian cancer [21]. Lee et al. showed a statistically significant correlation between nuclear β-catenin expression and high-grade serous ovarian cancer [23]. The protooncogene, frequently rearranged in advanced T-cell lymphomas-1 (FRAT1), which inhibits phosphorylation of β-catenin, was found to be overexpressed in serous ovarian cancer and was strongly correlated with the accumulation of cytoplasmic β-catenin, leading to an increase in nuclear β-catenin [21]. Pygo, oneof the co-activators that binds to β-catenin is a necessary component of tumor cell growth and is widely expressed in ovarian cancer, both in cell lines and in primary tumor tissue [33]. RNA expression of BCL9/LGS, also a co-activator,is common in both epithelial ovarian cancer and normal ovaries. Upregulation of these co-activators is further evidence that the Wnt pathway plays a pivotal role in the tumorigenesis of ovarian cancer.

Intercellular interactions

Cells undergoing EMT are known to lose E-cadherin and gain vimentin expression, resulting in tumor cell invasion and metastasis [34]. Epithelial ovarian cancer cells also undergo a mesenchymal to epithelial transition (MET) because the normal ovarian surface epithelium is mesenchymally derived. This dynamic process has been termed EMP (epithelial to mesenchymal plasticity). It is thought that both transitions are equally important for metastasis formation and that the “metastable” state is actually when the cells transition between the two states [34]. Metastatic epithelial ovarian cancer cells adhere to the interstitial collagen of the peritoneal cavity via integrins. Cell–matrix and cell– cell adhesions are paramount to this process and are mediated by integrins and E-cadherins. Integrin engagement has been linked to increased internalization of E-cadherin [31]. In epithelial cancer, the MET component dominates, unlike other epithelial cell-derived cancers where the EMT component dominates; therefore, E-cadherin expression is increased with malignant transformation in ovarian cancer [31]. E-cadherin-based adherens junctions are stabilized by β-catenin, and the loss of stability in the junctions may cause an increase in cytoplasmic and/or nuclear β-catenin. Integrins have also been suggested to inhibit GSK3β, elevate levels of nuclear β-catenin, and increase β-catenin-regulated promoter activation. Burkhalter et al.
showed that an inhibitor of β-catenin and TCF-4, a member of the TCF/LEF transcription factor family, reduced cellular invasion [31]. Most of the regulation of the Wnt pathway ultimately leads to an accumulation or depletion of β-catenin in the nucleus, or affects the binding of nuclear β-catenin to TCF/LEF, which determines whether apoptosis can occur. It is important to note that the transcriptional regulatory activity of β-catenin is also controlled by factors other than Wnt signaling. One example of Wnt-independent regulation of β-catenin is through E-cadherin expression, which selectively depletes the transcriptionally active pool of β-catenin [35]. This is especially significant as epithelial ovarian cancer cells are known to undergo MET which causes an increase in E-cadherin.

Extracellular factors

Not only have membranous and intercellular components of theWnt pathway been found to be upregulated in epithelial ovarian cancer, but extracellular activators also are upregulated. These factors specifically include Wnt-1,Wnt-2b,Wnt-5a, and Wnt-11 [30]. Ricken et al. reported the possibility that Wnt-5a could be involved in ovarian carcinogenesis [18]. This study used RT-PCR on RNA from five ovarian cancer cell lines and confirmed the expression of transcripts for Wnt-2b, Wnt-5a and Wnt-11. Filho et al. showed that upregulation of Wnt-1 and Wnt-5a, detected by immunohistochemistry in patient samples, portended a significantly lower survival than ovarian cancer patient samples that did not have an upregulation of Wnt-1 and Wnt-5a [30].

Gene expression

Kumar et al. analyzed 1500 miRNAs to identify which ones were potentially different between A2780 (parental ovarian cancer cell line) and A2780.cp70 (cisplatin resistant cell line) and found changes in 11 miRNAs [36]. The microRNA data was validated by quantitative realtime PCR for these 11 miRNAs. Ingenuity Pathway Analysis (IPA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed for the 11 miRNAs and their targets to identify the pathways involved in cisplatin resistance. Not only was Wnt signaling one of the pathways identified, but so were MAPK and mTOR signaling pathways which both cross-talk with the Wnt pathway by causing the phosphorylation of GSK3β, blocking its ability to phosphorylate βcatenin to allow it to be ubiquitinated. Four gene expression datasets: Moffitt Cancer Center (MCC), Total Cancer Care (TCC), the Cancer Genome Atlas(TCGA),andMDAnderson (MDA) were analyzed, and only four pathways were noted to be differentially expressed between normal ovarian surface epithelium and ovarian cancer. One of these pathways is the “Cytoskeleton remodeling/TGF–Wnt pathway” [37]. The“Cytoskeleton remodeling/TGF/WNT” pathway was previously described as a common pathway created by the crosstalk between the TGF-β pathway and the Wnt pathway that is involved in cytoskeleton remodeling: cell–cell adhesion and cell–matrix adhesion [38]. This pathway has been associated with metastasis in various cancer types and is critical for cancer cell migration and invasion. The same group at H. Lee Mof fitt Cancer Center found that six common molecular signaling pathways were associated with chemoresistance and survival in ovarian cancer that included the TGF– Wnt pathway and specifically Wnt pathway activated by Wnt-2, one of the 19 Wnt ligands [39]. In addition, this group also used the same novel computer analysis technique to identify genes and molecular signaling pathways associated with cancer cell proliferation. Genes and pathways associated with cancer cell proliferation and survival were analyzed against the NCI 60 cell line-drug screening database to identify agents predicted to have pathway- and gene-specific activity. They identified 81 existing agents that could potentially be repurposed to target the TGF-Wnt pathway that are currently the focus of in vitro functional analyses [40].

Non-canonical pathways

Fig. 1. Non-canonical Wnt signaling pathways. In the planar cell polarity pathway Wnt–Frizzled complex binds to the Dsh-associated activator of morphogenesis (Daam1). This cascade of events leads to the activation of Rac and RhoA GTPases which mediate cell polarity. In the Wnt–Ca2+ pathway, the Wnt/Fzd/Dsh complex binds with a G protein, which leads to activation of calmodulin-dependent kinase II (CaMKII), protein kinase C (PKC), and the phosphatase calcineurin. This binding promotes the increase in intracellular calcium levels which stimulates other signaling pathways.

Fig.2.The canonical Wnt signaling pathway. (A)In the absence of Wnt ligand, β-catenin is degraded through interactions with Axin, APC and GSK3β “destruction complex”. β-Cateninis phosphorylated by the kinases CK1 (casein kinase 1) and GSK3β (glycogen synthase kinase 3β), followed by ubiquitylation and proteasomal degradation. Low cytoplasmic levels of βcatenin allow for the recruitment of the corepressor Groucho to LEF (lymphoid enhanced factor)–TCF (T-cell factor) transcription factors which ensures transcriptional repression. Dikkopf (DKK) family proteins, the Wnt-inhibitory factor (WIF), and the family of secreted Frizzled receptor proteins (SFRPs) all act as antagonists to the Wnt pathway. SFRP binds directly to the Wnt ligand or th eFrizzled receptor to inhibit Wnt binding to Frizzled. (B) In the presence of Wnt ligands, Wnt proteins bind to Frizzled/LRP6/LRP5 receptor complex at the cell surface. LRP6/LRP5 is phosphorylated by the kinases casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β). Dishevelled (Dsh) molecules are recruited to the plasma membrane to interact with Frizzled. Interaction of Axin with phosphorylated LRP6/LRP5 and Dsh leads to inactivation of the destruction complex. Degradation of β-catenin is inhibited. β-Catenin accumulates inthe cytoplasm and nucleus. β-Catenin forms a transcriptionally active complex with TCF/LEF by displacing Groucho and interacting with co-activators suchasBCL9/LGS (B-cell lymphoma 9/Legless) and Pygo (Pygopus) to promote transcription of target genes (Axin, CyclinD1, Survivin). β-Catenin is also a coactivator of CREB binding protein (CBP) which is the binding protein of the cAMP response element-binding protein (CREB). β-Catenin/CBP binds to Wnt-responsive element (WRE) and activates transcription. This leads to cell proliferation, survival, and self-renewal.

Potential therapeutic targets of the Wnt pathway in ovarian cancer

Identification of the specific membranous, intracellular, and extracellular components of the Wnt pathway gives insight to potential targets for therapy. There currently are several small molecules that have recently entered into phase I clinical trials that target the Wnt pathway (Table 2). In order for the Wnt protein to be secreted by the cell to act as a ligand it must first undergo fatty acyl modification. Once it undergoes palmiteolyation it is shepherded through the secretory pathway by Wntless chaperone protein. PORCN is the founding member of a 16-gene family with acyltransferase activity and Porcupine (Porcn) is the acyltransferase enzyme that adds the fatty acid to Wnt which is a crucial step in the secretion of the Wnt ligand. Without Porcn to catalyze this modification, the Wnt protein remains trapped inside the cell. Currently being studied in a phase 1 trial is the small molecule, LGK974 (Novartis Pharmaceuticals) that inhibits Porcn(NCT01352203) [41]. Drugs that specifically target the Wnt signaling pathway in the nucleus include the small molecule inhibitor, PRI-724, which specifically blocks the recruitment of β-catenin with its coactivator CBP which is the binding protein of the cAMP response element-binding protein CREB. βCatenin/CBP binds to Wnt-responsive element (WRE) and activates transcription; therefore, PRI-724 prevents activated transcription by aberrant Wnt signaling. This drug is being studied in solid tumors and myeloid malignancies (NCT01606579) [41]. Other pathways may cross-talk with the Wnt pathway. In Wnt signaling, Axin is a key scaffolding protein of the destruction complex of β-catenin, and Poly (ADP ribose) polymerases (PARPs) promote the ribosylation of Axin, thereby causing it to become degraded and no longer facilitate β-catenin destruction. If PARP is inhibited, Axin is stabilized, which allows it to degrade β-catenin [42]. There are several PARP inhibitors that are currently being used in clinical trials for ovarian cancer. In addition, preclinical studies have been carried out with XAV939, which is a small-molecule PARP inhibitor that targets tankyrases, a specific type of PARP. Huang et al. used a chemical genetic screen to identify the small molecule, XAV939, which selectively inhibits β-catenin mediated transcription. XAV939 was shown to stimulate β-catenin degradation by stabilizing Axin. They used a quantitative chemical proteomic approach to show that XAV939 stabilizes Axin by inhibiting tankyrase1 and tankyrase2.They showed that both tankyrase isoforms 1 and 2 stimulate Axin degradation through the ubiquitin–proteasome pathway [43]. JW55 (Tocris Bioscience) is a selective tankyrase 1 and 2 inhibitor which has been shown to inhibit the growth of cancer. JW55 inhibits the canonical Wnt signaling pathway in colon carcinoma cells that contained mutations either in the APC locus or in anallele of β-catenin [44]. Frizzled, oneof themembrane receptors that activates thepathway upon Wnt ligand binding, has been reported to be overexpressed in ovarian cancer. There are two drugs that specifically target the Fzd receptorthatarebeingevaluatedinclinicaltrials.OMP-18R5(OncoMed Pharmaceuticals/Bayer) is one of the Wnt-targeted compounds that is in clinical development (NCT01345201) [41]. It is a monoclonal antibody that targets Fzd receptors and blocks their association with Wnt ligands. This drug is being used in combination with the standard chemotherapy for breast, lung, pancreas, and colon cancer. Another drug, OMP-54F28, binds to and sequesters the Wnt ligand and is a fusion protein of the Fzd8 ligand-binding domain with the Fc region of a human immunoglobulin (OncoMed Pharmaceuticals/Bayer) (NCT01352203) [41]. There has been a growing trend in oncology to evaluate“repurposed” drugs which are drugs that have been used in the past for other purposes and are now being screened for their function as anticancer drugs. Several drugs have been shown to work through the Wnt pathway including the FDA-approved anti-helminth compound, niclosamide, non-steroidalanti-inflammatory drugs(NSAIDS), and two antipsychotic drugs: lithium and valproic acid. NSAIDS have been shown to cause degradation of TCF and inhibit Wnt target genes such as COX2. Although they do not target the Wnt pathway directly, they could be a potential anti-Wnt agent. Niclosamide inhibitsWnt/β-catenin pathway activation. In colorectal cancer, it was shown to downregulate Dvl2, a member of the Dsh protein family, which in turn decreased downstreamβ-catenin signaling [45]. Recently, niclosamide has been reported to target not only Wnt/β-catenin but also other signaling pathways involved in CSC maintenance such as NF-κB, Notch, ROS, mTORC1, and Stat3 [46,47]. Niclosamide has also been reported to inhibit Wnt/β-catenin signaling by inducing degradation of the Wnt surface receptor, LRP6 [48]. Our laboratory has seen an increase expression of LRP6 in ovarian cancer patients. Yo et al. identified a subset of chemoresistant ovarian tumor cells that fulfilled the definition of CSCs and subjected these cells to high-throughput drug screening using more than 1200 clinically approved drugs. Sixty-one potential compounds were identified on preliminary screening and after more stringent screening, niclosamide was found to be the best drug to selectively target ovarian CSCs both in vitro and in vivo [49].

Wnt/β-catenin pathway and CSC

TheWnt/β-catenin pathway is an important pathway in cell survival and has been implicated in the mechanism of chemoresistance of ovarian CSCs. CSCs are a subpopulation of tumor cells that possess characteristics associated with normal stem cells and have the ability to self-renew and differentiate. Wnt/β-catenin signaling plays an important role in the transcription of multidrug resistance genes such as ABCB1/MDR-1 [50]. Chemoresistance, which can be a result of the inhibition of apoptosis, has been reported to be associated with acquiring EMT in ovarian cancer cells [51,52]. Ovarian cancer cells undergoing EMT have stem-like properties that enable cancer cell dissemination and metastasis formation. A recent study done at Georgia Institute of Technology confirmed that metastasizing ovarian cancer cells taken from patients have a different molecular structure from primary tumor cells and display genetic signatures consistent with EMT [53]. The Wnt/ β-catenin pathway is one of the major signaling pathways thought to be involved in EMT and thus has been shown to play an integral role in metastasis.

Alterations affectingWnt pathway proteins on the cel lmembrane, in the cytoplasm, and in the nucleus have been shown to play important roles in the tumorigenesis of ovarian cancer. Pre-clinical studies have shown an upregulation of 5 of the 19 known Wnt ligands in ovarian cancer, which leads to increased activity of the Wnt pathway. Fzd is one of the membrane receptors that activates the pathway upon Wnt ligand binding. It has been reported to be overexpressed in ovarian cancer. Our laboratory has also seen an upregulation of LRP6 detected by immunohistochemistry (unpublished data). In ovarian cancer, an increase in nuclear β-catenin has been shown to be the result of an upregulation in the β-catenin gene itself and also mutations in the proteins necessary to degrade cytoplasmic β-catenin such as Axin2 and APC. The β-catenin destruction complex consists of Axin2, APC, and GSK3β, which must not be phosphorylated in order to cause βcatenin degradation. GSK3β is frequently phosphorylated in ovarian cancer through other pathways, such as PI3K, inhibiting its ability to degrade β-catenin. Upregulation of co-activators of β-catenin also contributes to the increase in transcription of the target genes. As many as 23 different target genes that lead to cell proliferation and survival, which is a result of nuclear β-catenin build-up, have been shown to be overexpressed in ovariancancer. Wntsignalingis activated in epithelial ovarian cancer, both directly through ligand activated upregulation of the pathway and through a ligand independent increase in nuclear β-catenin through cross-talk with other pathways. Recently, Yo et al. reported that niclosamide, which has been shown to have anti-Wnt activity inhibits growth in ovariantumor-initiatingcells[49].Morepre-clinicalstudies,specifically animal studies and mechanistic studies, are warranted to further investigate other Wnt inhibitors in ovarian cancer. The Wnt pathway is very complex, and further studies with targeted agents need to be done to see if inhibition of a single component of the pathway will be clinically useful. This paper supports the fact that the Wnt pathway shows promise as an effective target for anti-cancer therapy in ovarian cancer. As more efficacy data is collected from the phase 1 studies with Wnt inhibitors LGK974, OMP-54F28, OMP-18R5, and PRI724: NCT01352203, NCT01608867, NCT01345201, and NCT01606579 (www.clinicaltrials.gov), they should be considered as potential agents in the treatment of ovarian cancer. Given the fact that the Wnt pathway is involved in so many biological pathways, results from these studies will be important to determine if effective Wnt pathway inhibition will be excessively toxic to patients. Future directions for investigating the Wnt pathway in ovarian cancer should include genetic sequencing of ovarian cancer patients with the aim of targeting those patients who specifically have upregulation of Wnt pathway target genes. More quantitative data is needed to specifically look at the mechanisms of these drugs in patients by performing qPCR on tissue obtained before and after treatment. The Wnt pathway should be investigated as a potential target in the development of new drugs for ovarian cancer as a single agent and in combination with chemotherapy or other targeted agents.

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7.10.3 Wnt Signaling in the Niche Enforces Hematopoietic Stem Cell Quiescence and Is Necessary to Preserve Self-Renewal In Vivo

Fleming HE1Janzen VLo Celso CGuo JLeahy KMKronenberg HMScadden DT.
Cell Stem Cell. 2008 Mar 6; 2(3):274-83

Wingless (Wnt) is a potent morphogen demonstrated in multiple cell lineages to promote the expansion and maintenance of stem and progenitor cell populations. Pharmacologic modification of Wnt signaling has been shown to increase hematopoietic stem cells (HSC). We explored the impact of Wnt signaling in vivo, specifically within the context of the HSC niche. Using an osteoblast-specific promoter to drive the expression of a pan-inhibitor of canonical Wnt signaling, Dickkopf1 (Dkk1), we noted changes in trabecular bone and in HSC. Wnt signaling was inhibited in HSC and the cells exhibited reduced p21Cip1 expression, increased cell cycling and a progressive decline in regenerative function after transplantation. This effect was microenvironment-determined, but irreversible if the cells were transferred to a normal host. Wnt pathway activation in the niche is required to preserve the reconstituting function of endogenous hematopoietic stem cells.

The regulation of hematopoietic stem cell function is a complex and balanced process that requires coordinated input from inherent HSC programs and moderating signals provided by the surrounding microenvironment. Together, these signals permit the maintenance of the stem cell pool for the life of the organism, while also allowing for sufficient steady-state and injury-responsive blood cell production. These somewhat dichotomous aspects of HSC function require mechanisms that both preserve a quiescent population of stem cells and also promote their activation, expansion, differentiation and circulation under appropriate conditions (Akala and Clarke, 2006Scadden, 2006). The morphogen family of signaling molecules has been identified as a prominent player in the function of numerous stem cell types, including the hematopoietic lineage. The wingless (Wnt) pathway has been studied extensively in the context of hematopoiesis, and the combined impact of multiple family members binding to a range of receptors leads to activation of canonical and non-canonical signaling pathways (Nemeth and Bodine, 2007). Canonical signals are mediated by TCF/LEF transcription factor activity (Daniels and Weis, 2005), and are considered to be largely dependent on the accumulation of nuclear β- (and/or γ-) catenin (Nemeth and Bodine, 2007).Wnt signals have been implicated in mammalian hematopoiesis by studies not intended to assess normal physiology in which Wnt activation had a strong expansive effect on reconstituting HSCs and multipotent progenitors (Baba et al, 2006Murdoch et al, 2003Reya et al, 2003Trowbridge et al, 2006). With enforced, persistent Wnt activation, however, engineered mice developed hematopoietic failure with impaired differentiation of HSC (Kirstetter et al, 2006Scheller et al, 2006). In contrast, deletion of members of the Wnt / β-catenin cascade under homeostatic conditions had little to no effect on blood cell production by HSCs (Cobas et al, 2004Jeannet et al, 2007Koch et al, 2007), raising the question of what physiological role, if any, Wnt signaling has on this cell type. Some of the variation observed may reflect differing influences exerted by canonical versus non-canonical Wnt signals, particularly given a recent report indicating that Wnt5a can modulate canonical signals mediated by Wnt3a (Nemeth et al, 2007). Wnt signals are also regulated by a host of soluble inhibitors that may interact directly with Wnt ligands, such as the frizzled-related proteins (sFRP) or by preventing Wnt binding to its receptors (Kawano and Kypta, 2003). The Dickkopf (Dkk) family of Wnt inhibitors falls into this latter category, by binding the Wnt coreceptor LRP5/6 in combination with a Kremen receptor, and leading to internalization of the complex (Mao et al, 2001Mao et al, 2002). In order to specifically examine the impact of Wnt activation in an in vivomicroenvironment that has been shown to regulate HSC number and function, we utilized mice engineered to overexpress the Wnt inhibitor, Dkk1, under control of the osteoblast specific 2.3kb fraction of the collagen1α promoter. This promoter has been previously shown to direct transgene expression to osteoblastic cells, resulting in changes in the number and function of HSCs (Calvi et al, 2001Calvi et al, 2003)

We noted very little overt phenotype in the hematopoietic compartment of the Dkk1 tg mice at steady-state, and confirmed that transgene expression did not extend to the primitive hematopoietic fraction itself. Clear alterations of bone morphology were observed, however, including a 20% decrease in trabecular bone (manuscript in preparation). Despite the absence of a steady-state hematopoetic phenotype, TCF/LEF activity was specifically reduced within the HSC-containing fraction of Dkk1 transgenic mice, and stem cell function was altered under specific conditions. For example, a highly significant defect in the maintenance of reconstitution potential of HSC was observed, either in settings of serial transplant, or following secondary transplantation of wildtype donor cells previously used to reconstitute Dkk1 tg hosts. In agreement with the functional data, HSC populations had a marked reduction of cells within the G0 fraction of the cell cycle, and displayed enhanced sensitivity to 5-fluorouracil treatment. Wnt signals therefore appear to participate in mediating HSC quiescence in vivo, a result that was largely unpredicted from previous studies, although recent analysis of Hmgb3 mutant mice also supports this conclusion (Nemeth et al., 2006). Our results highlight the importance of studying the impact of a signaling pathway over long-term experiments, and in a physiologic context when seeking to resolve the effects of manipulations on HSC function. In that context, Wnt signaling plays an unanticipated role in maintaining HSC quiescence, which may underlie its requirement in preserving the self-renewing capability of HSC.

Osteoblast expression of Dkk1 does not affect blood or marrow primitive hematopoietic cell populations at steady state

The Wnt inhibitor, Dkk1, has been shown to play an important role in bone formation during development (Niehrs, 2006), and is normally expressed by osteoblasts (Grotewold et al., 1999MacDonald et al., 2004), hence may have regulatory roles as part of the endosteal HSC niche. To examine the impact of Wnt inhibition on hematopoietic stem cells localized to the periendosteal region, Dkk1 was overexpressed within osteoblastic lineage cells under the control of the truncated 2.3kb collagen 1α promoter (manuscript in preparation). Resulting Col1α2.3-Dkk1 transgenic (Dkk1 tg) mice were backcrossed for at least 5 generations to the C57Bl/6 background and examined for bone and blood phenotypic alterations. No significant differences in peripheral white or red blood cell counts were observed (figure S1a). Bone marrow (BM) and spleen cellularity were also unchanged when Dkk1 tg mice and their littermates were compared, although a slight but not significant trend towards reduced body weight and BM cellularity was apparent in transgenic mice (figure S1b and data not shown). In contrast, significant alterations in bone morphology were observed, as is reported elsewhere (manuscript in preparation, and (Li et al, 2006)) Of note, trabecular bone volume was reduced by approximately 20%, whereas cortical bone was unaffected in Dkk1 tg mice (data not shown). Trabecular bone has been shown by us and others to affect HSC number and function (Adams et al, 2007Calvi et al, 2003Jung et al, 2007Zhang et al, 2003). A panel of antibodies using 7 different flurochromes was used for multiparametric analysis of primitive precursors within the BM of Dkk1 tg mice and their littermates, including populations of LT-HSC, ST-HSC, CMP, GMP, MEP and CLP (figure 1a,c). Subpopulations containing primitive HSCs were not significantly altered at steady-state (figure 1b). However, additional cell surface markers revealed a slight but significant increase in the population containing phenotypically-defined common lymphoid progenitors (figure 1d). The calculated absolute cell numbers based on these frequencies indicated a similar pattern of results (figure S2). Despite the elevation of early lymphoid progenitors in the BM of Dkk1 tg mice, no significant changes were observed in the relative proportion of early B lineage progenitor subsets in the BM (data not shown).

Seven color FACS analysis of primitive populations in wt and Dkk1-tg BM nihms-240191-f0001

Seven color FACS analysis of primitive populations in wt and Dkk1-tg BM nihms-240191-f0001

Seven color FACS analysis of primitive populations in wt and Dkk1-tg BM


Figure 1 Seven color FACS analysis of primitive populations in wt and Dkk1-tg BM

BM from Dkk1 tg and littermates was assayed by multiparameter FACS for relative proportion of primitive HSC populations. BM was stained with antibodies against Lineage markers, cKit, Sca-1, CD34, Flk2, CD16/32 and CD127 and gated as shown in panels (A) and (C). At least 10 mice per genotype were compared, over at least three separate experiments. The proportion of BM corresponding to the HSC-containing LK+S+ fraction (A, blue gate) is shown in (B, left axis), and is sub-sectioned according to CD34 and Flk2 expression to yield phenotypic assessments of LT-HSC and ST-HSC fractions (B, right axis). More differentiated progenitors gated in the LK+S− population (A, left, green gate) were sub-sectioned based on CD16/32 and CD34 expression to compare CMP, GMP and MEP progenitors as shown in (C, left panel). Frequencies of each population, from the same samples quantified for HSC frequency in (B) are shown in (D, left axis). The CLP fraction, gated on LKloSlo in (A, red gate), and gated further on CD127+ cells in (C, right panel) are quantified in (D, right axis). Significance was determined by a Student’s 2-tailed T-test. Error bars indicate the SE of the mean.

Dkk1-tg HSCs exhibit impaired Wnt signaling in a non-cell autonomous manner

To confirm that the transgenic expression of Dkk1 leads to the inhibition of Wnt/βcatenin signaling in the Dkk1 tg mice, HSC-containing populations were isolated from Dkk1 transgenic mice that had been intercrossed with the Topgal reporter strain. In these Topgal mice (DasGupta and Fuchs, 1999), multiple TCF/LEF binding sites have been inserted to control the expression of the reporter gene, β-galactosidase. Reporter activity using this construct has been shown to correlate with canonical Wnt signaling. Of note, TCF/LEF transcription has recently been shown to proceed even with the combined loss of β-catenin and γ-catenin, suggesting that canonical Wnt signals can be transduced by alternate intermediates (Jeannet et al, 2007). Reporter activity was examined within the LK+S+ (Lineage-cKit+Sca1+), HSC-containing population, and the LK+S− population which is devoid of LT-HSC potential. When the Wnt reporter activity detected in each of these populations was compared, a dramatic reduction (>100 fold reduction) in β-catenin activation was observed in the HSC-containing LK+S+ population isolated from Dkk1 tg mice (figure 2a). A more modest reduction (<5 fold reduction) was observed in the less-actively signaling LK+S− fraction. This finding indicates that despite the unchanged frequency of phenotypically-defined HSC-containing populations in unmanipulated Dkk1 tg animals, there is evidence that these cells are molecularly altered by osteoblast expression of the Wnt inhibitor. These data provide evidence for direct inhibition of Wnt signaling in the HSC population in addition to any effects that might be mediated by decreased trabecular bone mass. Wnt signaling is regulated, in part, via a negative feedback loop by TCF/LEF-dependent transcription of endogenous Dkk1 (Niida et al, 2004). Consistent with the decrease in Topgal reporter activity, expression of endogenous Dkk1 was also inhibited in the LK+S+ population of Dkk1 tg mice (figure 2b). Using primers specific for the Dkk1 tg, and in comparison its expression in wt and Dkk1 tg tibea, sorted LK+S+ cells do not express the Dkk1 transgene (figure 2c). Together, these results confirm that Dkk1 tg mice inhibit Wnt signaling specifically within the HSC compartment in a non-cell autonomous manner.

Assessment of canonical Wnt signal activity in HSC-containing populations of Dkk1-tg mice nihms-240191-f0002

Assessment of canonical Wnt signal activity in HSC-containing populations of Dkk1-tg mice nihms-240191-f0002

Assessment of canonical Wnt signal activity in HSC-containing populations of Dkk1-tg mice


Figure 2 Assessment of canonical Wnt signal activity in HSC-containing populations of Dkk1-tg mice

Functional impact of the Dkk1 transgene on BM reconstitution

Analysis of stem/progenitor activity cannot rely exclusively on the quantitation of precursors according to phenotypically-defined parameters. Using functional measures, we detected a consistent defect in multilineage and myeloid colony formation on a per cell basis in BM isolated from Dkk1 transgenic mice (figure 3a). This result was despite the absence of significant alteration of myeloid and more primitive progenitors by immunophenotype, possibly reflecting the elevated lymphoid fraction, whose progeny are not read out under these culture conditions. In vitro methods such as the CFU assay offer an entry-level analysis of hematopoietic activity, however functional reconstitution in vivo more accurately examines true HSC function (Purton and Scadden, 2007). Therefore, in order to better assess the functional capacity of HSCs isolated from the Dkk1 transgenic environment, BM was transplanted from wt or Dkk1 tg littermates with an equivalent dose of competing marrow from congenic donor mice into lethally irradiated recipients. Donor marrow was isolated from a single wt or transgenic mouse to assess any individual-to-individual variation. Following six months of engraftment, no significant changes in reconstitution were observed across the groups of recipients receiving BM isolated from individual wt or Dkk1 tg environments, although a range of reconstitution capacity was apparent in both groups (figure S3a). Using a limiting dilution assay to determine the frequency of repopulating cells present in BM isolated from individual Dkk1-expressing animals revealed a two-fold elevation in the number of functional reconstituting HSCs (Figure 3b). These transplant results indicate that cells isolated from the Dkk1-epressing niche are capable of reconstituting irradiated recipients, and appear to be present at a higher frequency when Wnt has been inhibited in this location. An important additional parameter to test when investigating HSC function is their longevity, or ability to respond to repeated rounds of expansion stress. To assay the longevity of HSCs isolated from Dkk1 tg mice, noncompetitive serial transplants were performed. As expected from the previous transplant experiments, Dkk1 tg BM was able to completely reconstitute wt irradiated recipients (data not shown).

Functional assessment of HSCs isolated from Dkk1-tg mice


Figure 3 Functional assessment of HSCs isolated from Dkk1-tg mice

(A) BM from 8 pairs of wt and Dkk1-tg mice was plated in methylcellulose with growth factors (SCF, IL-3, IL-6, Epo) and scored for CFU-C (combined scoring for BFU-E, CFU-GM and CFU-GEMM colonies) after 12 days. All live colonies of more than 30 cells were counted for each of three wells plated per sample. Data are shown as mean colonies per well for each of 8 mice studied over three individual experiments. Significance was determined using a two-tailed Student’s T test. (B) Limiting dilution experiments were performed using three doses of test marrow (CD45.1) transplanted with 5×105 competing cells (CD45.2) into groups of at least 9 recipients (CD45.2) per dose. Test marrow was isolated from two wt and two Dkk1-tg mice, and the Dkk1-tg donors shown here were transplanted into separate groups of irradiated recipients. Data points are plotted as the percent of recipients per group that did not exhibit at least 1% multi-lineage PB engraftment at 6 months (percent unreconstituted). LT-HSC frequency and significance were determined using Poisson statistics: wt, 1 in 63,00 (circles) vs tg, 1 in 31,500 or 1: 37,000 (squares); p<0.02. Similar results were obtained in an independent assessment of two Dkk1-tg donors. (C) Non-competitive serial transplants were initiated by transplanting 1×106 whole BM pooled from three wt or Dkk1-tg donors (CD45.1) into each of 10 irradiated recipients (CD45.2). Secondary and tertiary transplants were performed after 14 weeks of engraftment by pooling BM from 3-4 reconstituted recipients to transplant 1×106 whole BM into new groups of 10 irradiated CD45.2 recipients. The Kaplan-Meier survival graph depicts the survival of tertiary recipients, mice receiving BM from Dkk1-tg mice (solid line) or wt controls (dashed line). Similar results were obtained in an independent assessment of 2 wt and 2 Dkk1-tg mice. (D) Prior to transplant into tertiary recipients, BM from 5 secondary recipients of both genotypes was assayed by FACS for the frequency of LT-HSCs (LK+S+CD34loFlk2−). Error bars indicated SD of the mean, and significance was determined by a two-tailed T test

Effect of temporary exposure to endosteal Dkk1 on HSC function


Figure 4 Transplant analysis of HSC function following residence in a Dkk1-tg environment

Wnt-inhibited HSC-containing populations are less quiescent


Figure 5 Examination of cell cycle status of primitive BM in wt and Dkk1-tg mice


Figure 6 Gene expression by quantitative PCR of sorted primitive populations

Understanding the role of specific signals in the varied regulatory functions of HSC activities is crucial for designing and developing therapeutic interventions involving these cells. The impact of the Wnt family on the expansion and regulation of hematopoietic cells has been examined in a variety of studies. However, the physiologic effects of this pathway remain somewhat ill-defined with often contradicting results. Some have demonstrated that Wnt cascade activation promotes the proliferation of HSCs and their progeny while maintaining at least short-term functional activity (Baba et al, 2006Murdoch et al, 2003Reya et al, 2003;Trowbridge et al, 2006). Others, employing persistent genetic activation of the pathway, have also demonstrated an increase in proliferation of cells with an HSC immunophenotype, but with marked impairment of HSC differentiation resulting in animal death (Kirstetter et al, 2006Scheller et al, 2006). However, induced deletion of β-catenin, the primary downstream mediator of the Wnt cascade resulted in no apparent impact on HSC activity, even in a reconstitution assay that required expansion of β-catenin null transplanted HSCs (Cobas et al, 2004). Furthermore, recent combined deletions of both β-catenin and its homologue, γ-catenin, also maintain HSC function under steady-state and primary reconstitution conditions (Jeannet et al, 2007Koch et al, 2007).All of these studies have either assayed Wnt activity in broad over- or under-stimulation settings, and the manipulations have been performed on the HSCs themselves, or broadly applied to recipient animals. The context in which morphogens are present is highly relevant to their effect and not previously studied for Wnt effects on hematopoiesis (Trowbridge et al., 2006). Indeed, Wnt ligands can modulate signaling initiated by other Wnt family members, underscoring the concept that context, and different signaling intermediates may have a strong impact on functional outcome (Nemeth et al, 2007).

In the present study, we have established a system that permits the analysis of localized Wnt inhibition, offering the opportunity to assay the impact of chronic or temporary exposure to this inhibited environment. In particular, we have directed expression of the Wnt inhibitor, Dkk1, to a cell population that has been previously demonstrated to exert a regulatory function over HSC activity, and which normally express Dkk-1, albeit at lower levels (Grotewold et al,1999MacDonald et al, 2004). It should be noted that while an increasing number of reports suggest that phenotypically-identified HSCs inhabit additional physical locations within the bone marrow environment (Hooper et al, 2007Scadden, 2006), the promoter used in our study has proven to functionally impact the number and activity of HSCs when used to direct modifying signal expression to a population of osteoblastic cells. Given that expression of Dkk1 also results in alterations to bone morphology itself, there is likely to be a dual effect of Dkk1: one altering the niche architecture and the other affecting Wnt signaling in stem/progenitor cells. Our studies demonstrated an effect of Dkk1 overexpression by non-HSCs on Wnt signaling in hematopoietic stem/progenitors, suggesting that this is at least a contributing factor to the phenotype observed. This observation that TCF/LEF reporter activity is reduced, as is expression of endogenous Dkk1, itself a Wnt signaling target (Niida et al, 2004) in BM cells of the transgenic mice indicates altered canonical Wnt signaling. It does not rule out that Dkk1 may exert additional Wnt-independent functions. The results presented here also indicate that the reduced longevity of HSCs does not require constant exposure to exogenous Dkk1, given that we were unable to detect Dkk1 tg expression within populations of primitive hematopoietic cells, and therefore the functional impact on transplanted cells is observed in a Dkk tg-free environment. It is important to note that transplantation of whole BM populations is generally not effective at engrafting non-hematopoietic cells (Koc et al, 1999).

Wnt mediates HSC quiescence and maintains reconstitution function in vivo

The results presented here establish a role for Wnt, in the maintenance of a quiescent fraction of functional HSCs in BM. This was associated with evidence of increased stem cells on limit dilution transplant analysis. However the ability of the same cells to function after serial rounds of transplantation was drastically reduced. The ability of stem cells to persist under the stress conditions of transplantation requires self-renewal capability that is compromised after Dkk1 exposure

The studies of inducible deletion of β- and γ-catenin noted that they were dispensable for HSC function, however did not include sequential transplants out to the extent where we observed our most dramatic phenotype (Cobas et al, 2004Jeannet et al, 2007Koch et al, 2007) Alternatively, it is possible that Dkk1 interferes with HSC function through a process that does not depend on β- or γ-catenin signaling (Jeannet et al, 2007Niehrs, 2006).

Our results emphasize the importance of studying pathways within the context of other signals present in the natural microenvironment, and underscore the potential for unanticipated functional roles. It is clear that different combinations of signals may have a range of effects depending on the context in which they are received. Indeed, we observed an impact of Wnt-inhibition on the activation of the Notch target, Hes-1, raising the possibility that Notch and Wnt coordinate in vivo to maintain quiescence of HSCs, rather than participating in expansive and/or self-renewal functions (Duncan et al, 2005). Notably, elevated Hes-1 and p21 expression have recently been shown to correlate with the maintenance of quiescence and repopulating function of primitive HSCs (Yu et al, 2006). We noted a highly specific impact of the Dkk1 tg on the stem cell enriched LK+S+ fraction in Wnt-dependent pathway activation and inhibition and the Notch target, Hes-1, or the cell cycle regulator, p21 expression.

The effects of Dkk1 on cell cycling were unanticipated given previous reports of constitutively active β-catenin inducing increased stem/progenitor cell proliferation (Kirstetter et al, 2006Scheller et al, 2006). However, others found that with deletion of the chromatin binding protein, Hmgb3, Wnt signaling was increased, yet stem cells more readily returned to quiescence after 5-FU challenge than controls. (Nemeth et al, 2006) Both increased and decreased activation of the pathway may therefore alter HSC cycling kinetics. This may again be due to the context differences observed with a microenvironmentally-provided signal in the current study contrasted with cell autonomous activation of the pathway in the prior reports. Alternatively, it may be an example of the complex effects of morphogens, which have dose-dependent actions (Delaney et al, 2005Kielman et al, 2002MacDonald et al, 2004). It may be that there is a bi-phasic response of cell cycling to the Wnt pathway and that proper control of stem cell quiescence requires a fine-tuned modulation of intermediate Wnt signaling intensity. This has implications for the potential use of Wnts as mediators of stem cell expansion ex vivo and for interruption of this pathway as an anti-leukemic intervention.

In sum, niche related expression of Dkk1 reveals a role for Wnt signaling in the physiologic regulation of the hematopoietic compartment, altering stem cell cycling and longevity following repeated expansion, or self-renewal. The phenotype observed was sufficiently distinct from what cell-autonomous modifications of the pathway would have predicted to argue for niche specific modeling of exogenous factors’ effects on stem cells. This may be particularly true for members of the locally acting morphogen group of cell modifiers.

7.10.4 Wnt.β-Catenin Signaling in Development and Disease

Clevers H1.
Cell. 2006 Nov 3; 127(3):469-80.

A remarkable interdisciplinary effort has unraveled the WNT (Wingless and INT-1) signal transduction cascade over the last two decades. Wnt genes encode small secreted proteins that are found in all animal genomes. Wnt signaling is involved in virtually every aspect of embryonic development and also controls homeostatic self-renewal in a number of adult tissues. Germline mutations in the Wnt pathway cause several hereditary diseases, and somatic mutations are associated with cancer of the intestine and a variety of other tissues.

The mouse wnt1 gene, originally named Int-1, was identified in 1982 by Nusse and Varmus as a preferential integration site for the Mouse Mammary Tumor Virus in virally induced breast tumors ( Nusse and Varmus, 1982). When sequenced, the Wnt1 proto-oncogene was seen to encode a secreted protein that is cysteine rich. Subsequently, Drosophila wingless (wg), which controls segment polarity during larval development ( Nüsslein-Volhard and Wieschaus, 1980), was shown to be a fly homolog of Wnt1 ( Rijsewijk et al., 1987). Segmentation of the epidermis of wg mutant fly embryos is severely impaired as evidenced by abnormalities in the overlying ventral cuticle. In contrast to the wild-type cuticle, which exhibits alternating denticle and naked belts, the wg cuticle is completely covered with denticles. Fly embryos carrying mutations in the porcupinedishevelled, and armadillo genes display similar cuticle abnormalities to wgmutant embryos, whereas mutations in shaggy/zeste-white 3 cause the opposite phenotype, a naked cuticle. Epistatic analysis of cuticle structure in double mutants indicated that these genes constituted the core of a new signal transduction cascade ( Siegfried et al., 1992Noordermeer et al., 1994 and Peifer et al., 1994).

In 1989, McMahon and Moon (McMahon and Moon, 1989) observed a duplication of the body axis inXenopus following injection of mouse Wnt1 mRNA into ventral blastomeres of embryos at the 4-cell stage. This observation supported the notion that Wnt signaling was shared between vertebrates and invertebrates and, moreover, provided a rapid and convenient assay to study components of the Wnt pathway in vertebrates. Axis duplication was also induced by Dishevelled (Dsh), β-catenin (the vertebrate homolog of armadillo), and a dominant-negative version of glycogen synthase kinase 3 (GSK3), the vertebrate homolog of shaggy/zeste-white 3 ( Dominguez et al., 1995Guger and Gumbiner, 1995 and He et al., 1995). Although long elusive, the specific Wnt signal that triggers axis induction in Xenopus was identified as Wnt11 by Heasman and colleagues last year ( Tao et al., 2005).

The combined observations made in Drosophila and Xenopus delineated a highly conserved signaling pathway, activated by secreted Wnt proteins. Independent of these studies, the adenomatous polyposis coli (APC) gene was discovered in a hereditary cancer syndrome termed familial adenomatous polyposis (FAP) ( Kinzler et al., 1991 and Nishisho et al., 1991). Soon after, the large cytoplasmic APC protein was found to interact with β-catenin ( Rubinfeld et al., 1993 and Su et al., 1993). This observation provided the first connection between the Wnt pathway and human cancer.

Genome sequencing has since revealed that mammalian species have roughly 20 secreted Wnt proteins, which can be divided into 12 conserved Wnt subfamilies. Of these, only 6 subfamilies have counterparts in ecdysozoan animals such as Drosophila and Caenorhabditis. In contrast, at least 11 of the Wnt subfamilies occur in the genome of a cnidarian (the sea anemone Nematostella vectensis). This finding suggests that some Wnt subfamilies were lost during the evolution of the ecdysozoan lineage but more importantly reveals that a complex inventory of Wnt factors was present in multicellular animals well before the Cambrian explosion (550 million years ago). Thus, comparative genomic analysis underscores the crucial role that Wnt genes play in organismal patterning throughout the animal kingdom ( Kusserow et al., 2005).

Currently, three different pathways are believed to be activated upon Wnt receptor activation: the canonical Wnt/β-catenin cascade, the noncanonical planar cell polarity (PCP) pathway, and the Wnt/Ca2+ pathway. Of these three, the canonical pathway is best understood and is the primary subject of this review. For recent comprehensive overviews on the other Wnt signaling pathways, the reader is referred to Katoh (2005) and Kohn and Moon (2005). This review discusses how Wnt proteins are produced and secreted and how they activate the canonical Wnt signaling pathway in recipient cells. Further, the review examines the roles of the canonical Wnt pathway in development, tissue self-renewal, and cancer.

Wnt Protein Secretion

Wnt proteins are characterized by a high number of conserved cysteine residues. Although Wnt proteins carry an N-terminal signal peptide and are secreted, they are relatively insoluble. This insolubility has been attributed to a particular protein modification, cysteine palmitoylation, which is essential for Wnt function (Willert et al., 2003). Hofmann (2000) reported that a Drosophila gene required in the Wnt-secreting cell, termed porcupine, displays homology to acyl-transferases, enzymes that acylate a variety of substrates in the endoplasmic reticulum. Thus, porcupine and its worm homolog mom-1 are believed to encode the enzyme that is responsible for Wnt palmitoylation ( Zhai et al., 2004).

Recently, Banziger et al. (2006) and Bartscherer et al. (2006) uncovered in Drosophila another conserved gene that is essential for Wnt secretion, named wntless (wls) and evenness interrupted (evi), respectively. The gene encodes a seven-pass transmembrane protein that is conserved from worms (mom-3) to man (hWLS). In the absence of Wls/evi, Wnts are retained inside the cell that produces them. The Wntless protein resides primarily in the Golgi apparatus, where it colocalizes and physically interacts with Wnts. A genetic screen in C. elegans revealed that the retromer, a multiprotein complex involved in intracellular trafficking and conserved from yeast to man, is also essential for Wnt secretion and for the generation of a Wnt gradient ( Coudreuse et al., 2006). An attractive hypothesis is that the retromer complex is involved in recycling a Wnt cargo receptor (such as Wntless) between the default secretory pathway and a compartment dedicated to Wnt secretion (see Figure 1).




Figure 1. Wnt Secretion

To be secreted, Wnt proteins in the endoplasmic reticulum (ER) need to be palmitoylated by the action of Porcupine. Wnt proteins also require Wntless (Wls/Evi) in order to be routed to the outside of the cell. Loading onto lipoprotein particles may occur in a dedicated endo/exocytic compartment. The retromer complex may shuttle Wls between the Golgi and the endo/exocytic compartment.

Wnt is thought to act as a morphogen (that is, a long-range signal whose activity is concentration dependent) (reviewed in Logan and Nusse, 2004). However, it is unclear how these long-range gradients are generated. It is conceivable that the palmitoyl moiety constrains movement away from membranes or lipid particles. Thus, Wnts may be tethered to intercellular transport vesicles or lipoprotein particles (Panakova et al., 2005). Alternatively, Wnts may be transported by cytonemes, which are long, thin filopodial processes. Additionally, studies in Drosophila suggest a role for extracellular heparan sulfate proteoglycans (HSPG) in the transport or stabilization of Wnt proteins. For instance, flies carrying mutations in Dally, a GPI-anchored HSPG, or in genes encoding enzymes that modify HSPGs resemblewingless mutants (reviewed in Lin, 2004).

Receptors, Agonists, and Antagonists for Wnt

Wnts bind Frizzled (Fz) proteins, which are seven-pass transmembrane receptors with an extracellular N-terminal cysteine-rich domain (CRD) (Bhanot et al., 1996). The Wnt-Fz interaction appears promiscuous, in that a single Wnt can bind multiple Frizzled proteins (e.g., Bhanot et al., 1996) and vice versa. In binding Wnt, Fzs cooperate with a single-pass transmembrane molecule of the LRP family known as Arrow inDrosophila ( Wehrli et al., 2000) and LRP5 and -6 in vertebrates ( Pinson et al., 2000 and Tamai et al., 2000). The transport of Arrow/LRP5/6 to the cell surface is dependent on a chaperone called Boca inDrosophila and Mesd in mice ( Culi and Mann, 2003 and Hsieh et al., 2003). And consistent with a role of the Boca/Mesd chaperone in the transport of Arrow/LRP5/6 transport, mutations in Boca and Mesdresemble loss of Arrow/LRP5/6. Although it has not been formally demonstrated that Wnt molecules form trimeric complexes with LRP5/6 and Frizzled, surface expression of both receptors is required to initiate the Wnt signal.

Derailed, a transmembrane tyrosine kinase receptor from the RYK subfamily, is an unusual Wnt receptor.Drosophila Wnt5 controls axon guidance in the central nervous system. Embryos lacking Dwnt-5 resemble those lacking Derailed, that is, they generate aberrant neuronal projections across the midline ( Yoshikawa et al., 2003). Derailed binds DWnt-5 through its extracellular WIF (Wnt inhibitory factor) domain. Signaling events downstream of this alternative Wnt receptor remain unclear. Somewhat unexpectedly, the Derailed kinase domain may be dispensable for signaling. Lu et al. (2004) propose that, unlike the Drosophila Ryk homolog Derailed, mammalian Ryk functions as a coreceptor along with Fz. Mammalian Ryk binds Dishevelled to activate the canonical Wnt/β-catenin signaling pathway. Another tyrosine kinase receptor, Ror2, harbors a Wnt binding CRD motif. Wnt5a can engage Ror2 to inhibit the canonical Wnt signaling pathway, although paradoxically Wnt5a can also activate the canonical pathway by directly engaging Fz4 (Mikels and Nusse, 2006) and Fz5 ( He et al., 1997).

At least two types of proteins that are unrelated to Wnt factors activate the Frizzled/LRP receptors. One of these factors is the cysteine-knot protein Norrin, which is mutated in Norrie disease, a developmental disorder characterized by vascular abnormalities in the eye and blindness. Norrin binds with high affinity to Frizzled-4 and activates the canonical signaling pathway in an LRP5/6-dependent fashion (Xu et al., 2004). Other factors that activate the canonical Wnt signaling pathway are R-spondins, which are thrombospondin domain-containing proteins. In Xenopus, R-spondin-2 is a Wnt agonist that synergizes with Wnts to activate β-catenin ( Kazanskaya et al., 2004). Human R-spondin-1 has been found to strongly promote the proliferation of intestinal crypt cells, a process which involves the stabilization of β-catenin (Kim et al., 2005). Indeed, studies in cultured cells demonstrate that R-spondins can physically interact with the extracellular domains of LRP6 and Fzd8 and activate Wnt reporter genes ( Nam et al., 2006).

The secreted Dickkopf (Dkk) proteins inhibit Wnt signaling by direct binding to LRP5/6 (Glinka et al., 1998). Through this interaction, Dkk1 crosslinks LRP6 to another class of transmembrane molecules, the Kremens (Mao et al., 2002), thus promoting the internalization and inactivation of LRP6. An unrelated secreted Wnt inhibitor, Wise, also acts by binding to LRP (Itasaki et al., 2003), as does the WISE family member SOST (Li et al., 2005 and Semenov et al., 2005).

Soluble Frizzled-Related Proteins (SFRPs) resemble the ligand-binding CRD domain of the Frizzled family of Wnt receptors (Hoang et al., 1996). WIF proteins are secreted molecules with similarity to the extracellular portion of the Derailed/RYK class of transmembrane Wnt receptors (Hsieh et al., 1999). SFRPs and WIFs are believed to function as extracellular Wnt inhibitors (reviewed in Logan and Nusse, 2004) but, depending on context, may also promote signaling by Wnt stabilization or by facilitating Wnt secretion or transport.

Canonical Wnt Signaling

Once bound by their cognate ligands, the Fz/LRP coreceptor complex activates the canonical signaling pathway (Figure 2). Fz can physically interact with Dsh, a cytoplasmic protein that functions upstream of β-catenin and the kinase GSK-3. Wnt signaling controls phosphorylation of Dsh (reviewed in Wallingford and Habas, 2005). However, it remains unclear whether the binding of Wnt to Fz regulates a direct Fz-Dsh interaction, nor is it known how Dsh phosphorylation is controlled or how phosphorylated Dsh functions in Wnt signal transduction.



Figure 2. Canonical Wnt Signaling

(Left panel) When Wnt receptor complexes are not bound by ligand, the serine/threonine kinases, CK1 and GSK3α/β, phosphorylate β-catenin. Phosphorylated β-catenin is recognized by the F box/WD repeat protein β-TrCP, a component of a dedicated E3 ubiquitin ligase complex. Following ubiquitination, β-catenin is targeted for rapid destruction by the proteasome. In the nucleus, the binding of Groucho to TCF (T cell factor) inhibits the transcription of Wnt target genes. (Right panel) Once bound by Wnt, the Frizzled(Fz)/LRP coreceptor complex activates the canonical signaling pathway. Fz interacts with Dsh, a cytoplasmic protein that functions upstream of β-catenin and the kinase GSK3β. Wnt signaling controls phosphorylation of Dishevelled (Dsh). Wnts are thought to induce the phosphorylation of LRP by GSK3β and casein kinase I-γ (CK1γ), thus regulating the docking of Axin. The recruitment of Axin away from the destruction complex leads to the stabilization of β-catenin. In the nucleus, β-catenin displaces Groucho from Tcf/Lef to promote the transcription of Wnt target genes.

Recent studies have indicated that the coreceptor LRP5/6 interacts with Axin through five phosphorylated PPP(S/T)P repeats in the cytoplasmic tail of LRP (Davidson et al., 2005 and Zeng et al., 2005). Wnts are thought to induce the phosphorylation of the cytoplasmic tail of LRP, thus regulating the docking of Axin. GSK3 phosphorylates the PPP(S/T)P motif, whereas caseine kinase I-γ (CK1γ) phosphorylates multiple motifs close to the GSK3 sites. CK1γ is unique within the CK1 family in that it is anchored in the membrane through C-terminal palmitoylation. Both kinases are essential for signal initiation. It remains presently debated whether Wnt controls GSK3-mediated phosphorylation of LRP5/6 (Zeng et al., 2005) or whether CK1γ is the kinase regulated by Wnt (Davidson et al., 2005). When bound to their respective membrane receptors, Dsh and Axin may cooperatively mediate downstream activation events by heterodimerization through their respective DIX (Dishevelled-Axin) domains.

The Cytoplasmic Destruction Complex

The central player in the canonical Wnt cascade is β-catenin, a cytoplasmic protein whose stability is regulated by the destruction complex. The tumor suppressor protein Axin acts as the scaffold of this complex as it directly interacts with all other components—β-catenin, the tumor suppressor protein APC, and the two kinase families (CK1α, -δ, -ɛ and GSK3α and -β [reviewed in Price, 2006]). When WNT receptor complexes are not engaged, CK1 and GSK3α/β sequentially phosphorylate β-catenin at a series of highly conserved Ser/Thr residues near its N terminus (Figure 2). Phosphorylated β-catenin is then recognized by the F box/WD repeat protein β-TrCP, a component of a dedicated E3 ubiquitin ligase complex. As a consequence, β-catenin is ubiquitinated and targeted for rapid destruction by the proteasome (Aberle et al., 1997). Note that the CK1 and GSK3 kinases perform paradoxical roles in the Wnt pathway. At the level of the LRP coreceptor they act as agonists, whereas in the destruction complex they act as antagonists

Although genetic observations imply an essential role for APC in the destruction complex, there is no consensus on its specific molecular activity. APC has a series of 15 and 20 amino acid repeats with which it interacts with β-catenin. Three Axin-binding motifs are interspersed between these β-catenin-binding motifs. Increasing the expression of Axin in cancer cells that lack APC restores the activity of the destruction complex, implying that APC is only essential when Axin levels are limiting. Quantitatively, Axin indeed appears to be the limiting factor (Lee et al., 2003) and may be the key scaffolding molecule that promotes the rapid assembly and disassembly of the destruction complex.

Given that CK1, Dsh, β-TrCP, and GSK3 participate in other signaling pathways, low levels of Axin may insulate the Wnt pathway from changes in the abundance or activity of these signaling components. It has been proposed that APC is required for efficient shuttling and loading/unloading of β-catenin onto the cytoplasmic destruction complex. Both APC and Axin can themselves be phosphorylated by their associated kinases, which changes their affinity for other components of the destruction complex. Our understanding of the relevance of these phosphorylation events in the regulation of Wnt signaling remains incomplete. For a comprehensive discussion of the kinases in the Wnt pathway, the reader is referred to a recent review (Price, 2006)

β-catenin plays a second role in simple epithelia, that is, as a component of adherens junctions. It is an essential binding partner for the cytoplasmic tail of various cadherins, such as E-cadherin (Peifer et al., 1992). Unlike the signaling pool of β-catenin, the pool that is bound to the adherens junction is highly stable. It is currently unclear whether the adhesive and signaling properties of β-catenin are interconnected. In a likely scenario, newly synthesized β-catenin first saturates the pool that is part of the adhesion junction, which never becomes available for signaling. “Excess,” free cytoplasmic β-catenin protein is then efficiently degraded by the APC complex. It is only this second, highly unstable pool that is subject to regulation by Wnt signals. In support of this model, these two functions of β-catenin are separately performed by two different β-catenin homologs in C. elegans ( Korswagen et al., 2000).

Upon receptor activation by WNT ligands, the intrinsic kinase activity of the APC complex for β-catenin is inhibited. It is unclear how this occurs, but it likely involves the Wnt-induced recruitment of Axin to the phosphorylated tail of LRP and/or to Fz-bound Dsh. As a consequence, stable, nonphosphorylated β-catenin accumulates and translocates into the nucleus, where it binds to the N terminus of LEF/TCF (lymphoid enhancer factor/T cell factor) transcription factors (Behrens et al., 1996Molenaar et al., 1996 and van de Wetering et al., 1997).

It has been suggested that protein phosphatases may regulate β-catenin stability as antagonists of the serine kinases (reviewed in Price, 2006). For example, heterotrimeric PP2A is required for the elevation of β-catenin levels that is dependent on Wnt. Moroever, PP2A can bind Axin and APC, suggesting that it might function to dephosphorylate GSK3 substrates. If and how PP2A activity is regulated by Wnt signals remains to be resolved.

Crystallographic studies are starting to provide insights into the structure of the destruction complex. The central region of β-catenin (to which most partners bind) was the first component of the pathway to be crystallized. It consists of 12 armadillo repeats, which adopt a superhelical shape with a basic groove running along its length. Subsequently, structural interactions of Axin, APC, E-cadherin, and TCF with β-catenin have been visualized (Choi et al., 2006, and references therein). APC, E-cadherin, and TCF bind the central part of the basic groove in a mutually exclusive fashion. Despite very limited conservation of primary sequence in the respective interaction domains, the modes of binding are structurally very similar. Axin utilizes a helix that occupies the groove formed by the third and fourth armadillo repeats of β-catenin. Axin binding precludes the simultaneous interaction with other β-catenin partners in this region. Based on this observation, it is suggested that a key function of APC is to remove phosphorylated β-catenin from the active site of the complex (Xing et al., 2003). In a further study, the structure of Axin bound to APC (Spink et al., 2000) was solved. These studies form stepping stones to a better understanding of the dynamics of the destruction complex. Unfortunately, biochemical studies of the destruction complex in its different activation states are sorely lacking.

Nuclear Events

Upon stabilization by Wnt signals, β-catenin enters the nucleus to reprogram the responding cell (Figure 3). There is no consensus on the mechanism by which β-catenin travels between the cytoplasm and the nucleus. In many cases, cells that undergo Wnt signaling may actually display an overall rise in β-catenin protein without a clear nuclear preference. β-catenin’s nuclear import is independent of the Nuclear Localization Signal/importin machinery. β-catenin itself is a close relative of importin/karyopherins and directly interacts with nuclear pore components. Two proteins, Tcf and Pygopus are proposed to anchor β-catenin in the nucleus, although β-catenin can still localize to the nucleus in the absence of either of the two (reviewed in Staedeli et al., 2006). β-catenin can also be actively transported back to the cytoplasm, by either an intrinsic export signal or as cargo of Axin (Cong and Varmus, 2004) or APC (Rosin-Arbesfeld et al., 2000) that shuttle between cytoplasm and nucleus.



Transactivation of Wnt Target Genes


Figure 3. Transactivation of Wnt Target Genes

The β-catenin/Tcf complex interacts with a variety of chromatin-remodeling complexes to activate transcription of Wnt target genes. The recruitment of β-catenin to Tcf target genes affects local chromatin in several ways. Bcl9 acts as a bridge between Pygopus and the N terminus of β-catenin. Evidence suggests that this trimeric complex is involved in nuclear import/retention of β-catenin (Townsley et al., 2004), but it may also be involved in the ability of β-catenin to activate transcription (Hoffmans et al., 2005). The C terminus of β-catenin also binds to coactivators such as the histone acetylase CBP, Hyrax, and Brg-1 (a component of the SWI/SNF chromatin-remodeling complex).

Whereas the fly and worm genomes both encode a single Tcf protein, the vertebrate genome harbors fourTcf/Lef genes. Tcf factors bind their cognate motif in an unusual fashion, i.e., in the minor groove of the DNA helix, while inducing a dramatic bend of over 90°. Tcf target sites are highly conserved between the four vertebrate Tcf/Lef proteins and Drosophila Tcf. These sites resemble AGATCAAAGG ( van de Wetering et al., 1997). Wnt/TCF reporter plasmids such as pTOPflash ( Korinek et al., 1997), widely used to measure Wnt pathway activation, consist of concatamers of 3–10 of these binding motifs cloned upstream of a minimal promoter. The four vertebrate TCF/LEF differ dramatically in their embryonic and adult expression domains, yet they are highly similar biochemically, explaining the extensive redundancy unveiled in double knockout experiments (as in Galceran et al., 1999).

In the absence of Wnt signals, Tcf acts as a transcriptional repressor by forming a complex with Groucho/Grg/TLE proteins (Cavallo et al., 1998 and Roose et al., 1998). The interaction of β-catenin with the N terminus of Tcf (Behrens et al., 1996Molenaar et al., 1996 and van de Wetering et al., 1997) transiently converts it into an activator, translating the Wnt signal into the transient transcription of Tcf target genes. To accomplish this, β-catenin physically displaces Groucho from Tcf/Lef (Daniels and Weis, 2005). The recruitment of β-catenin to Tcf target genes affects local chromatin in several ways. Its C terminus is a potent transcriptional activator in transient reporter gene assays (van de Wetering et al., 1997). It binds coactivators such as the histone acetylase CBP and Brg-1, a component of the SWI/SNF chromatin remodeling complex (reviewed in Staedeli et al., 2006). A recent study implies that the human and fly homologs of yeast Cdc37 (Parafibromin and Hyrax, respectively) also interact with the C-terminal transactivation domain of β-catenin to activate target gene transcription (Mosimann et al., 2006). Cdc37 is a component of the PAF complex. In yeast the PAF complex directly interacts with RNA polymerase II to regulate transcription initiation and elongation.

Two dedicated, nuclear partners of the TCF/β-catenin complex, Legless/Bcl9 and Pygopus, were recently found in genetic screens in Drosophila ( Kramps et al., 2002Parker et al., 2002 and Thompson et al., 2002). Mutations in these genes result in phenotypes similar to wingless, and overexpression of both genes promotes TCF/β-catenin activity in mammalian cells ( Thompson et al., 2002). Bcl9 bridges Pygopus to the N terminus of β-catenin. The formation of this trimeric complex has been implicated in nuclear import/retention of β-catenin ( Townsley et al., 2004) but may also directly contribute to the ability of β-catenin to transactivate transcription ( Hoffmans et al., 2005). Although most if not all Wnt signaling events in Drosophila appear to be dependent on Bcl9 and Pygopus, it is currently unclear if this holds true in vertebrate development.

Tcf itself can be regulated by phosphorylation. The MAP kinase-related protein kinase NLK/Nemo (Ishitani et al., 1999) phosphorylates Tcf, thereby decreasing the DNA-binding affinity of the β-catenin/Tcf complex and inhibiting transcriptional regulation of Wnt target genes. In C. elegans, LIT-1/NLK-dependent phosphorylation results in PAR-5/14-3-3- and CRM-1-dependent nuclear export of POP-1/Tcf ( Meneghini et al., 1999 and Lo et al., 2004). And lastly, a recent study utilizing chromatin immunoprecipitations suggests that APC, independent of its role in the cytoplasmic destruction complex, acts on chromatin to facilitate CtBP-mediated repression of Wnt target genes in normal, but not in colorectal cancer cells ( Sierra et al., 2006).

Wnt Target Genes

Loss of components of the Wnt pathway can produce dramatic phenotypes that affect a wide variety of organs and tissues. A popular view equates Wnt signaling with maintenance or activation of stem cells (Reya and Clevers, 2005). It should be realized, however, that Wnt signals ultimately activate transcriptional programs and that there is no intrinsic restriction in the type of biological event that may be controlled by these programs. Thus, Wnt signals may promote cell proliferation and tissue expansion but also control fate determination or terminal differentiation of postmitotic cells. Sometimes, these disparate events, proliferation and terminal differentiation, can be activated by Wnt in different cell types within the same structure, such as the hair follicle or the intestinal crypt (Reya and Clevers, 2005).

Numerous Tcf target genes have been identified in diverse biological systems. These studies tend to focus on target genes involved in cancer, as exemplified by the wide interest in the Wnt target genes cMyc and Cyclin D1. For a comprehensive, updated overview of Tcf target genes, the reader is referred to the Wnt homepage (http://www.stanford.edu/∼rnusse/wntwindow.html). The Wnt pathway has distinct transcriptional outputs, which are determined by the developmental identity of the responding cell, rather than by the nature of the signal. In other words, the majority of Wnt target genes appear to be cell type specific. It is not clear whether “universal” Wnt/Tcf target genes exist. The best current candidates in vertebrates are Axin2/conductin (Jho et al., 2002) and SP5 (Weidinger et al., 2005). As noted (Logan and Nusse, 2004), Wnt signaling is autoregulated at many levels. The expression of a variety of positive and negative regulators of the pathway, such as Frizzleds, LRP and HSPG, Axin2, and TCF/Lef are all controlled by the β-catenin/TCF complex.

Wnt Signaling in Self-Renewing Tissues in Adult Mammals

Wnt signaling not only features in many developmental processes; in some self-renewing tissues in mammals it remains essential throughout life. It is this aspect of Wnt signaling that is intricately connected to the development of disease. The examples discussed below illustrate how the Wnt pathway is involved in adult tissue self-renewal. Mutations in the Wnt pathway tip the homoeostatic balance in these tissues to cause pathological conditions such as disturbances in skeletal bone mass or cancer.


Figure 4. Self-Renewing Tissues in the Adult Mammal

Current evidence indicates that the Wnt cascade is the dominant force in controlling cell fate along the crypt-villus axis. In neonatal mice lacking Tcf4, the differentiated villus epithelium appears unaffected, but the crypt progenitor compartment is entirely absent (Korinek et al., 1998). This implies that physiological Wnt signaling is required for the establishment of this progenitor compartment.

Hair Follicle

Multipotent epidermal stem cells reside in the bulge region of the hair follicle (Figure 4). Bulge stem cells can generate all hair lineages but also the sebocytes and even the stem cells of the interfollicular epidermis (Alonso and Fuchs, 2003). To form a hair, cells migrate downward from the bulge through the outer root sheath. At the base of the hair, the cells enter a transit-amplifying compartment termed the germinative matrix where they undergo terminal differentiation in the precortex compartment of the hair.

Hematopoietic System

Hematopoietic stem cells (HSCs) are the best studied stem cells in mammals. A number of studies have implicated the Wnt signaling pathway as an important regulator of hematopoietic stem and progenitor cells. HSCs themselves as well as the bone marrow microenvironment can produce Wnt proteins. Indeed, Tcf reporters are active in HSCs in their native microenvironment.


In postnatal and adult life, osteoblasts produce bone matrix, whereas osteoclasts resorb the matrix. Bone density is determined by the relative activities of these two cell types. Gain-of-function mutations in the human LRP5 gene occur in bone diseases, indicating that canonical Wnt signaling may regulate bone mass. This observation has motivated genetic studies in mouse models, which generally confirm the importance of this signaling pathway in bone homeostasis, primarily as a positive regulator of the osteoblast lineage. Similar to humans carrying the gain-of-function LRP5G171V mutation, transgenic mice expressing this allele in osteoblasts display increased bone density and elevated numbers of active osteoblasts (reviewed in Hartmann, 2006).

Wnt Signaling in Cancer

Colon Cancer

The APC gene was among the first tumor suppressors to be cloned. A germline APC mutation is the genetic cause of a hereditary cancer syndrome termed Familiar Adenomatous Polyposis (FAP) (Kinzler et al., 1991 and Nishisho et al., 1991). FAP patients inherit one defective APC allele and as a consequence develop large numbers of colon adenomas, or polyps, in early adulthood. Polyps are benign, clonal outgrowths of epithelial cells in which the second APC allele is inactivated. Inevitably, some of these polyps progress into malignant adenocarcinoma. Loss of both APC alleles occurs in the large majority of sporadic colorectal cancers (Kinzler and Vogelstein, 1996). Mutational inactivation of APC leads to the inappropriate stabilization of β-catenin (Rubinfeld et al., 1996Figure 4). Indeed, Tcf reporter constructs, normally transcribed only upon Wnt signaling, are inappropriately transcribed in APC mutant cancer cells through the action of constitutive complexes between β-catenin and the intestinal TCF family member Tcf4 (Korinek et al., 1997). In rare cases of colorectal cancer where APC is not mutated, Axin2 is mutant (Liu et al., 2000), or activating (oncogenic) point mutations in β-catenin remove its N-terminal Ser/Thr destruction motif (Morin et al., 1997). Of note, patients with hereditary Axin2 mutations display a predisposition to colon cancer (Lammi et al., 2004).

In intestinal epithelial cells in which APC is mutated, the constitutive β-catenin/Tcf4 complex activates a genetic program in crypt stem/progenitor cells (van de Wetering et al., 2002). In the crypt, the Wnt signaling gradient drives expression of this genetic program to maintain progenitor cell proliferation. The Wnt gradient also controls expression of the EphB/EphrinB sorting receptors and ligands (Battle et al., 2002). The resulting EphB/EphrinB countergradients establish crypt-villus boundaries as well as position the Paneth cells at the bottom of the crypt. Several EphB genes are initially upregulated as Wnt/Tcf4 target genes in early adenomas, but their expression is lost upon cancer progression (Batlle et al., 2005) apparently as the result of a selection process. Activating Wnt pathway mutations are not restricted to cancer of the intestine. Loss-of-function mutations in Axin have also been found in hepatocellular carcinomas, whereas oncogenic β-catenin mutations occur in a wide variety of solid tumors (reviewed inReya and Clevers, 2005).

Several animal models exist for FAP. Dove and colleagues first described the multiple intestinal neoplasia(min) mouse, which carries a stop codon in APC (Apcmin). Unlike FAP patients, Apcmin mice develop adenomas predominantly in the small intestine ( Su et al., 1992). Several additional Apc knockout models have been generated in mice. Invariably, these mice develop neoplastic lesions but they may differ in tumor incidence and tissue type in which tumors first appear. In a recent elegant study, the Wnt cascade was mutationally activated in adult mice by conditional deletion of Apc ( Sansom et al., 2004). Within days, villi were entirely populated by crypt-like cells, demonstrating the direct link between active Wnt signaling and the proliferation of crypt progenitors, which when unrestrained results in cancer. Zebrafish that are mutant in Apc resemble the mouse models in that heterozygous mutants develop adenomas in organs of endodermal origin including the intestine. These fish may prove useful for genetic screens for genes that modify cancer risk ( Haramis et al., 2006).

Hair Follicle Tumors


Drawing from the parallels between self-renewal and cancer in the gut and hair follicle, the effects of Wnt pathway components on hematopoietic progenitors predict that Wnt deregulation may contribute to hematological malignancies. Indeed, a recent report suggests that leukemic growth of both myeloid and lymphoid lineages is dependent on Wnt signaling. Granulocyte-macrophage progenitors from Chronic Myelogenous Leukemia patients and blast crisis cells from patients resistant to therapy display active Wnt signaling as demonstrated by Tcf reporter activity and the accumulation of nuclear β-catenin (Jamieson et al., 2004).

Over the last 20 years, a detailed outline of the canonical Wnt pathway has emerged. Although it is likely that most core components of the pathway have now been identified, much remains to be learned about the biochemical events that connect these components. Many of the gaps in our knowledge are due to the notorious difficulties in the production of purified Wnt proteins. Few good Wnt antibodies exist and, 25 years after the cloning of Wnt1, its structure remains unknown. The routing and the coincident posttranslational modifications of Wnt proteins in the secreting cell are incompletely understood. And the rules that dictate the movement of Wnt proteins between cells remain uncertain. However, a procedure to produce soluble Wnt has recently been developed (Willert et al., 2003), which creates avenues to address many of these issues.

The components of the destruction complex have been long known, yet the biochemistry of its activity has remained elusive. APC is an essential component of the destruction complex, but what is its biochemical activity? How relevant is Dsh for the coupling of Wnt receptors to the destruction complex? And what mechanism inhibits the phosphorylation of β-catenin by the destruction complex when a Wnt signal is being transduced?

In addition, a multitude of proposed pathway components, not discussed here, may activate, modify, or inhibit Wnt signaling or may be involved in crosstalk to other pathways. An updated, comprehensive list of these putative components and interactions appears on http://www.stanford.edu/∼rnusse/wntwindow.html. Often based on single studies, these candidate components remain to be independently confirmed.

Wnt signaling ultimately controls developmental fates through the transcription of cell type-specific programs of Tcf target genes. Recent developments in array-based technology allow detailed analysis of the nuclear transcriptional response to Wnt signals. With these technologies, it is expected that the dissection of the gene programs in various developmental or pathological events will provide a wealth of insight into the biology of these processes.

7.10.5 Wnt.β-Catenin Signaling. Components, Mechanisms, and Diseases

MacDonald BT1Tamai KHe X.
Dev Cell. 2009 Jul; 17(1):9-26

Signaling by the Wnt family of secreted glycolipoproteins via the transcription co-activator β-catenin controls embryonic development and adult homeostasis. Here we review recent progresses in this so-called canonical Wnt signaling pathway. We discuss Wnt ligands, agonists and antagonists and their interactions with Wnt receptors. We also dissect critical events that regulate β-catenin stability from Wnt receptors to the cytoplasmic β-catenin destruction complex, and nuclear machinery that mediates β-catenin-dependent transcription. Finally we highlight some key aspects of Wnt/β-catenin signaling in human diseases including congenital malformations, cancer and osteoporosis and potential therapeutic implications.

Signaling by the Wnt family of secreted glycolipoproteins is one of the fundamental mechanisms that direct cell proliferation, cell polarity and cell fate determination during embryonic development and tissue homeostasis (Logan and Nusse, 2004). As a result, mutations in the Wnt pathway are often linked to human birth defects, cancer and other diseases (Clevers, 2006). A critical and most studied Wnt pathway is canonical Wnt signaling, which functions by regulating the amount of the transcriptional co-activator β-catenin that controls key developmental gene expression programs. This review focuses on our current understanding of Wnt/β-catenin signaling, drawing mainly from genetic, developmental and biochemical analyses in Drosophila, Xenopus, mice and humans. For more comprehensive and historic perspective we refer readers to earlier reviews (Clevers, 2006Logan and Nusse, 2004) and the Wnt homepage (www.stanford.edu/~rnusse/wntwindow.html). The nematode Caenorhabditis elegans exhibits similar but also divergent Wnt/β-catenin pathways, which are covered elsewhere (Mizumoto and Sawa, 2007) and in the accompanying review (Kimble 2009). Wnt also activates a number of non-canonical signaling pathways that are independent of β-catenin and have been recently reviewed (Seifert and Mlodzik, 2007Wang and Nathans, 2007).

The central logic of Wnt/β-catenin signaling has emerged from two decades of studies (Figure 1). In the absence of Wnt, cytoplasmic β-catenin protein is constantly degraded by the action of the Axin complex, which is composed of the scaffolding protein Axin, the tumor suppressor adenomatous polyposis coli gene product (APC), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3). CK1 and GSK3 sequentially phosphorylate the amino terminal region of β-catenin, resulting in β-catenin recognition by β-Trcp, an E3 ubiquitin ligase subunit, and subsequent β-catenin ubiquitination and proteasomal degradation (He et al., 2004). This continual elimination of β-catenin prevents β-catenin from reaching the nucleus, and Wnt target genes are thereby repressed by the DNA-bound T cell factor/lymphoid enhancer factor (TCF/LEF) family of proteins (Figure 1a). The Wnt/β-catenin pathway is activated when a Wnt ligand binds to a seven-pass transmembrane Frizzled (Fz) receptor and its co-receptor, low-density lipoprotein receptor related protein 6 (LRP6) or its close relative LRP5. The formation of a likely Wnt-Fz-LRP6 complex together with the recruitment of the scaffolding protein Dishevelled (Dvl) results in LRP6 phosphorylation and activation and the recruitment of the Axin complex to the receptors. These events lead to inhibition of Axin-mediated β-catenin phosphorylation and thereby to the stabilization of β-catenin, which accumulates and travels to the nucleus to form complexes with TCF/LEF and activates Wnt target gene expression (Figure 1b).

Overview of Wnt.β-catenin signaling nihms196288f1

Overview of Wnt.β-catenin signaling nihms196288f1

Overview of Wnt/β-catenin signaling

Figure 1 Overview of Wnt/β-catenin signaling

Wnt ligands and biogenesis

Wnts are conserved in all metazoan animals. In mammals, complexity and specificity in Wnt signaling are in part achieved through 19 Wnt ligands, which are cysteine rich proteins of approxiamately 350-400 amino acids that contain an N-terminal signal peptide for secretion. Murine Wnt3a represents the first purified and biochemically characterized Wnt protein (Willert et al., 2003) owing to its relatively efficient secretion (in contrast to most other Wnt proteins). In addition to N-linked glycosylation, which is required for Wnt3a secretion (Komekado et al., 2007), Wnt3a undergoes two types of lipid modifications that likely account for the hydrophobicity and poor solubility of Wnt proteins (Hausmann et al., 2007). The first reported lipididation was the addition of palmitate to cysteine 77 (Willert et al., 2003). Its mutation had minimal effect on Wnt3a secretion but diminished the ability of Wnt3a to activate β-catenin signaling (Galli et al., 2007;Komekado et al., 2007Willert et al., 2003). The second identified lipididation was a palmitoleoyl attached to serine 209, and its mutation resulted in Wnt3a accumulation in the endoplasmic reticulum (ER) and failure in secretion (Takada et al., 2006).

Drosophila Wingless (Wg) is the Wnt molecule most investigated in vivo (Hausmann et al., 2007). These studies plus work in nematodes have identified genes that regulate Wnt biogenesis and secretion. Porcupine (Porc) encodes a multipass transmembrane ER protein that contains an O-acyl transferase domain suggesting a role in Wg lipid modification (Hausmann et al., 2007). Porc deficiency results in Wg and Wnt3a accumulation in the ER and diminished Wnt3a palmitoleoylation at serine 209 (Takada et al., 2006), suggesting that Porc is responsible for this particular lipidation. Whether Porc or a distinct acyltransferase is involved in Wnt3a palmitoylation at cysteine 77 remains unknown.

Two additional proteins/protein complexes were identified for Wg/Wnt secretion: Wntless (Wls), also known as Evenness interrupted (Evi) or Sprinter (Srt), in Drosophila and the retromer complex in nematodes (Hausmann et al., 2007). Wls is a multipass transmembrane protein that localizes to the Golgi, endocytic compartments and the plasma membrane, and is essential for Wg secretion. The retromer complex, which is composed of five subunits, was defined first in yeast. It mediates membrane protein trafficking between endosomes and the Golgi apparatus (Hausmann et al., 2007). Several groups recently reported that the retromer complex is required for retrieval/recycling of Wls from the endosome to the Golgi (Belenkaya et al., 2008Franch-Marro et al., 2008bPan et al., 2008aPort et al., 2008Yang et al., 2008), likely mediated by direct interaction between Wls and the retromer Vps35 subunit. Loss of retromer function causes Wls to be degraded in the lysosomes and results in reduction of Wls and thus Wnt secretion. These studies led to an emerging picture of Wnt biogenesis (Figure 2). Wnt is glycosylated and lipid modified by Porc in the ER, and is escorted by Wls from the Golgi to the plasma membrane for secretion. Wls is recycled by endocytosis and trafficked back to Golgi by the retromer. Note that porcwls and retromer mutants largely phenocopywg/wnt mutants in flies and worms, attesting their dedicated roles in Wnt biogenesis.

Wnt biogenesis and secretion nihms196288f2

Wnt biogenesis and secretion nihms196288f2

Wnt biogenesis and secretion


Figure 2  Wnt biogenesis and secretion

Wnt extracellular distribution and movement

Wnt proteins can function as morphogens that are capable of both short and long range signaling, as best demonstrated for Wg. Wg lipidation raises the issue of its diffusion and distribution through the aqueous extracellular space. Indeed purified Wnt3a exhibits increased activity via artificial liposomal packaging (Morrell et al., 2008). Two distinct Wg secretory pathways for short and long range signaling have been speculated but not fully substantiated. Wg may form multimers to bury lipid modifications inside (Katanaev et al., 2008), or bind to lipoprotein particles, which may be involved in Wg long range signaling (Panakova et al., 2005) (Figure 2). The membrane microdomain protein reggie-1/flotillin-2 specifically promotes Wg long-range secretion (Katanaev et al., 2008). The Wg receptors (see below) and heparan sulfate proteoglycans (HSPGs) such as Dally and Dally-like protein have important roles in the Wg morphogen concentration via regulating Wg degradation, diffusion, endocytosis/transcytosis, and may function in Wg signaling as potential low-affinity co-receptors (Lin, 2004). Note that reggie-1/flotillin-2, lipoprotein particles, Dally and Dally-like protein are important analogously for secreted Hedgehog morphogen, which is also lipid modified (Katanaev et al., 2008Lin, 2004Panakova et al., 2005).

Wnt receptors: Frizzled and LRP5/6

Two distinct receptor families are critical for Wnt/β-catenin signaling (Figure 3): the Frizzled (Fz or Fzd) seven-pass transmembrane receptors (Logan and Nusse, 2004) and the LDL receptor-related proteins 5 and 6 (LRP5 and LRP6) (He et al., 2004). The Wnt-receptor relationship is best illustrated for Wg, which binds toDrosophila Fz2 (Dfz2) and Dfz1 with high affinity (1-10 nM) and requires either Fz in a redundant manner (Logan and Nusse, 2004). Wg reception also absolutely depends on Arrow, the LRP5/6 homolog (He et al., 2004). The mammalian genome harbors 10 Fz genes, most of which have variable capacities to activate β-catenin signaling when co-overexpressed with Wnt and LRP5/6 (e.g., Binnerts et al., 2007) and functional redundancy among Fz members is likely prevalent (Logan and Nusse, 2004). Between the two LRPs, LRP6 plays a more dominant role and is essential for embryogenesis whereas LRP5 is dispensable for embryogenesis but critical for adult bone homeostasis. Nonetheless LRP5 and LRP6 are partially redundant as their functions together are required for mouse gastrulation (He et al., 2004). Most data, including Wnt binding to LRP5/6 and Wnt1-Fz8-LRP6 complex formation in vitro and observations that engineered Fz-LRP5/6 proximity is sufficient to activate β-catenin signaling (Cong et al., 2004Holmen et al., 2005;Tolwinski et al., 2003), support the model that Wnt induces the formation of Fz-LRP5/6 complex (He et al., 2004) (Figure 1). But unambiguous demonstration of this receptor complex in vivo is lacking. It is noteworthy that Wnt3a palmitoylation (at cysteine 77) is important for binding to both Fz and LRP6 (Cong et al., 2004Komekado et al., 2007), explaining in part the importance of this lipid modification

Secreted Wnt antagonists and agonists nihms196288f3

Secreted Wnt antagonists and agonists nihms196288f3

Secreted Wnt antagonists and agonists

Figure 3 Secreted Wnt antagonists and agonists

A particular Wnt may activate β-catenin and/or non-canonical pathways depending on the receptor complement (van Amerongen et al., 2008). Fz function is involved in β-catenin and non-canonical pathways. The Fz-LRP5/6 co-receptor model stipulates that a Wnt-Fz pair capable of recruiting LRP5/6 activates the β-catenin pathway, consistent with the specific requirement of LRP5/6 in Wnt/β-catenin signaling (He et al., 2004). However some evidence suggests that LRP6 antagonizes non-canonical Wnt signaling in vivo, possibly via competing for Wnt ligands (Bryja et al., 2009) or an unknown mechanism (Tahinci et al., 2007). Other Wnt receptors exist such as Ryk and ROR2, which are not required for, but in some cases may antagonize, Wnt/β-catenin signaling (van Amerongen et al., 2008).

Wnt antagonists and agonists

Several secreted protein families antagonize or modulate Wnt/β-catenin signaling (Figure 3). sFRPs (secreted Frizzled related proteins), and WIF (Wnt inhibitory protein) bind to Wnt, and in the case of sFRPs, also to Fz (Figure 3), and thereby function as Wnt antagonists for both β-catenin and non-canonical signaling (Bovolenta et al., 2008). Loss-of-function studies in mice have revealed significant redundancy for the sFRP genes (Satoh et al., 2008). The Wnt-binding property suggests that sFRPs and WIF may also regulate Wnt stability and diffusion/distribution extracellularly beyond just Wnt inhibitors. Some sFRPs have been shown to have Wnt-independent activity such as regulators of extracellular proteinases (Bovolenta et al., 2008).

Two distinct classes of Wnt inhibitors are the Dickkopf (Dkk) family and the Wise/SOST family (Figure 3). Dkk proteins, exemplified by Dkk1, are LRP5/6 ligands/antagonists and are considered specific inhibitors for Wnt/β-catenin signaling. Although two different models for Dkk1 action have been proposed (Mao et al., 2002Semenov et al., 2001), recent biochemical and genetic studies (Ellwanger et al., 2008Semenov et al., 2008Wang et al., 2008) have argued against the model that Dkk1 inhibits Wnt signaling via inducing LRP6 internalization/degradation through transmembrane Kremen (Krm) proteins (Mao et al., 2002). Dkk1 disruption of Wnt-induced Fz-LRP6 complex remains a more likely mechanism (Semenov et al., 2001), with Krm playing a minor modulatory role in specific tissues (Ellwanger et al., 2008). Wise and SOST constitute another family of LRP5/6 ligands/antagonists (Itasaki et al., 2003Li et al., 2005Semenov et al., 2005). Like Dkk1, SOST is able to disrupt Wnt-induced Fz-LRP6 complex in vitro (Semenov et al., 2005). Both Dkk1 and SOST are strongly implicated in human diseases (see below).

Shisa proteins represent a distinct family of Wnt antagonists (Figure 3), which trap Fz proteins in the ER and prevent Fz from reaching the cell surface, thereby inhibiting Wnt signaling cell-autonomously (Yamamoto et al., 2005). Shisa proteins also antagonize FGF (fibroblast growth factor) signaling by trapping FGF receptors in the ER. Other Wnt antagonists with multivalent activities exist. Xenopus Cerberus binds to and inhibits Wnt as well as Nodal and BMP (bone morphogenetic protein) (Piccolo et al., 1999), and IGFBP-4 (Insulin-like growth-factor-binding protein-4) antagonizes Wnt signaling via binding to both Fz and LRP6, in addition to modulating IGF signaling (Zhu et al., 2008).

Norrin and R-spondin (Rspo) proteins are two families of agonists for Wnt/β-catenin signaling (Figure 3). Norrin is a specific ligand for Fz4 and acts through Fz4 and LRP5/6 during retinal vascularization (Xu et al., 2004). Rspo proteins exhibit synergy with Wnt, Fz and LRP6 (Kazanskaya et al., 2004Kim et al., 2005;Nam et al., 2006Wei et al., 2007), and show genetic interaction with LRP6 during embryogenesis (Bell et al., 2008), but their mechanism of action is controversial. Results that Rspo binds to both Fz and LRP6 (Nam et al., 2006), to LRP6 primarily (Wei et al., 2007), or to neither (Kazanskaya et al., 2004) have been reported. Another model suggests that Rspo is a ligand for Krm and antagonizes Dkk/Krm-mediated LRP6 internalization (Binnerts et al., 2007), but this seems unlikely given that Krm1 and Krm2 double knockout mice are viable and do not exhibit Rspo mutant phenotypes, and Rspo activates β-catenin signaling in cells lacking both Krm genes (Bell et al., 2008Ellwanger et al., 2008). Rspo genes are often co-expressed with and depend on Wnt for expression (Kazanskaya et al., 2004), and may represent a means of positive feedback that reinforces Wnt signaling. Mutations in Norrin and Rspo genes cause distinct hereditary diseases (see below).

Wnt signaling

Wnt-off state: β-catenin phosphorylation/degradation by the Axin complex

Cytosolic β-catenin phosphorylation/degradation and its regulation by Wnt are the essence of Wnt signaling (Figure 1). The scaffolding protein Axin uses separate domains to interact with GSK3, CK1α, and β-catenin and coordinates sequential phosphorylation of β-catenin at serine 45 by CK1α and then at threonine 41, serine 37 and serine 33 by GSK3 (Kimelman and Xu, 2006). β-catenin phosphorylation at serine 33 and 37 creates a binding site for the E3 ubiquitin ligase β-Trcp, leading to β-catenin ubiquitination and degradation (Figure 4). Mutations of β-catenin at and surrounding these serine and threonine residues are frequently found in cancers, generating mutant β-catenin that escapes phosphorylation and degradation (Table 1). Axin also contains an RGS (regulator of G protein signaling) domain that interacts with APC, a large multifunctional scaffolding protein that itself binds β-catenin. These core Axin complex components (Kimelman and Xu, 2006) share a common goal of ensuring β-catenin phosphorylation and degradation. Indeed both APC and Axin are tumor suppressor genes, and APC mutations are particularly prevalent in colorectal cancer (Table 1).

Regulation of Axin complex assembly for β-catenin degradation

Figure 4 Regulation of Axin complex assembly for β-catenin degradation

Table 1 Human diseases associated with mutations of the Wnt signaling components

Several aspects of the Axin complex deserve further discussion. (i) In addition to β-catenin, GSK3 and CK1 also phosphorylate Axin and APC, leading to increased association of Axin and APC with β-catenin and thus enhanced β-catenin phosphorylation/degradation (Huang and He, 2008Kimelman and Xu, 2006) (Figure 4). (ii) Two abundant serine/threonine phosphatases, PP1 and PP2A, both of which associate with Axin and/or APC, counteract the action of GSK3 and/or CK1 in the Axin complex. Thus PP1 dephosphorylates Axin and promotes the disassembly of the Axin complex (Luo et al., 2007), whereas PP2A dephosphorylates β-catenin (Su et al., 2008), each resulting in reduced β-catenin degradation (Figure 4). One should note that PP2A may have multiple and opposing roles in the Wnt pathway depending on the particular associated regulatory subunits and substrates (Kimelman and Xu, 2006). (iii) The assembly of the Axin complex appears to be multivalent and robust. In fly embryos that are null for Axin, expression, at physiological levels, of Axin mutants lacking either the APC-, GSK3-, or β-catenin-binding domain restores a significant degree of normal patterning, implying a quasi-functional Axin complex assembly via multivalent interactions; furthermore, some of these Axin deletion mutants can complement each other and restore fly viability, possibly via Axin dimerization or multimerization (Peterson-Nedry et al., 2008). Indeed Axin has multiple potential dimerization domains (Luo et al., 2005) and the Axin DIX domain may form multimeric polymers (Schwarz-Romond et al., 2007a). (iv) Axin concentration is exceedingly low compared to other components in Xenopus oocytes, indicating that Axin is rate limiting for the complex assembly. This feature may ensure that changes in the Axin protein level will not fluctuate the availability of GSK3 (or other components) for non-Wnt functions, thereby further insulating Wnt and other signaling events (Lee et al., 2003). It is unknown, however, whether the drastic difference between the concentration of Axin versus the other components applies universally, and whether different cells employ quantitative differences in the ratio of Axin and other components to shape their unique Wnt response kinetics (such as the speed and level of β-catenin accumulation). Indeed in Drosophila photoreceptors, APC appears to be present at minimal levels such that a 50% reduction alters the graded Wg response (Benchabane et al., 2008).

Other proteins such as WTX (Wilms tumor gene on the X chromosome) may have roles in β-catenin degradation. Loss of WTX and activating β-catenin mutations seem to have non-overlapping occurrence in Wilms tumor (a pediatric kidney cancer) (Rivera et al., 2007). WTX binds to β-catenin, Axin, APC and β-Trcp to promote β-catenin ubiquitination, although its biochemical role remains unknown (Major et al., 2007). Another Axin-binding protein Diversin can facilitate β-catenin degradation via recruiting CK1ε to phosphorylate β-catenin (Schwarz-Romond et al., 2002).

APC function and APC-Axin cross regulation

The biochemical nature of APC has been enigmatic. A recent study suggested that APC protectsβ-catenin from dephosphorylation by PP2A thereby enhancing β-catenin phosphorylation/degradation (Su et al., 2008) (Figure 4), consistent with the observation that Axin overexpression causes β-catenin degradation even in cells lacking APC function (Behrens et al., 1998). Surprisingly APC (upon phosphorylation by CK1/GSK3) and Axin bind to and compete for the same β-catenin interaction interface, leading to a proposal that APC acts as a “ratchet” to remove phosphorylated β-catenin from Axin for ubiquitination and for making Axin available for a further round of β-catenin phosphorylation (Kimelman and Xu, 2006Xing et al., 2003). A different model was proposed based on differential β-catenin binding affinity by unphosphorylated versus phosphorylated APC (Ha et al., 2004). APC has also been shown to promote β-catenin nuclear export and to act as a chromatin-associated suppressor for β-catenin target genes, thus functioning in the nucleus (see below).

Another paradoxical observation is that APC has a positive function in physiological and ectopic Wg/Wnt signaling through the promotion of Axin degradation (Lee et al., 2003Takacs et al., 2008) (Figure 4). One model suggests that this represents a fail-safe mechanism to buffer dramatic β-catenin fluctuations when APC levels vary (Lee et al., 2003). Thus a decrease in the APC level results in higher Axin amounts, compensating for β-catenin degradation. APC-mediated Axin degradation depends on the APC amino terminal domain that is not involved inβ-catenin degradation (Takacs et al., 2008). It is intriguing that colon cancer cells are rarely null for APC but rather retain the amino terminal half, and may have hijacked a part of this fail-safe regulation for tumorigenesis. Conversely Axin can also facilitate APC degradation upon overexpression (Choi et al., 2004), constituting perhaps the other side of the Axin-APC regulation circuit (Figure 4). Mechanisms for Axin and APC degradation, which are proteosome-dependent, have not been characterized.

Wnt-on state

Activation of Wnt receptors

Wnt signaling requires both Fz and LRP6 (or LRP5), likely through a Wnt-induced Fz-LRP6 complex (Figure 1). Wnt-induced LRP6 phosphorylation is a key event in receptor activation (Tamai et al., 2004). LRP6, LRP5 and Arrow each have five reiterated PPPSPxS motifs (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to a heterologous receptor to result in constitutive β-catenin signaling (MacDonald et al., 2008Tamai et al., 2004Zeng et al., 2005). These dually phosphorylated PPPSPxS motifs are docking sites for the Axin complex (Davidson et al., 2005;Tamai et al., 2004Zeng et al., 2005), thereby recruiting Axin to LRP6 upon Wnt stimulation (Mao et al., 2001) (Figure 5).

Models of Wnt receptor activation nihms196288f5

Models of Wnt receptor activation nihms196288f5

Models of Wnt receptor activation


Figure 5 Models of Wnt receptor activation

The kinases responsible for PPPSPxS phosphorylation have been identified unexpectedly as GSK3 and CK1 (Davidson et al., 2005Zeng et al., 2005). Although one study argued that only CK1 phosphorylation is Wnt-induced (Davidson et al., 2005), most available data support that Wnt induces PPPSP phosphorylation (Binnerts et al., 2007Khan et al., 2007Pan et al., 2008bWei et al., 2007), which is carried out by GSK3 and primes xS phosphorylation by CK1, thereby leading to dually induced phosphorylation (Zeng et al., 2005) (Figure 5). Although potential involvement of additional kinases cannot be ruled out, experiments in GSK3α/β null cells indicate that GSK3 accounts for most, if not all, PPPSP phosphorylation (Zeng et al., 2008Zeng et al., 2005). As in β-catenin phosphorylation, Axin-bound GSK3 appears to mediate LRP6 phosphorylation (Zeng et al., 2008). Thus PPPSPxS phosphorylation exhibits a mirror image of β-catenin phosphorylation in sequential order, in priming requirement, and importantly in functionality, but apparently by the same Axin-GSK3 complex (Huang and He, 2008) (Figure 5). This unusual mechanism, using the same kinase complex for both positive and negative regulation, is reminiscent of another morphogenetic pathway, Hedgehog signaling in Drosophila (Price, 2006), and implies a simple view that Wnt signaling regulates the two opposing activities of the Axin-GSK3 complex. One caveat is that GSK3 is genetically defined as a negative regulator of β-catenin signaling. The positive requirement of GSK3 in LRP6 activation is demonstrated when a membrane-tethered GSK3 inhibitory peptide blocks Wnt signaling (Zeng et al., 2008).

Fz function is required for Wnt-induced LRP6 phosphorylation, and forced Fz-LRP6 association is sufficient to trigger LRP6 phosphorylation (Zeng et al., 2008). Fz function is usually linked to Dsh/Dvl (Wallingford and Habas, 2005), a cytoplasmic scaffolding protein that may directly interact with Fz (Wong et al., 2003). Indeed Fz-Dvl interaction and Dvl function are critical for Wnt-induced LRP6 phosphorylation (Bilic et al., 2007Zeng et al., 2008). As Dvl interacts with Axin (Wallingford and Habas, 2005), and is required for Axin recruitment to the plasma membrane during Wg signaling (Cliffe et al., 2003) or in Fz overexpression (Zeng et al., 2008), one model stipulates that Fz-Dvl recruitment of the Axin-GSK3 complex initiates LRP6 phosphorylation by GSK3 (Zeng et al., 2008) (Figure 5).

Several features of Wnt receptor activation deserve further discussion. (i) The observation that Axin is required for LRP6 phosphorylation, and phosphorylated LRP6 in turn recruits Axin suggests a positive feed-forward loop, potentially amplifying and ensuring the phosphorylation of all five PPPSPxS motifs (Figure 5). Indeed the phosphorylation of these motifs relies on the presence of one another, and LRP6 activity is particularly sensitive to the PPPSPxS copy number (MacDonald et al., 2008Wolf et al., 2008). This may explain the distinct roles of Fz and LRP6/Arrow in the “initiation” (which requires both Fz and Arrow) and “amplification” (which requires Arrow only) during Wg signaling (Baig-Lewis et al., 2007) (Figure 5a). (ii) Wnt-induced clustering of Fz-LRP6 receptor has been reported that critically depend on Dvl, Axin and GSK3 for formation (see below) (Bilic et al., 2007Schwarz-Romond et al., 2007a). Although unambiguous evidence for such aggregation under physiological conditions without overexpression remains to be shown, this “signalsome” model (Figure 5b) and the “initiation-amplification” model (Figure 5a) together provide a spatial and temporal framework for understanding Wnt receptor activation. (iii) Wnt also induces LRP6 phosphorylation by CK1γ outside the PPPSPxS motifs, in particular in a conserved S/T cluster amino-terminal to the first PPPSPxS motif (Davidson et al., 2005). This region upon phosphorylation binds to GSK3 (Piao et al., 2008), potentially accounting for observed LRP6-GSK3 interaction (Mi et al., 2006Zeng et al., 2005). The significance of this S/T cluster to LRP6 function has not been investigated in the intact receptor, but these results imply multiple interaction interfaces among LRP6, Axin and GSK3. (iv) Wnt may also “activate” Fz, which is structurally related to G-protein coupled receptors (GPCRs). Some genetic and pharmacological evidence suggests that trimeric G proteins, specifically the Gαo and Gαq, are required downstream of Fz and probably upstream of Dvl in Wnt/β-catenin signaling (Katanaev et al., 2005Liu et al., 2001Liu et al., 2005). Whether G proteins are involved in Wnt/Fz/Dvl-regulated LRP6 phosphorylation is unknown.

Dvl is involved in Wnt/β-catenin and other Wnt/Fz-dependent pathways and has numerous putative binding partners (Wallingford and Habas, 2005). For example CK1ε (or CK1δ) binds to Dvl and is a potent activator of β-catenin signaling, possibly via phosphorylating Dvl, LRP6 and/or the Axin complex (Price, 2006) (Figure 5). PP2A also associates with Dvl but has a positive or negative influence on Wnt signaling depending on the associated regulatory subunit (Kimelman and Xu, 2006). In addition Dvl is subjected to proteasomal degradation via distinct ubiquitination pathways (Angers et al., 2006Simons et al., 2005). Some of these Dvl regulation events have been suggested to switch Dvl between β-catenin and non-canonical pathways. Despite these progresses, the mechanism by which Dvl acts in Wnt/β-catenin signaling remains enigmatic. Two recent findings suggest potential new insights. (i) Polymerization/aggregation of Dvl (and Axin). Fz-Dvl and Dvl-Axin interactions are relatively weak (Schwarz-Romond et al., 2007bWong et al., 2003). However Dvl and Axin each harbor a homologous DIX domain that exhibit dynamic polymerization (Schwarz-Romond et al., 2007a). This unusual property is proposed to allow Dvl and Axin to form large aggregates that facilitate weak but dynamic protein interactions (Figure 5b). Indeed Wnt-induced receptor clustering requires an intact Dvl DIX domain (Bilic et al., 2007Schwarz-Romond et al., 2007a). It is unclear whether Wnt regulates DIX-dependent polymerization, and perhaps in a related manner, Fz-Dvl or Dvl-Axin interaction. (ii) Dvl stimulation of phosphatidylinositol 4,5-bisphosphate [PtdIns (4,5)P2 or PIP2] production by sequential actions of phosphatidylinositol 4-kinase type II (PI4KIIα) and phosphatidylinositol-4-phosphate 5-kinase type I (PIP5KI) (Pan et al., 2008b). Wnt induces Dvl, via the DIX domain, to bind to and activate PIP5K, and the resulting PIP2 production is suggested to promote LRP6 clustering and phosphorylation, although the underlying mechanism remains unclear (Figure 5c). Given that PIP2 has pleiotropic functions in cells including receptor endocytosis (see below), other potential mechanisms for PIP2 in LRP6 phosphorylation remain to be explored. Nonetheless Dvl DIX polymerization and stimulation of PIP2 may act in concert to ensure LRP6 clustering/phosphorylation/activation.

Other regulatory events at or proximal to Wnt receptors

A cytoplasmic protein in vertebrates, referred to as Caprin-2, binds to LRP6 and facilitates LRP6 phosphorylation by GSK3 (Ding et al., 2008). Caprin-2 has an oligomerization domain that may enhance LRP6 aggregation, and Caprin-2 additionally may also associate with both GSK3 and Axin and promote LRP6-Axin-GSK3 complex formation (Ding et al., 2008). Besides the requirement of Dvl, recruitment of Axin to the receptor complex may involve a giant protein (600 kD), Macf1 (microtubule actin cross-linking factor 1) (Chen et al., 2006). Macf1 is a member of the spectraplakin family of proteins that link the cytoskeleton to junctional proteins. Defective gastrulation in Macf1−/− mouse embryos phenotypically resembles Lrp5/6−/− double knockout mutants. On Wnt stimulation Macf1 associates with the Axin complex (including APC) in the cytosol and with LRP6 and the Axin complex (but not APC) in the membrane fraction (Chen et al., 2006), and may shuttle Axin to LRP6 (Figure 5). This Macf1 function may be vertebrate-specific as Drosophila Macf1 (shortstop) mutants do not exhibit wg-related phenotypes. …

Inhibition of β-catenin phosphorylation

How receptor activation leads to inhibition of β-catenin phosphorylation remains uncertain, and available data suggest possible parallel mechanisms. In the LRP6-centric view, as constitutively activated forms of LRP6 fully activate β-catenin signaling in an apparently Fz and Dvl-independent manner (He et al., 2004), LRP6 represents the key output whereas Fz and Dvl act upstream to control LRP6 activation. On the other hand, Dsh overexpression in Drosophila or recombinant Dvl in Xenopus egg extracts can activate β-catenin signaling presumably in the absence of Arrow/LRP6 (Salic et al., 2000Wehrli et al., 2000), and so does a GPCR-Fz chimeric protein in response to the GPCR ligand (Liu et al., 2001). These results argue that Fz/Dvl may activate β-catenin signaling independent of LRP6. The fact that nematodes have a related Wnt/β-catenin pathway (Kimble 2009) but have no LRP6 homolog may be consistent with this notion. Perhaps inDrosophila and vertebrates Wnt signaling components exist under sub-optimal levels and the two parallel branches need to operate together to counteract efficient β-catenin phosphorylation/degradation, whereas over-activation of either branch is sufficient to stabilize β-catenin. …

β-catenin nuclear function

β-catenin nuclear/cytoplasmic shuttling and retention

β-catenin stabilization results in its higher nuclear levels, but how β-catenin is shuttled to and retained in the nucleus is not well understood (Henderson and Fagotto, 2002Stadeli et al., 2006). Earlier studies suggested that β-catenin enters the nucleus in an NLS (nuclear localization signal)- and importin-independent fashion by interacting directly with nuclear pore proteins (Henderson and Fagotto, 2002). β-catenin also exits the nucleus via export involving APC (Henderson and Fagotto, 2002), Axin (Cong and Varmus, 2004), and RanBP3 (Ran binding protein 3), which binds to β-catenin in a Ran-GTP dependent manner (Hendriksen et al., 2005). Live cell imaging suggests that while Axin and APC can enrich β-catenin in the cytoplasm and TCF and β-catenin co-activators (BCL9 and Pygopus, see below) increase nuclear β-catenin, they do not accelerate the export or import rate of β-catenin, thereby arguing for their roles in β-catenin retention rather than shuttling (Krieghoff et al., 2006). Thus β-catenin nuclear and cytoplasmic partitioning is likely the dynamic sum of both shuttling and retention between the two compartments via multiple mechanisms. ….


The TCF/LEF family of DNA-bound transcription factors is the main partner for β-catenin in gene regulation (Arce et al., 2006Hoppler and Kavanagh, 2007). TCF represses gene expression by interacting with the repressor Groucho (TLE1 in human), which promotes histone deacetylation and chromatin compaction; Wnt-induced β-catenin stabilization and nuclear accumulation leads TCF to complex with β-catenin, which appears to displace Groucho (Daniels and Weis, 2005) and recruits other co-activators for gene activation (Figure 1). While a single TCF gene is found in Drosophila and worm, four TCF genes, TCF1, LEF1, TCF3 and TCF4, exist in mammals. Alternative splicing and promoter usage produce a large number of TCF variants with distinct properties (Arce et al., 2006Hoppler and Kavanagh, 2007). TCF proteins are HMG (high mobility group) DNA-binding factors, and upon binding to a DNA consensus sequence referred to as the Wnt responsive element (WRE), CCTTTGWW (W represents either T or A), they cause significant DNA bending that may alter local chromatin structure. A genome-wide analysis in colon cancer cells suggests that TCF4/β-catenin target genes are frequently “decorated” with multiple WREs, most of which are located at large distances from transcription start sites (Hatzis et al., 2008). Some TCF1 and TCF4 splicing variants harbor a second DNA-binding domain called C-clamp, which recognizes an additional GC element downstream of the typical WRE, allowing regulation of different sets of target genes (Atcha et al., 2007). These similarities and differences, combined with overlapping and unique expression patterns, underlie in part distinct and sometimes redundant functions of vertebrate/mammalian TCF genes. ….

Three major strategies exist to regulate TCF/β-catenin transcription. (i) Alternative promoter usage in TCF-1 and LEF-1 genes produces dnTCF-1/dnLEF-1, which lack the amino-terminal β-catenin-binding domain and thus act as the endogenous dominant negative TCF/LEF (Arce et al., 2006Hoppler and Kavanagh, 2007). Indeed the TCF-1 locus acts as an intestinal tumor suppressor primarily due to the production of dnTCF-1, which antagonizes TCF-4 in stem cell renewal. (ii) Nuclear antagonists Chibby and ICAT bind to β-catenin and disrupt β-catenin/TCF and β-catenin/co-activator interactions and promote β-catenin nuclear export (Li et al., 2008Tago et al., 2000). Besides these devoted inhibitors, many DNA-binding transcription factors interact with β-catenin or TCF and antagonize TCF/β-catenin-dependent transcription (Supplemental Table 1). For example, KLF4 inhibition of β-catenin transcriptional activation is important for intestinal homeostasis and tumor suppression (Zhang et al., 2006). (iii) Post-translational modifications of TCF/LEF exist including phosphorylation, acetylation, sumoylation, and ubiquitination/degradation (Arce et al., 2006Hoppler and Kavanagh, 2007). For instance, TCF-3 phosphorylation by CK1ε and LEF-1 phosphorylation by CK2 enhances their binding to β-catenin and diminishes LEF-1 binding to Groucho/TLE, whereas LEF-1 and TCF-4 phosphorylation by NLK (Nemo-like kinase) leads to less LEF/TCF/β-catenin complex binding to DNA and to LEF-1/TCF-4 degradation. LEF-1 and TCF-4 sumoylation (by the SUMO ligase PIASy) represses LEF-1 activity by targeting it to nuclear bodies but enhances TCF-4/β-catenin transcription, while CBP-mediated acetylation of TCF results in decreased TCF/β-catenin-binding in Drosophila and increased TCF nuclear retention in nematodes, both leading to transcriptional repression. These diverse modifications are often specific to individual TCF/LEF proteins, conferring differential regulation.

β-catenin associated co-activators

A plethora of β-catenin associated co-activators have been identified. These multi-protein complexes include BCL9 and Pygopus (Pygo), Mediator (for transcription initiation), p300/CBP and TRRAP/TIP60 histone acetyltransferases (HATs), MLL1/2 histone methyltransferases (HMTs), the SWI/SNF family of ATPases for chromatin remodeling, and the PAF1 complex for transcription elongation and histone modifications (Mosimann et al., 2009Willert and Jones, 2006) (Figure 6). While the central Arm-repeats of β-catenin associate with TCF, and the amino-terminal Arm-repeat binds to BCL9, most of the co-activator complexes interact with the β-catenin carboxyl terminal portion (Figure 6), creating a dazzling interplay between β-catenin and the transcriptional apparatus and the chromatin. Indeed TCF/β-catenin binding to WREs leads to histone acetylation in a CBP-dependent manner over a significant genomic distance (30 kb), suggesting that local TCF/β-catenin recruitment results in widespread chromatin modifications (Parker et al., 2008). …

Nuclear TCF.β-catenin co-activator complexes  nihms196288f6

Nuclear TCF.β-catenin co-activator complexes nihms196288f6

Nuclear TCF/β-catenin co-activator complexes


Figure 6  Nuclear TCF/β-catenin co-activator complexes


Unlike most co-activators that have general roles in transcription, BCL9 and Pygo in Drosophila are specifically required for β-catenin-dependent transcription and their biochemical functions proposed provide a glimpse of the complexity of TCF/β-catenin-coactivator interactions (Mosimann et al., 2009). (i) BCL9 and Pygo function as a “chain of activators” (Hoffmans et al., 2005). β-catenin binding to BCL9 recruits Pygo, which also interacts with Mediator (Carrera et al., 2008) (Figure 6); (ii) Pygo is constitutively nuclear and may have a role in recruiting/retaining BCL9/β-catenin in the nucleus upon Wg/Wnt signaling (Brembeck et al., 2004Townsley et al., 2004); (iii) Pygo also co-occupies chromatin loci with and via TCF in the absence of Wg signaling (despite a lack of direct TCF-Pygo interaction), and may help capture BCL9/β-catenin for TCF at the onset of Wg signaling (de la Roche and Bienz, 2007); (iv) Pygo has a PHD (plant homology domain) that binds preferentially to dimethylated H3K4 upon interaction with BCL9 (Fiedler et al., 2008). This “histone code” recognition leads to the speculation that Pygo/BCL9 act during the transition from gene silencing to Wnt-induced transcription by participating in histone methylation changes. Alternatively Pygo/BCL9-binding to dimethylated H3K4 may provide a separate β-catenin anchor on chromatin, thereby freeing TCF for interaction with Groucho to pause/terminate transcription (Mosimann et al., 2009); (v) Pygo function is not required when Groucho activity is absent, suggesting that Pygo acts as an anti-repressor (Mieszczanek et al., 2008). Therefore either a single biochemical mechanism of Pygo underlies these diverse observations, or multiple functional properties of Pygo participate in β-catenin signaling. …

Nuclear functions of “cytoplasmic” Wnt signaling components

APC also acts directly on chromatin/WREs to antagonize β-catenin-mediated gene activation via promoting the exchange of co-activators with co-repressors in a stepwise and oscillating manner, as such exchange does not occur in APC mutant cancer cells (Sierra et al., 2006). How APC is recruited to chromatin is a mystery but is unlikely due to β-catenin/TCF, because APC and TCF bind to β-catenin in a mutually exclusive manner. GSK3 and β-Trcp also appear to be associated with the WRE in a cyclic fashion that synchronizes with APC but is opposite to that of β-catenin/co-activators, suggesting that they may have negative roles in TCF/β-catenin-mediated transcription (Sierra et al., 2006). Some studies have also suggested that Dvl is observed in the nucleus (Itoh et al., 2005Torres and Nelson, 2000) and that nuclear Dvl is a component of the TCF/β-catenin complex and facilitates TCF/β-catenin interaction in conjunction with the c-Jun transcription factor (Gan et al., 2008). …

β-catenin-mediated repression and other transcriptional events

Wnt signaling, via the TCF/β-catenin complex, also represses transcription. Note that this is distinct from TCF-mediated repression in the absence of β-catenin. One mechanism is competitive repression, through which TCF/β-catenin displaces or inhibits other DNA-binding transcription activators (Kahler and Westendorf, 2003Piepenburg et al., 2000). Another mechanism is direct repression via TCF/β-catenin binding to the canonical WREs by recruiting co-repressors (Jamora et al., 2003Theisen et al., 2007). A third mechanism is revealed by a novel TCF binding element, AGAWAW, which specifically mediates TCF/β-catenin repression in Drosophila (Blauwkamp et al., 2008). There is evidence that β-catenin is capable of recruiting co-repressors including Groucho/TLE and histone deacetylases (Olson et al., 2006), but the mechanism by which β-catenin recruits co-activators versus co-repressors is unknown. The involvement of co-factors (Theisen et al., 2007) or distinct TCF/β-catenin configurations offers potential explanations. A less understood aspect of β-catenin signaling is that many DNA-binding transcription factors, in addition to TCF/LEF, interact with β-catenin to either activate or repress transcription (Supplemental Table 1b). These β-catenin partners in principle expand significantly the gene expression programs that are regulated by Wnt/β-catenin signaling, but further substantiation of their roles in mediating Wnt signaling is required.

Wnt/β-catenin target genes and Wnt pathway self-regulation

As Wnt/β-catenin signaling regulates proliferation, fate specification and differentiation in numerous developmental stages and adult tissue homeostasis, Wnt target genes are diverse (Vlad et al., 2008) and cell- and context-specific (Logan and Nusse, 2004). An emerging feature is that Wnt signaling components including Fz, LRP6, Axin2, TCF/LEF, Naked (a Dvl antagonist), Dkk1, and Rspo, are often regulated positively or negatively by TCF/β-catenin (Chamorro et al., 2005Kazanskaya et al., 2004Khan et al., 2007Logan and Nusse, 2004). Wnt induction of Axin2, Dkk1 and Naked and suppression of Fz and LRP6 constitute negative feedback loops that dampen Wnt signaling, and the suppression of Fz and LRP6 also enhances Wg/Wnt gradient formation over longer distances (Logan and Nusse, 2004). On the contrary, Wnt induction of Rspo and TCF/LEF genes constitute positive feed-forward circuits that reinforce Wnt signaling, a feature that has been exploited during colon carcinogenesis (Arce et al., 2006Hoppler and Kavanagh, 2007). These various Wnt pathway self-regulatory loops are mostly utilized in a cell-specific manner, affording additional complexity in the control of amplitude and duration of Wnt responses. …

Wnt/β-catenin signaling in diseases and potential therapeutics

Give the critical roles of Wnt/β-catenin signaling in development and homeostasis it is no surprise that mutations of the Wnt pathway components are associated with many hereditary disorders, cancer and other diseases (Table 1). …

LRP5 activity correlates with bone mass likely via regulation of osteoblast (bone forming cell) proliferation, whereas SOST and DKK1, which are specifically expressed in osteocytes, negatively regulates bone mass by antagonizing LRP5. …

Association of deregulated Wnt/β-catenin signaling with cancer has been well documented, particularly with colorectal cancer (Polakis, 2007) (Table 1). Constitutively activated β-catenin signaling, due to APC deficiency or β-catenin mutations that prevent its degradation, leads to excessive stem cell renewal/proliferation that predisposes cells to tumorigenesis. Indeed APC deletion or β-catenin activation in stem cells is essential for intestinal neoplasia (Fuchs, 2009). Blocking β-catenin signaling for cancer treatment has thus generated significant interests. Indeed the beneficial effect of non-steroidal anti-inflammatory drugs (NSAIDS) in colorectal cancer prevention and therapy has been attributed partially to the perturbation of TCF/β-catenin signaling through the ability of NSAIDS to inhibit Prostaglandin E2 production, which enhances TCF/β-catenin-dependent transcription (Castellone et al., 2005Shao et al., 2005). Small molecules that disrupt TCF/β-catenin (Lepourcelet et al., 2004) or β-catenin/co-activator (CBP) interaction (Emami et al., 2004) and thereby block TCF/β-catenin signaling have been described. The task of disrupting TCF/β-catenin interaction specifically, however, is a difficult one since β-catenin interacts with TCF and other binding partners such as APC, Axin and E-cadherin via the same or overlapping interface (Barker and Clevers, 2006). Another potential therapeutic target is the kinase CDK8, which, as a Mediator subunit, is often amplified in and is required for β-catenin-dependent transcription and proliferation of colon cancer cells (Firestein et al., 2008Morris et al., 2008). A new class of small molecules that inhibits β-catenin signaling has recently be identified (Chen et al., 2009), which via an unknown mechanism stabilizes the Axin protein, thereby promoting β-catenin degradation even in cancer cells that lack APC function. As discussed above, since Axin protein levels are the rate-limiting step for β-catenin degradation, manipulation of Axin stabilization represents a promising therapeutic strategy.

Many cancers that do not harbor mutations in the Wnt pathway nonetheless rely on autocrine Wnt signaling for proliferation or survival (Barker and Clevers, 2006). In fact APC mutant colon cancer cells maintain their dependence on Wnt and epigenetically silence the expression of secreted Wnt antagonists (He et al., 2005;Suzuki et al., 2004). Therefore targeting Wnt signaling upstream of TCF/β-catenin is also an important therapeutic option. Reagents against Wnt proteins such as antibodies (He et al., 2005) or a secreted Fz extracellular domain (DeAlmeida et al., 2007), which act outside the cancer cells to block Wnt-receptor interaction, show promise in certain experimental settings, as do small molecule and peptide inhibitors that antagonize Fz-Dvl interaction (Shan et al., 2005Zhang et al., 2009). Small molecules have also been identified that inhibit Porcupine and thus prevent Wnt lipidation and secretion (Chen et al., 2009). We will likely see additional molecular and chemical agents that can interfere with different steps of Wnt/β-catenin signaling, whose complexity presents many potential therapeutic targets. The challenge will be ensuring that these agents target cancer cells without damaging normal tissue homeostasis.

Since the discovery of the Wnt-1 gene 27 years ago (Nusse and Varmus, 1982), Wnt/β-catenin signaling has cemented its role as a key regulatory system in biology. Studies of different animal models and human diseases have established a complex Wnt signaling network far beyond a linear pathway, with many components having multiple distinct roles and acting in different cellular compartments, and many modulators feeding into and cross-regulating within this network. The patterns of dynamic and kinetic protein phosphorylation/modification and complex assembly/disassembly are beginning to emerge. Challenges and excitement both lie ahead. (i) Novel regulators will likely continue to be identified using classical genetic, molecular, modern genomic and proteomic approaches. (ii) New analytical and imaging technologies should enable us to dissect and visualize the dynamic signaling events in vivo and to shed light on the cell biological aspects of Wnt signaling, including where, when and how signaling occurs inside the cell. (iii) Although we have obtained significant structural information on individual domains and protein interaction interfaces, atomic structures of protein complexes such as the Axin complex and ligand-receptor complexes remain daunting challenges. (iv) Additional specific small molecular inhibitors or activators with defined targets and mechanisms would provide not only leads for therapeutics but also research tools to manipulate the Wnt pathway in precise temporal and spatial manners. (v) Integration of vast amounts of information into quantitative models will allow us to predict the behavior and to study the robustness and evolvability of Wnt signaling in various biological contexts. (vi) The Wnt responsive transcriptome remains a gold mine for digging into Wnt-regulated biology. Unfolding examples include Wnt regulation of intestinal and hair follicle development/homeostasis, which has provided significant insights into stem cell biology and cancer pathogenesis (Clevers, 2006Fuchs, 2009). As β-catenin is a co-activator for other transcription factors in addition to TCF/LEF, comparative analyses of Wnt responsive transcription programs that depend on TCF/LEF versus others will likely uncover further complexity of Wnt-regulated gene expression. (vii) β-catenin and APC are also key components in the E-cadherin cell adhesion complex and the microtubule network, but how Wnt/β-catenin signaling interacts with these cellular structures remains poorly understood. In addition, the involvement of the primary cilium, a centrosome- and microtubule-based protrusive organelle in vertebrate cells, in Wnt/β-catenin versus non-canonical Wnt signaling remains an intriguing but debated topic (Gerdes et al., 2009).

Since the discovery of the Wnt-1 gene 27 years ago (Nusse and Varmus, 1982), Wnt/β-catenin signaling has cemented its role as a key regulatory system in biology. Studies of different animal models and human diseases have established a complex Wnt signaling network far beyond a linear pathway, with many components having multiple distinct roles and acting in different cellular compartments, and many modulators feeding into and cross-regulating within this network. The patterns of dynamic and kinetic protein phosphorylation/modification and complex assembly/disassembly are beginning to emerge. Challenges and excitement both lie ahead. (i) Novel regulators will likely continue to be identified using classical genetic, molecular, modern genomic and proteomic approaches. (ii) New analytical and imaging technologies should enable us to dissect and visualize the dynamic signaling events in vivo and to shed light on the cell biological aspects of Wnt signaling, including where, when and how signaling occurs inside the cell. (iii) Although we have obtained significant structural information on individual domains and protein interaction interfaces, atomic structures of protein complexes such as the Axin complex and ligand-receptor complexes remain daunting challenges. (iv) Additional specific small molecular inhibitors or activators with defined targets and mechanisms would provide not only leads for therapeutics but also research tools to manipulate the Wnt pathway in precise temporal and spatial manners. (v) Integration of vast amounts of information into quantitative models will allow us to predict the behavior and to study the robustness and evolvability of Wnt signaling in various biological contexts. (vi) The Wnt responsive transcriptome remains a gold mine for digging into Wnt-regulated biology. Unfolding examples include Wnt regulation of intestinal and hair follicle development/homeostasis, which has provided significant insights into stem cell biology and cancer pathogenesis (Clevers, 2006Fuchs, 2009). As β-catenin is a co-activator for other transcription factors in addition to TCF/LEF, comparative analyses of Wnt responsive transcription programs that depend on TCF/LEF versus others will likely uncover further complexity of Wnt-regulated gene expression. (vii) β-catenin and APC are also key components in the E-cadherin cell adhesion complex and the microtubule network, but how Wnt/β-catenin signaling interacts with these cellular structures remains poorly understood. In addition, the involvement of the primary cilium, a centrosome- and microtubule-based protrusive organelle in vertebrate cells, in Wnt/β-catenin versus non-canonical Wnt signaling remains an intriguing but debated topic (Gerdes et al., 2009).

Finally the study of Wnt signaling in human diseases, and in stem cell biology and regeneration holds promises for translational medicine. In addition to cancer and osteoporosis, both of which will likely see Wnt signaling-based therapeutics moving into clinical trials or even clinics in the near future, potential links between neurological diseases (De Ferrari and Moon, 2006) and a Schizophrenia susceptibility gene product (Mao et al., 2009) to Wnt/β-catenin signaling offer new hopes for the treatment of neurological and psychiatric disorders. Manipulation of Wnt signaling for stem cell regulation also offers exciting opportunities for regenerative medicine (Clevers, 2006Fuchs, 2009Goessling et al., 2009Willert et al., 2003). A better understanding of Wnt/β-catenin signaling will have broad impact on biology and medicine.

7.10.6 Wnt.β-Catenin Signaling. Turning the Switch

Cadigan KM1.
Dev Cell. 2008 Mar; 14(3):322-3

The regulation of many targets of the Wnt/β-catenin signaling pathway is thought to occur through a transcriptional switch that is achieved by β-catenin binding to TCF transcription factors. Recent work indicates that β-catenin’s intrinsic affinity for TCF is not sufficient for the switch to occur.

The Wnt/β-catenin signaling pathway plays many crucial roles in specifying cell fates during animal development and in regenerating adult tissues. In addition, this pathway is linked to several pathological states, most notably colorectal cancer. Many of the transcriptional responses to Wnt/β-catenin signaling are mediated by the TCF/LEF-1 (TCF) family of transcription factors. Several TCFs are known to repress Wnt targets in the absence of signaling, but upon pathway activation, β-catenin enters the nucleus and binds to TCF on the target chromatin, creating a transcriptional activation complex. Is β-catenin’s intrinsic affinity for TCF sufficient to switch TCF from the repression to the activation state? Two recent papers shed some light on this question. One reports that two previously characterized co-repressor subunits bind to β-catenin and are required to stabilize the β-catenin-TCF interaction. The other suggests that this interaction may be regulated by ubiquitination of APC, a well-known negative regulator of the Wnt/β-catenin pathway.

The first report from Li and Wang (2008) concerns Transducin β-like protein 1 (TBL1) and TBL1-related protein (TBLR1). These proteins are components of the SMRT-nuclear receptor corepressor (N-CoR) complex, where they have been shown to recruit E3 ubiquitin ligases to facilitate the replacement of corepressors with coactivators (Perissi et al., 2004). Similarly, the Drosophila homolog of TBL1, known as Ebi, facilitates proteosomal degradation of the fly N-CoR homolog SMRTER ( Tsuda et al., 2002). In addition, TBL1 binds to the E3 ubiquitin ligase components Siah-1 and Skp1 to promote β-catenin degradation ( Matsuzawa and Reed, 2001). Despite the extensive connections between TBL1, TBLR1, and proteosomal degradation, Li and Wang (2008) found no evidence for these proteins influencing β-catenin turnover in their system. In addition, the proteosome does not appear to contribute to the function of TBL1 and TBLR1 in promoting Wnt/β-catenin signaling.

Using siRNA, Li and Wang found that TBL1 and TBLR1 are required for activation of several targets by Wnt signaling in cell culture. Both proteins interact with β-catenin in coimmunoprecipitation assays. When TBL1 or TBLR1 are depleted, the pathway still promotes nuclear accumulation of β-catenin, but its recruitment to Wnt response element (WRE) chromatin is dramatically reduced. Conversely, TBL1 and TBLR1 are recruited to several WREs in a Wnt- and β-catenin-dependent manner. Thus, binding of β-catenin, TBL1, and TBLR1 to WREs is mutally dependent. Interestingly, TBL1 (but not TBLR1) can be immunoprecipitated by TCF4, and TBL1 is present at some WREs even in the absence of Wnt stimulation. This suggests a model where interactions between TBL1, TCFs, and β-catenin reinforce the complex on WREs, which is required for subsequent recruitment of transcriptional coactivators necessary to activate target gene expression (see Figure 1).



Role of TBL1-TBLR1 and Trabid in TCF-β-Catenin Gene Regulation


Figure 1. Speculative Model on the Role of TBL1-TBLR1 and Trabid in TCF-β-Catenin Gene Regulation

In the absence of signaling (top panel), a TCF-corepressor complex silences target gene expression. When Wnt signaling causes nuclear accumulation of β-catenin (bottom panel), TBL1 and TBLR1 help recruit β-catenin to TCF at target loci, which nucleates a complex of transcriptional coactivators. APC can inhibit the TCF-β-catenin complex, and Trabid’s positive role in the pathway can be explained by its ability to regulate the ubiquitination state of APC.

This report extends the importance of TBL1 and TBLR1 in Wnt/β-catenin gene regulation in two important ways. First, the key findings were reproduced in Drosophila cell culture. Second, the authors demonstrate that depletion of TBL1 or TBLR1 greatly reduced activation of Wnt targets in a well-characterized colorectal cell line lacking functional APC. This decrease in target gene activation had a striking effect on the ability of these cells to grow on soft agar. In addition, the invasive nature of head and squamous cell carcinoma cells transfected with β-catenin was greatly curtailed by TBL1 or TBLR1 knockdown, as was the growth of these cells into tumors in nude mice. These results clearly demonstrate both the evolutionary conservation of these factors in the pathway and suggest that strategies to interfere with their function might have great therapeutic value.

While most reports (and reviews) focus on the TCF transcriptional switch from the “OFF” to the “ON” state, it is also interesting to consider how the switch works in reverse. For example, a colorectal cell line lacking functional APC can be stably transfected with an inducible full-length APC gene. Without induction, β-catenin is bound to WREs and Wnt target expression is high. Upon induction of APC, Jones and coworkers found that β-catenin and coactivators are rapidly replaced by corepressors at the WRE (Sierra et al., 2006). Interestingly, APC transiently occupies the WRE during this switch. A recent report from Bienz and coworkers (Tran et al., 2008) suggests that ubiquitination of APC may influence its ability to regulate the TCF-β-catenin complex.

This group identified an APC-interacting protein they call Trabid, which contains three tandem Npl4 zinc (NZF) fingers and an ovarian tumor (OTU) domain. They demonstrated that the OTU domain contains a deubiquitylating (DUB) activity that shows marked preference for K63-linked ubiquitin chains. When Trabid is depleted from cells by siRNA, activation of several Wnt targets is reduced, and rescue experiments indicate that both the NZF and OTU domains are required for Trabid’s positive role in Wnt signaling. Epitasis analysis indicates that Trabid is required downstream of β-catenin stabilization but is dispensible for TCF fusion proteins containing transactivation domains. This suggests that Trabid may influence the formation or dynamics of TCF-β-catenin complexes.

7.10.7 Wnt–β-catenin signaling

Tetsu Akiyama
Cyokine & GF Rev Dec 1, 2000; 11(4):273–282

The Wnt/Wingless signaling transduction pathway plays an important role in both embryonic development and tumorigenesis. β-Catenin, a key component of the Wnt signaling pathway, interacts with the TCF/LEF family of transcription factors and activates transcription of Wnt target genes. Recent studies have revealed that a number of proteins such as, the tumor suppressor APC and Axin are involved in the regulation of the Wnt signaling pathway. Furthermore, mutations in APC or β-catenin have been found to be responsible for the genesis of human cancers.

7.10.8 Extracellular modulators of Wnt signaling

Boudin E1Fijalkowski IPiters EVan Hul W.
Semin Arthritis Rheum. 2013 Oct; 43(2):220-40

Objectives: The Wnt signaling pathway is a key pathway in various processes, including bone metabolism. In this review, current knowledge of all extracellular modulators of the canonical Wnt signaling in bone metabolism is summarized and discussed. Methods: The PubMed database was searched using the following keywords: canonical Wnt signaling, β-catenin bone metabolism, BMD, osteoblast, osteoporosis, Wnt, LRPs, Frizzleds, sFRPs, sclerostin or SOST, dickkopfs, Wif1, R-spondins, glypicans, SOST-dc1 and kremen, all separately as well as in different combinations.
Results: Canonical Wnt signaling is considered to be one of the major pathways regulating bone formation. Consequently, a large number of studies were performed to elucidate the role of numerous proteins in canonical Wnt signaling and bone metabolism. These studies led to the identification of novel modulators of the pathway like the R-spondin and glypican protein families. Furthermore novel insights are gained in the regulatory role of the different Wnt proteins. Finally, due to its function in bone formation, the pathway is an interesting target for the development of therapeutics for osteoporosis and other bone diseases. In this review, we discuss the promising results of the Wnt modulators sclerostin, Dkk1 and sFRP1 as targets for osteoporosis treatment.
Conclusion: The increasing number of studies into the exact function of all proteins in the canonical Wnt pathway in general and in bone metabolism already led to novel insights in the regulation of the canonical Wnt pathway. In this review we covered the current knowledge of all extracellular modulators of canonical Wnt signaling.

Fig 1. Activators and inhibitors of the Wnt/b-catenin signaling pathway.
(a) Lipid-modified Wnt protein (green; palmitoleoyl group is shown in red) binds to Frizzled CRD, LRP6 b-propellers 1–2 and/or 3–4, and triggers downstream signaling. CRD of Wnt receptor Frizzled8
is shown in light blue, four b propellers of  co-receptor LRP6 are shown in dark blue. Hinge region between b-propellers 1–2 and 3–4 is shown as a blue dot. Dimeric signaling activator Norrin (monomers
shown in magenta and grey) binds specifically to Frizzled4 (grey) and LRP6 b-propellers 1–2. Dotted lines represent interactions between molecules where crystal structures of the complexes are absent.
(b) Extracellular inhibitors bind to Wnt co-receptor LRP6 or Wnt and prevent them from triggering signalling. Both Sclerostin and Dickkopf  (Dkk) contain an Asn-X-Ile motif (peptide shown as
connected yellow spheres) recognized by LRP6 b-propeller 1.  The C-terminal domain of Dkk1 (red) binds to LRP6 b-propeller 3.  WIF1 (pink; WIF1-bound DPPC, light blue) and secreted Frizzled
related protein 3 (sFRP3 CRD, teal) prevent signaling by binding to Wnts. WIF1 binds to HS chains of HSPGs (grey). Sclerostin (as well as other activators and inhibitors) bind to HS-mimic, heparin.
Signaling inhibitor 5T4/Wnt-activated inhibitory factor 1 (WAIF1, purple) acts via unknown binding partners.

Fig 2. Atomic details of Wnt recognition and signaling inhibition.
(a) Zoom-in view of the palmitoleoyl binding site in the CRD of Frizzled8. Molecules are colored as in Figure 1. The Ser187-linked palmitoleoyl group is shown as connected red spheres. Frizzled8 CRD
residues forming the hydrophobic groove are shown as sticks (carbon, blue; oxygen, red) and numbered. Boundaries of the lipid-binding groove are marked with grey lines.
(b) WIF domain of WIF-1 forms a hydrophobic pocket which accommodates DPPC (carbon, grey; oxygen, red; phosphorus, orange; nitrogen, blue). The head group of DPPC is exposed to the solvent
and located proximal to the putative Wnt3a binding site.
(c) The first b propeller of LRP6 recognizes an evolutionarily conserved tripeptide motif Asn-X-Ile (X, variable amino acid) present in two inhibitors of  Wnt signaling, Dickkopf1 and Sclerostin. A peptide
derived from human Sclerostin (residues Leu115–Arg121) is shown as sticks (carbon, yellow; oxygen, red; nitrogen, blue).

Regulation of Wnt signaling by R-spondin and its receptors.

(a) Transmembrane ubiquitin (Ub, shown in grey) E3 ligases ZNRF3 (brown) and RNF43 (red) ubiquitinylate Frizzled thus promoting its endocytosis and inhibition of
Wnt signalling. Cytoplasmic regions of both ligases contain RING domains required for ubiquitinylation. The extracellular domains of ZNRF3 form weak dimers in
solution (protomers are shown in brown and grey, respectively; [36]).
(b) R-spondin 1 (RSPO1, green) forms a ternary complex with RNF43 and LGR5 (blue) [35]. Endocytosis of RNF43 and ZNRF3 in complex with RSPOs and LGRs
4–6 prevents ubiquitinylation of Frizzled and promotes Wnt signaling. Dotted lines represent interactions between molecules where crystal structures of complexes
are not determined.

Conclusions and future perspectives

Tremendous progress has been made in structural studies of  Wnt signaling receptors and modulators during the past five years. A series of structures of the Wnt co-receptor LRP6, agonists and
antagonists, and, remarkably, the first crystal structure of a Wnt family member, Wnt8, in complex with the Frizzled8 CRD, provide invaluable insights into the basic mechanisms of Wnt
signaling activation and regulation. In 2012, a novel mechanism of Wnt signaling regulation was discovered which centers on the interactions of the ZNRF3/RNF43 E3 ubiquitin ligases,
the R-spondins and LGR4/5/6.

7.10.9 FOXO3a modulates WNT.β-catenin signaling and suppresses epithelial-to-mesenchymal transition in prostate cancer cells

Liu H1Yin J1Wang H2Jiang G3Deng M1Zhang G2Bu X2Cai S4Du J5He Z6.
Cell Signal. 2015 Mar; 27(3):510-8


  • FOXO3a inhibits β-catenin expression through transactivating miR-34b/c.
  • FOXO3a direct binds to β-catenin.
  • FOXO3a inhibits β-catenin/TCF transcriptional activity.
  • FOXO3a inhibit EMT in prostate cancer cells.
  • β-catenin as a regulator of FOXO3a-mediated suppression of EMT.

Emerging evidence has revealed a negative correlation between Forkhead box-O (FOXO) expression and prostate cancer grade and spread, indicating its role as a suppressor of prostate cancer metastasis. However, there is still incomplete understanding about the role of FOXO transcription factors in prostate cancer progression. In this investigation, we demonstrate that FOXO3a significantly inhibits the expression β-catenin in prostate cancer cells. The mechanism of inhibiting β-catenin expression involves the FOXO3a-mediated transactivated microRNA-34b/c, which consequently suppressed β-catenin mRNA expression by targeting the untranslated regions (UTRs) of β-catenin. Additionally, FOXO3a can directly bind to β-catenin, and competes with TCF for interaction with β-catenin, thereby inhibiting β-catenin/TCF transcriptional activity and reducing the expression of β-catenin target genes. Furthermore, prostate cancer cells expressing FOXO3a shRNAs display mesenchymal characteristics, including enhanced cell migration and differential regulation of the EMT markers, whereas knockdown of β-catenin results in reversal of shFOXO3a-mediated EMT phenotypic changes. Collectively, these observations demonstrated that FOXO3a inhibits malignant phenotypes that are dependent on β-catenin-dependent modulation of EMT-related genes, and provided fresh insight into the mechanisms by which a FOXO3a-miR-34b/c axis restrains canonical β-catenin signaling cascades in prostate cancer cell.

Fig.1. FOXO3a activation correlates with downregulation of β-catenin expression in prostate cancer cells. (A) PC3 and DU145 cells were treated with LY294002 for 48h, and Western blot was performed to assess p-FOXO3a, total FOXO3a, and β-catenin expression compared with that of the control cells.(B,C) The PC3 and DU145 cells were transfected with FOXO3a overexpressing and si-FOXO3a knockdown vectors; the mRNA expression (B) and protein expression (C) of β-catenin were assessed by real-time RT-PCR and Western blot, respectively. (D) PC3 cells were transfected with FOXO3a overexpressing vector, immunofluorescence images from PC3 cells stained for FOXO3aand β-catenin. DNA is stained with 4,6-diamidino2-phenylindole (DAPI, blue).Data were presentedasmeans± SDof three independent experiments. *Significant difference from control values with P b 0.05.

Fig.2.FOXO3a inhibits β-catenin expression by modulating miR-34 expression. (A) The miR-34b/c promoter contains consensus FOXO binding sites. miR-34b and miR-34c are encoded by one primary transcript (BC021736). Putative FOXO binding sites were identified at positions−1518,−1512,−1223,and−185 relative to the transcription start site.(B)FOXO3abinds to the BC021736 promoter in vivo. PC3 cells were infected with pCMV-FOXO3a. DNA-bound proteins were crosslinked to chromatin, and FOXO3a was immunoprecipitated with an antibody directed against FOXO3a. Rabbit IgG immune serum was used as IP control. Immunoprecipitated DNA-fragments were amplified by PCR with primers specific for theputative FOXO3 a consensus binding sites(−1518/12,−1223,−185) or a control region.Data are plotted aspercentage ofinput DNA ± SD. (C, D)The PC3 cells were transfectedwith FOXO3a overexpressing(C)andsi-FOXO3aknockdownvectors(D),themRNAexpressionofmiR-34wereassessedbyreal-timeRT-PCR.(E)RealtimeRT-PCRanalysesofβ-cateninmRNAexpression levelswereperformedinPC3 cells 48h after transfectionwith control,miR-34b, ormiR-34cmimics. (F)ThePC3 cells were transfected with pCMV-FOXO3a, anti-miR-34c, pCMVFOXO3a and anti-miR-34c, respectively; or shFOXO3a, miR-34c mimics, shFOXO3a and miR-34c mimics, respectively, the protein expression of FOXO3a and β-catenin were analyzed by Western blot. Data were presented as means± SD of three independent experiments. *Significant difference from control valueswith P < 0.05.

Fig.3. FOXO3a binds to β-catenin, reduces binding of β-Catenin to TCF, and inhibits β-Catenin/TCF-dependent transcription. (A) Total protein extracts of PC3 and DU145 cells were subjected to IP using FOXO3a antibody or control IgG, followed by IB with β-cateninantibody (upper panels). Reciprocal IP was done using β-catenin antibody or control IgG, followed by IB with the FOXO3a antibody (lower panels). (B) Lysates of PC3 cells that stably express FOXO3a or a control vector were subjected to IPusing FOXO3a antibodies, followed by IB with β-catenin antibody.(C) Lysates of PC3 cells that stably express FOXO3a or a control vector were subjected to IP using TCF4 antibodies, followed by IB with β-catenin antibody. Reciprocal IP was done using β-catenin antibody or control IgG, followed by IB with the TCF4 antibody. (D) TOP flash and FOP flash firefly luciferase expression vectors were co-transfected with control, pCMV-FOXO3a, and pCMV-β-catenin plasmid in PC3 cells, and TOP flash activity was measured. (E) PC3 cells were transfected with pCMV-FOXO3a plasmid, or FOXO3 as hRNA, the differential expression of potential β-catenin target genes are shown in the heat map.Data were presented as means±SD of three independent experiments.**Significant difference from control values with P<b 0.01


Aurelian Udristioiu


Aurelian Udristioiu

Lab Director at Emergency County Hospital Targu Jiu

Some studies showed that patients with cancer make
antibodies against p53 proteins, but the frequency and
magnitude of this response is still under debate (Vojtesek
et al., 1995). However, a large number of patients with
cancer did produce p53-reactive T cells (Van der Burg et
al., 2001).
The results from these studies served as a good
reason to attempt the vaccination of patients using p53-
derived peptides, and a several clinical trials are currently
in progress. The most advanced work used a long
synthetic peptide mixture derived from p53 (p53-SLP; ISA
Pharmaceuticals, Bilthoven, the Netherlands) (Speetjens
et al., 2009; Shangary et al., 2008; Van der Burg et al.,
* The vaccine is delivered in the adjuvant setting
and induces T helper type cells.

UPDATE 10/10/2021

WNT/β-catenin pathway activation correlates with immune exclusion across human cancers

Source: Luke JJ, Bao R, Sweis RF, Spranger S, Gajewski TF. WNT/β-catenin Pathway Activation Correlates with Immune Exclusion across Human Cancers. Clin Cancer Res. 2019;25(10):3074-3083. doi:10.1158/1078-0432.CCR-18-1942



The T cell-inflamed phenotype correlates with efficacy of immune-checkpoint blockade while non-T cell-inflamed tumors infrequently benefit. Tumor-intrinsic WNT/β-catenin signaling mediates immune exclusion in melanoma, but association with the non-T cell-inflamed tumor microenvironment in other tumor types is not well understood.


Using The Cancer Genome Atlas (TCGA), a T cell-inflamed gene expression signature segregated samples within tumor types. Activation of WNT/β-catenin signaling was inferred using three approaches: somatic mutations or somatic copy number alterations (SCNAs) in β-catenin signaling elements including CTNNB1, APC, APC2, AXIN1, AXIN2; pathway prediction from RNAseq gene expression; and inverse correlation of β-catenin protein levels with the T cell-inflamed gene expression signature.


Across TCGA, 3137/9244 (33.9%) tumors were non-T cell-inflamed while 3161/9244 (34.2%) were T cell-inflamed. Non-T cell-inflamed tumors demonstrated significantly lower expression of T cell inflammation genes relative to matched normal tissue, arguing for loss of a natural immune phenotype. Mutations of β-catenin signaling molecules in non-T cell-inflamed tumors were enriched three-fold relative to T cell-inflamed tumors. Across 31 tumors, 28 (90%) demonstrated activated β-catenin signaling in the non-T cell-inflamed subset by at least one method. This included target molecule expression from somatic mutations and/or SCNAs of β-catenin signaling elements (19 tumors, 61%), pathway analysis (14 tumors, 45%), and increased β-catenin protein levels (20 tumors, 65%).


Activation of tumor-intrinsic WNT/β-catenin signaling is enriched in non-T cell-inflamed tumors. These data provide a strong rationale for development of pharmacologic inhibitors of this pathway with the aim of restoring immune cell infiltration and augmenting immunotherapy.


Immunotherapies targeting immune checkpoints have contributed to a marked improvement in treatment outcomes in patients with advanced cancer. In melanoma, anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) and anti-programmed death 1 (PD-1) antibodies have demonstrated robust response rates with years of durability in some patients(,) and improvement in overall survival(,). Significant clinical activity of PD-1-targeting agents has led to FDA approval in multiple additional cancer entities. Despite this broad activity, only a subset of patients benefits from treatment within each cancer subtype, and molecular mechanisms to explain primary resistance in these patients remain incompletely understood.

High expression of specific immune cell genes in the tumor microenvironment, described as the T cell-inflamed phenotype, has been observed to correlate with response to multiple immunotherapies including therapeutic vaccines and checkpoint blocking antibodies(,). Conversely, the non-T cell inflamed tumor microenvironment appears to closely associate with lack of clinical benefit to immunotherapy, particularly with anti-PD-1 antibodies(,). Categorization of tumors using transcriptional profiles marking the T cell-inflamed gene expression signature is advantageous as it can define biologically relevant patient sub-populations and set a framework in which to investigate hypothetical mechanisms for primary immunotherapy resistance.

Although multiple molecular mechanisms could theoretically disfavor a T cell-inflamed microenvironment, several lines of investigation have indicated that specific oncogenic molecular aberrations can be sufficient to drive this immune exclusion phenotype in some cases. Tumor cell-intrinsic WNT/β-catenin signaling in melanoma was the first somatic alteration associated with the non-T cell-inflamed tumor microenvironment in patients, and was demonstrated to be causal using a genetically-engineered mouse model(). The mechanism of this effect appears to be through transcriptional repression of key chemokine genes that leads to lack of Batf3-lineage dendritic cell recruitment and subsequent failure to prime and recruit CD8+ T cells(,). This effect is dominant in the tumor microenvironment and leads to loss of therapeutic efficacy of checkpoint blockade, tumor antigen vaccination, and adoptive T cell transfer immunotherapy approaches preclinically(,). While the above studies of tumor-intrinsic WNT/β-catenin signaling have been evaluated in the context of melanoma, the impact of this pathway in driving the non-T cell-inflamed tumor microenvironment in other tumor types are increasingly being recognized. In syngeneic murine models of B16F10 melanoma, 4T1 mammary carcinoma, Neuro2A neuroblastoma, and Renca renal adenocarcinoma, blocking β-catenin pathway signaling via RNA interference resulted in influx of CD8+ T cells and increase in interferon-γ-associated gene targets(). Subsequent combination with immunotherapy yielded complete regressions in the majority of treated animals. More broadly, roles for WNT/β-catenin signaling impacting on the immune system via development and function, active immune exclusion by tumor cells and cancer immunosurveillance are being recognized and accepted across cancer types().

To investigate the influence of WNT/β-catenin signaling across cancers, we performed an integrative analysis of The Cancer Genome Atlas (TCGA) separating individual tumors by T cell-inflamed status and identifying β-catenin pathway activation on three levels. We find that most tumor types within TCGA are enriched for activation of WNT/β-catenin signaling in non-T cell-inflamed tumors. These observations suggest pharmacologic targeting of this pathway could have broad implications for improving immunotherapy efficacy.

Editors note:  Although the majority of mutations in the WNT signaling pathway in cancer have been in the APC gene, this study, although bioinformatic in nature, shows good correlate between other pathway mutations and immune infiltrate. It is interesting to also note that tumor utational burden is the approved biomarker for immune checkpoint inhibitor efficacy.

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Hypoxia Inducible Factor 1 (HIF-1)

Writer and Curator: Larry H Bernstein, MD, FCAP

7.9  Hypoxia Inducible Factor 1 (HIF-1)

7.9.1 Hypoxia and mitochondrial oxidative metabolism

7.9.2 Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability

7.9.3 Hypoxia-Inducible Factors in Physiology and Medicine

7.9.4 Hypoxia-inducible factor 1. Regulator of mitochondrial metabolism and mediator of ischemic preconditioning

7.9.5 Regulation of cancer cell metabolism by hypoxia-inducible factor 1

7.9.6 Coming up for air. HIF-1 and mitochondrial oxygen consumption

7.9.7 HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption

7.9.8 HIF-1. upstream and downstream of cancer metabolism

7.9.9 In Vivo HIF-Mediated Reductive Carboxylation

7.9.10 Evaluation of HIF-1 inhibitors as anticancer agents



7.9.1 Hypoxia and mitochondrial oxidative metabolism

Solaini G1Baracca ALenaz GSgarbi G.
Biochim Biophys Acta. 2010 Jun-Jul; 1797(6-7):1171-7

It is now clear that mitochondrial defects are associated with a large variety of clinical phenotypes. This is the result of the mitochondria’s central role in energy production, reactive oxygen species homeostasis, and cell death. These processes are interdependent and may occur under various stressing conditions, among which low oxygen levels (hypoxia) are certainly prominent. Cells exposed to hypoxia respond acutely with endogenous metabolites and proteins promptly regulating metabolic pathways, but if low oxygen levels are prolonged, cells activate adapting mechanisms, the master switch being the hypoxia-inducible factor 1 (HIF-1). Activation of this factor is strictly bound to the mitochondrial function, which in turn is related with the oxygen level. Therefore in hypoxia, mitochondria act as [O2] sensors, convey signals to HIF-1directly or indirectly, and contribute to the cell redox potential, ion homeostasis, and energy production. Although over the last two decades cellular responses to low oxygen tension have been studied extensively, mechanisms underlying these functions are still indefinite. Here we review current knowledge of the mitochondrial role in hypoxia, focusing mainly on their role in cellular energy and reactive oxygen species homeostasis in relation with HIF-1 stabilization. In addition, we address the involvement of HIF-1 and the inhibitor protein of F1F0 ATPase in the hypoxia-induced mitochondrial autophagy.

Over the last two decades a defective mitochondrial function associated with hypoxia has been invoked in many diverse complex disorders, such as type 2 diabetes [1] and [2], Alzheimer’s disease [3] and [4], cardiac ischemia/reperfusion injury [5] and [6], tissue inflammation [7], and cancer [8][9][10],[11] and [12].

The [O2] in air-saturated aqueous buffer at 37 °C is approx. 200 μM [13]; however, mitochondria in vivo are exposed to a considerably lower [O2] that varies with tissue and physiological state. Under physiological conditions, most human resting cells experience some 5% oxygen tension, however the [O2] gradient occurring between the extracellular environment and mitochondria, where oxygen is consumed by cytochrome c oxidase, results in a significantly lower [O2] exposition of mitochondria. Below this oxygen level, most mammalian tissues are exposed to hypoxic conditions  [14]. These may arise in normal development, or as a consequence of pathophysiological conditions where there is a reduced oxygen supply due to a respiratory insufficiency or to a defective vasculature. Such conditions include inflammatory diseases, diabetes, ischemic disorders (cerebral or cardiovascular), and solid tumors. Mitochondria consume the greatest amount (some 85–90%) of oxygen in cells to allow oxidative phosphorylation (OXPHOS), which is the primary metabolic pathway for ATP production. Therefore hypoxia will hamper this metabolic pathway, and if the oxygen level is very low, insufficient ATP availability might result in cell death [15].

When cells are exposed to an atmosphere with reduced oxygen concentration, cells readily “respond” by inducing adaptive reactions for their survival through the AMP-activated protein kinase (AMPK) pathway (see for a recent review [16]) which inter alia increases glycolysis driven by enhanced catalytic efficiency of some enzymes, including phosphofructokinase-1 and pyruvate kinase (of note, this oxidative flux is thermodynamically allowed due to both reduced phosphorylation potential [ATP]/([ADP][Pi]) and the physiological redox state of the cell). However, this is particularly efficient only in the short term, therefore cells respond to prolonged hypoxia also by stimulation of hypoxia-inducible factors (HIFs: HIF-1 being the mostly studied), which are heterodimeric transcription factors composed of α and β subunits, first described by Semenza and Wang [17]. These HIFs in the presence of hypoxic oxygen levels are activated through a complex mechanism in which the oxygen tension is critical (see below). Afterwards HIFs bind to hypoxia-responsive elements, activating the transcription of more than two hundred genes that allow cells to adapt to the hypoxic environment [18] and [19].

Several excellent reviews appeared in the last few years describing the array of changes induced by oxygen deficiency in both isolated cells and animal tissues. In in vivo models, a coordinated regulation of tissue perfusion through vasoactive molecules such as nitric oxide and the action of carotid bodies rapidly respond to changes in oxygen demand [20][21][22][23] and [24]. Within isolated cells, hypoxia induces significant metabolic changes due to both variation of metabolites level and activation/inhibition of enzymes and transporters; the most important intracellular effects induced by different pathways are expertly described elsewhere (for recent reviews, see [25][26] and [27]). It is reasonable to suppose that the type of cells and both the severity and duration of hypoxia may determine which pathways are activated/depressed and their timing of onset [3][6][10][12][23] and [28]. These pathways will eventually lead to preferential translation of key proteins required for adaptation and survival to hypoxic stress. Although in the past two decades, the discovery of HIF-1 by Gregg Semenza et al. provided a molecular platform to investigate the mechanism underlying responses to oxygen deprivation, the molecular and cellular biology of hypoxia has still to be completely elucidated. This review summarizes recent experimental data concerned with mitochondrial structure and function adaptation to hypoxia and evaluates it in light of the main structural and functional parameters defining the mitochondrial bioenergetics. Since mitochondria contain an inhibitor protein, IF1, whose action on the F1F0 ATPase has been considered for decades of critical importance in hypoxia/ischemia, particular notice will be dedicated to analyze molecular aspects of IF1 regulation of the enzyme and its possible role in the metabolic changes induced by low oxygen levels in cells.

Mechanism(s) of HIF-1 activation

HIF-1 consists of an oxygen-sensitive HIF-1α subunit that heterodimerizes with the HIF-1β subunit to bind DNA. In high O2 tension, HIF-1α is oxidized (hydroxylated) by prolyl hydroxylases (PHDs) using α-ketoglutarate derived from the tricarboxylic acid (TCA) cycle. The hydroxylated HIF-1α subunit interacts with the von Hippel–Lindau protein, a critical member of an E3 ubiquitin ligase complex that polyubiquitylates HIF. This is then catabolized by proteasomes, such that HIF-1α is continuously synthesized and degraded under normoxic conditions [18]. Under hypoxia, HIF-1α hydroxylation does not occur, thereby stabilizing HIF-1 (Fig. 1). The active HIF-1 complex in turn binds to a core hypoxia response element in a wide array of genes involved in a diversity of biological processes, and directly transactivates glycolytic enzyme genes [29]. Notably, O2 concentration, multiple mitochondrial products, including the TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, hence the cellular responses to O2 depletion [30] and [31]. Incidentally, impaired TCA cycle flux, particularly if it is caused by succinate dehydrogenase dysfunction, results in decreased or loss of energy production from both the electron-transport chain and the Krebs cycle, and also in overproduction of free radicals [32]. This leads to severe early-onset neurodegeneration or, as it occurs in individuals carrying mutations in the non-catalytic subunits of the same enzyme, to tumors such as phaeochromocytoma and paraganglioma. However, impairment of the TCA cycle may be relevant also for the metabolic changes occurring in mitochondria exposed to hypoxia, since accumulation of succinate has been reported to inhibit PHDs [33]. It has to be noticed that some authors believe reactive oxygen species (ROS) to be essential to activate HIF-1 [34], but others challenge this idea [35], therefore the role of mitochondrial ROS in the regulation of HIF-1 under hypoxia is still controversial [36]. Moreover, the contribution of functional mitochondria to HIF-1 regulation has also been questioned by others [37][38] and [39].


Major mitochondrial changes in hypoxia

Major mitochondrial changes in hypoxia

Fig. 1. Major mitochondrial changes in hypoxia. Hypoxia could decrease electron-transport rate determining Δψm reduction, increased ROS generation, and enhanced NO synthase. One (or more) of these factors likely contributes to HIF stabilization, that in turn induces metabolic adaptation of both hypoxic cells and mitophagy. The decreased Δψm could also induce an active binding of IF1, which might change mitochondrial morphology and/or dynamics, and inhibit mitophagy. Solid lines indicate well established hypoxic changes in cells, whilst dotted lines indicate changes not yet stated. Inset, relationships between extracellular O2concentration and oxygen tension.

Oxygen is a major determinant of cell metabolism and gene expression, and as cellular O2 levels decrease, either during isolated hypoxia or ischemia-associated hypoxia, metabolism and gene expression profiles in the cells are significantly altered. Low oxygen reduces OXPHOS and Krebs cycle rates, and participates in the generation of nitric oxide (NO), which also contributes to decrease respiration rate [23] and [40]. However, oxygen is also central in the generation of reactive oxygen species, which can participate in cell signaling processes or can induce irreversible cellular damage and death [41].

As specified above, cells adapt to oxygen reduction by inducing active HIF, whose major effect on cells energy homeostasis is the inactivation of anabolism, activation of anaerobic glycolysis, and inhibition of the mitochondrial aerobic metabolism: the TCA cycle, and OXPHOS. Since OXPHOS supplies the majority of ATP required for cellular processes, low oxygen tension will severely reduce cell energy availability. This occurs through several mechanisms: first, reduced oxygen tension decreases the respiration rate, due first to nonsaturating substrate for cytochrome c oxidase (COX), secondarily, to allosteric modulation of COX[42]. As a consequence, the phosphorylation potential decreases, with enhancement of the glycolysis rate primarily due to allosteric increase of phosphofructokinase activity; glycolysis however is poorly efficient and produces lactate in proportion of 0.5 mol/mol ATP, which eventually drops cellular pH if cells are not well perfused, as it occurs under defective vasculature or ischemic conditions  [6]. Besides this “spontaneous” (thermodynamically-driven) shift from aerobic to anaerobic metabolism which is mediated by the kinetic changes of most enzymes, the HIF-1 factor activates transcription of genes encoding glucose transporters and glycolytic enzymes to further increase flux of reducing equivalents from glucose to lactate[43] and [44]. Second, HIF-1 coordinates two different actions on the mitochondrial phase of glucose oxidation: it activates transcription of the PDK1 gene encoding a kinase that phosphorylates and inactivates pyruvate dehydrogenase, thereby shunting away pyruvate from the mitochondria by preventing its oxidative decarboxylation to acetyl-CoA [45] and [46]. Moreover, HIF-1 induces a switch in the composition of cytochrome c oxidase from COX4-1 to COX4-2 isoform, which enhances the specific activity of the enzyme. As a result, both respiration rate and ATP level of hypoxic cells carrying the COX4-2 isoform of cytochrome c oxidase were found significantly increased with respect to the same cells carrying the COX4-1 isoform [47]. Incidentally, HIF-1 can also increase the expression of carbonic anhydrase 9, which catalyses the reversible hydration of CO2 to HCO3 and H+, therefore contributing to pH regulation.

Effects of hypoxia on mitochondrial structure and dynamics

Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. The fusion/fission machineries are modulated in response to changes in the metabolic conditions of the cell, therefore one should expect that hypoxia affect mitochondrial dynamics. Oxygen availability to cells decreases glucose oxidation, whereas oxygen shortage consumes glucose faster in an attempt to produce ATP via the less efficient anaerobic glycolysis to lactate (Pasteur effect). Under these conditions, mitochondria are not fueled with substrates (acetyl-CoA and O2), inducing major changes of structure, function, and dynamics (for a recent review see [48]). Concerning structure and dynamics, one of the first correlates that emerge is that impairment of mitochondrial fusion leads to mitochondrial depolarization, loss of mtDNA that may be accompanied by altered respiration rate, and impaired distribution of the mitochondria within cells [49][50] and [51]. Indeed, exposure of cortical neurons to moderate hypoxic conditions for several hours, significantly altered mitochondrial morphology, decreased mitochondrial size and reduced mitochondrial mean velocity. Since these effects were either prevented by exposing the neurons to inhibitors of nitric oxide synthase or mimicked by NO donors in normoxia, the involvement of an NO-mediated pathway was suggested [52]. Mitochondrial motility was also found inhibited and controlled locally by the [ADP]/[ATP] ratio [53]. Interestingly, the author used an original approach in which mitochondria were visualized using tetramethylrhodamineethylester and their movements were followed by applying single-particle tracking.

Of notice in this chapter is that enzymes controlling mitochondrial morphology regulators provide a platform through which cellular signals are transduced within the cell in order to affect mitochondrial function [54]. Accordingly, one might expect that besides other mitochondrial factors [30] and [55] playing roles in HIF stabilization, also mitochondrial morphology might reasonably be associated with HIF stabilization. In order to better define the mechanisms involved in the morphology changes of mitochondria and in their dynamics when cells experience hypoxic conditions, these pioneering studies should be corroborated by and extended to observations on other types of cells focusing also on single proteins involved in both mitochondrial fusion/fission and motion.

Effects of hypoxia on the respiratory chain complexes

O2 is the terminal acceptor of electrons from cytochrome c oxidase (Complex IV), which has a very high affinity for it, being the oxygen concentration for half-maximal respiratory rate at pH 7.4 approximately 0.7 µM [56]. Measurements of mitochondrial oxidative phosphorylation indicated that it is not dependent on oxygen concentration up to at least 20 µM at pH 7.0 and the oxygen dependence becomes markedly greater as the pH is more alkaline [56]. Similarly, Moncada et al. [57] found that the rate of O2 consumption remained constant until [O2] fell below 15 µM. Accordingly, most reports in the literature consider hypoxic conditions occurring in cells at 5–0.5% O2, a range corresponding to 46–4.6 µM O2 in the cells culture medium (see Fig. 1 inset). Since between the extracellular environment and mitochondria an oxygen pressure gradient is established [58], the O2 concentration experienced by Complex IV falls in the range affecting its kinetics, as reported above.

Under these conditions, a number of changes on the OXPHOS machinery components, mostly mediated by HIF-1 have been found. Thus, Semenza et al. [59] and others thereafter [46] reported that activation of HIF-1α induces pyruvate dehydrogenase kinase, which inhibits pyruvate dehydrogenase, suggesting that respiration is decreased by substrate limitation. Besides, other HIF-1 dependent mechanisms capable to affect respiration rate have been reported. First, the subunit composition of COX is altered in hypoxic cells by increased degradation of the COX4-1 subunit, which optimizes COX activity under aerobic conditions, and increased expression of the COX4-2 subunit, which optimizes COX activity under hypoxic conditions [29]. On the other hand, direct assay of respiration rate in cells exposed to hypoxia resulted in a significant reduction of respiration [60]. According with the evidence of Zhang et al., the respiration rate decrease has to be ascribed to mitochondrial autophagy, due to HIF-1-mediated expression of BNIP3. This interpretation is in line with preliminary results obtained in our laboratory where the assay of the citrate synthase activity of cells exposed to different oxygen tensions was performed. Fig. 2 shows the citrate synthase activity, which is taken as an index of the mitochondrial mass [11], with respect to oxygen tension: [O2] and mitochondrial mass are directly linked.

Citrate synthase activity

Citrate synthase activity


Fig. 2. Citrate synthase activity. Human primary fibroblasts, obtained from skin biopsies of 5 healthy donors, were seeded at a density of 8,000 cells/cm2 in high glucose Dulbecco’s Modified Eagle Medium, DMEM (25 mM glucose, 110 mg/l pyruvate, and 4 mM glutamine) supplemented with 15% Foetal Bovine Serum (FBS). 18 h later, cell culture dishes were washed once with Hank’s Balanced Salt Solution (HBSS) and the medium was replaced with DMEM containing 5 mM glucose, 110 mg/l pyruvate, and 4 mM glutamine supplemented with 15% FBS. Cell culture dishes were then placed into an INVIVO2 humidified hypoxia workstation (Ruskinn Technologies, Bridgend, UK) for 72 h changing the medium at 48 h, and oxygen partial pressure (tension) conditions were: 20%, 4%, 2%, 1% and 0.5%. Cells were subsequently collected within the workstation with trypsin-EDTA (0.25%), washed with PBS and resuspended in a buffer containing 10 mM Tris/HCl, 0.1 M KCl, 5 mM KH2PO4, 1 mM EGTA, 3 mM EDTA, and 2 mM MgCl2 pH 7.4 (all the solutions were preconditioned to the appropriate oxygen tension condition). The citrate synthase activity was assayed essentially by incubating 40 µg of cells with 0.02% Triton X-100, and monitoring the reaction by measuring spectrophotometrically the rate of free coenzyme A released, as described in [90]. Enzymatic activity was expressed as nmol/min/mg of protein. Three independent experiments were carried out and assays were performed in either duplicate or triplicate.

However, the observations of Semenza et al. must be seen in relation with data reported by Moncada et al.[57] and confirmed by others [61] in which it is clearly shown that when cells (various cell lines) experience hypoxic conditions, nitric oxide synthases (NOSs) are activated, therefore NO is released. As already mentioned above, NO is a strong competitor of O2 for cytochrome c oxidase, whose apparent Km results increased, hence reduction of mitochondrial cytochromes and all the other redox centres of the respiratory chain occurs. In addition, very recent data indicate a potential de-activation of Complex I when oxygen is lacking, as it occurs in prolonged hypoxia [62]. According to Hagen et al. [63] the NO-dependent inhibition of cytochrome c oxidase should allow “saved” O2 to redistribute within the cell to be used by other enzymes, including PHDs which inactivate HIF. Therefore, unless NO inhibition of cytochrome c oxidase occurs only when [O2] is very low, inhibition of mitochondrial oxygen consumption creates the paradox of a situation in which the cell may fail to register hypoxia. It has been tempted to solve this paradox, but to date only hypotheses have been proposed [23] and [26]. Interestingly, recent observations on yeast cells exposed to hypoxia revealed abnormal protein carbonylation and protein tyrosine nitration that were ascribed to increased mitochondrially generated superoxide radicals and NO, two species typically produced at low oxygen levels, that combine to form ONOO [64]. Based on these studies a possible explanation has been proposed for the above paradox.

Finally, it has to be noticed that the mitochondrial respiratory deficiency observed in cardiomyocytes of dogs in which experimental heart failure had been induced lies in the supermolecular assembly rather than in the individual components of the electron-transport chain [65]. This observation is particularly intriguing since loss of respirasomes is thought to facilitate ROS generation in mitochondria [66], therefore supercomplexes disassembly might explain the paradox of reduced [O2] and the enhanced ROS found in hypoxic cells. Specifically, hypoxia could reduce mitochondrial fusion by impairing mitochondrial membrane potential, which in turn could induce supercomplexes disassembly, increasing ROS production[11].

Complex III and ROS production

It has been estimated that, under normoxic physiological conditions, 1–2% of electron flow through the mitochondrial respiratory chain gives rise to ROS [67] and [68]. It is now recognized that the major sites of ROS production are within Complexes I and III, being prevalent the contribution of Complex I [69] (Fig. 3). It might be expected that hypoxia would decrease ROS production, due to the low level of O2 and to the diminished mitochondrial respiration [6] and [46], but ROS level is paradoxically increased. Indeed, about a decade ago, Chandel et al. [70] provided good evidence that mitochondrial reactive oxygen species trigger hypoxia-induced transcription, and a few years later the same group [71] showed that ROS generated at Complex III of the mitochondrial respiratory chain stabilize HIF-1α during hypoxia (Fig. 1 and Fig. 3). Although others have proposed mechanisms indicating a key role of mitochondria in HIF-1α regulation during hypoxia (for reviews see [64] and [72]), the contribution of mitochondria to HIF-1 regulation has been questioned by others [35][36] and [37]. Results of Gong and Agani [35] for instance show that inhibition of electron-transport Complexes I, III, and IV, as well as inhibition of mitochondrial F0F1 ATPase, prevents HIF-1α expression and that mitochondrial reactive oxygen species are not involved in HIF-1α regulation during hypoxia. Concurrently, Tuttle et al. [73], by means of a non invasive, spectroscopic approach, could find no evidence to suggest that ROS, produced by mitochondria, are needed to stabilize HIF-1α under moderate hypoxia. The same authors found the levels of HIF-1α comparable in both normal and ρ0 cells (i.e. cells lacking mitochondrial DNA). On the contrary, experiments carried out on genetic models consisting of either cells lacking cytochrome c or ρ0 cells both could evidence the essential role of mitochondrial respiration to stabilize HIF-1α [74]. Thus, cytochrome c null cells, being incapable to respire, exposed to moderate hypoxia (1.5% O2) prevented oxidation of ubiquinol and generation of the ubisemiquinone radical, thus eliminating superoxide formation at Complex III [71]. Concurrently, ρ0 cells lacking electron transport, exposed 4 h to moderate hypoxia failed to stabilize HIF-1α, suggesting the essential role of the respiratory chain for the cellular sensing of low O2 levels. In addition, recent evidence obtained on genetic manipulated cells (i.e. cytochrome b deficient cybrids) showed increased ROS levels and stabilized HIF-1α protein during hypoxia [75]. Moreover, RNA interference of the Complex III subunit Rieske iron sulfur protein in the cytochrome b deficient cells, abolished ROS generation at the Qo site of Complex III, preventing HIF-1α stabilization. These observations, substantiated by experiments with MitoQ, an efficient mitochondria-targeted antioxidant, strongly support the involvement of mitochondrial ROS in regulating HIF-1α. Nonetheless, collectively, the available data do not allow to definitely state the precise role of mitochondrial ROS in regulating HIF-1α, but the pathway stabilizing HIF-1α appears undoubtedly mitochondria-dependent [30].

Overview of mitochondrial electron and proton flux in hypoxia

Overview of mitochondrial electron and proton flux in hypoxia

Overview of mitochondrial electron and proton flux in hypoxia


Fig. 3. Overview of mitochondrial electron and proton flux in hypoxia. Electrons released from reduced cofactors (NADH and FADH2) under normoxia flow through the redox centres of the respiratory chain (r.c.) to molecular oxygen (blue dotted line), to which a proton flux from the mitochondrial matrix to the intermembrane space is coupled (blue arrows). Protons then flow back to the matrix through the F0 sector of the ATP synthase complex, driving ATP synthesis. ATP is carried to the cell cytosol by the adenine nucleotide translocator (blue arrows). Under moderate to severe hypoxia, electrons escape the r.c. redox centres and reduce molecular oxygen to the superoxide anion radical before reaching the cytochrome c (red arrow). Under these conditions, to maintain an appropriate Δψm, ATP produced by cytosolic glycolysis enters the mitochondria where it is hydrolyzed by the F1F0ATPase with extrusion of protons from the mitochondrial matrix (red arrows).

Hypoxia and ATP synthase

The F1F0 ATPase (ATP synthase) is the enzyme responsible of catalysing ADP phosphorylation as the last step of OXPHOS. It is a rotary motor using the proton motive force across the mitochondrial inner membrane to drive the synthesis of ATP [76]. It is a reversible enzyme with ATP synthesis or hydrolysis taking place in the F1 sector at the matrix side of the membrane, chemical catalysis being coupled to H+transport through the transmembrane F0 sector.

Under normoxia the enzyme synthesizes ATP, but when mitochondria experience hypoxic conditions the mitochondrial membrane potential (Δψm) decreases below its endogenous steady-state level (some 140 mV, negative inside the matrix [77]) and the F1F0 ATPase may work in the reversal mode: it hydrolyses ATP (produced by anaerobic glycolysis) and uses the energy released to pump protons from the mitochondrial matrix to the intermembrane space, concurring with the adenine nucleotide translocator (i.e. in hypoxia it exchanges cytosolic ATP4− for matrix ADP3−) to maintain the physiological Δψm ( Fig. 3). Since under conditions of limited oxygen availability the decline in cytoplasmic high energy phosphates is mainly due to hydrolysis by the ATP synthase working in reverse [6] and [78], the enzyme must be strictly regulated in order to avoid ATP dissipation. This is achieved by a natural protein, the H+ψm-dependent IF1, that binds to the catalytic F1 sector at low pH and low Δψm (such as it occurs in hypoxia/ischemia) [79]. IF1 binding to the ATP synthase results in a rapid and reversible inhibition of the enzyme [80], which could reach about 50% of maximal activity (for recent reviews see [6] and [81]).

Besides this widely studied effect, IF1 appears to be associated with ROS production and mitochondrial autophagy (mitophagy). This is a mechanism involving the catabolic degradation of macromolecules and organelles via the lysosomal pathway that contributes to housekeeping and regenerate metabolites. Autophagic degradation is involved in the regulation of the ageing process and in several human diseases, such as myocardial ischemia/reperfusion [82], Alzheimer’s Disease, Huntington diseases, and inflammatory diseases (for recent reviews see [83] and [84], and, as mentioned above, it promotes cell survival by reducing ROS and mtDNA damage under hypoxic conditions.

Campanella et al. [81] reported that, in HeLa cells under normoxic conditions, basal autophagic activity varies in relation to the expression levels of IF1. Accordingly, cells overexpressing IF1 result in ROS production similar to controls, conversely cells in which IF1 expression is suppressed show an enhanced ROS production. In parallel, the latter cells show activation of the mitophagy pathway (Fig. 1), therefore suggesting that variations in IF1 expression level may play a significant role in defining two particularly important parameters in the context of the current review: rates of ROS generation and mitophagy. Thus, the hypoxia-induced enhanced expression level of IF1[81] should be associated with a decrease of both ROS production and autophagy, which is in apparent conflict with the hypoxia-induced ROS increase and with the HIF-1-dependent mitochondrial autophagy shown by Zhang et al. [60] as an adaptive metabolic response to hypoxia. However, in the experiments of Zhang et al. the cells were exposed to hypoxia for 48 h, whereas the F1F0-ATPase inhibitor exerts a prompt action on the enzyme and to our knowledge, it has never been reported whether its action persists during prolonged hypoxic expositions. Pertinent with this problem is the very recent observation that IEX-1 (immediate early response gene X-1), a stress-inducible gene that suppresses production of ROS and protects cells from apoptosis [85], targets the mitochondrial F1F0-ATPase inhibitor for degradation, reducing ROS by decreasing Δψm. It has to be noticed that the experiments described were carried out under normal oxygen availability, but it does not seem reasonable to rule out IEX-1 from playing a role under stress conditions as those induced by hypoxia in cells, therefore this issue might deserve an investigation also at low oxygen levels.

In conclusion, data are still emerging regarding the regulation of mitochondrial function by the F1F0 ATPase within hypoxic responses in different cellular and physiological contexts. Given the broad pathophysiological role of hypoxic cellular modulation, an understanding of the subtle tuning among different effectors of the ATP synthase is desirable to eventually target future therapeutics most effectively. Our laboratory is actually involved in carrying out investigations to clarify this context.

Conclusions and perspectives

The mitochondria are important cellular platforms that both propagate and initiate intracellular signals that lead to overall cellular and metabolic responses. During the last decades, a significant amount of relevant data has been obtained on the identification of mechanisms of cellular adaptation to hypoxia. In hypoxic cells there is an enhanced transcription and synthesis of several glycolytic pathway enzymes/transporters and reduction of synthesis of proteins involved in mitochondrial catabolism. Although well defined kinetic parameters of reactions in hypoxia are lacking, it is usually assumed that these transcriptional changes lead to metabolic flux modification. The required biochemical experimentation has been scarcely addressed until now and only in few of the molecular and cellular biology studies the transporter and enzyme kinetic parameters and flux rate have been determined, leaving some uncertainties.

Central to mitochondrial function and ROS generation is an electrochemical proton gradient across the mitochondrial inner membrane that is established by the proton pumping activity of the respiratory chain, and that is strictly linked to the F1F0-ATPase function. Evaluation of the mitochondrial membrane potential in hypoxia has only been studied using semiquantitative methods based on measurements of the fluorescence intensity of probes taken up by cells experiencing normal or hypoxic conditions. However, this approach is intrinsically incorrect due to the different capability that molecular oxygen has to quench fluorescence [86] and [87] and to the uncertain concentration the probe attains within mitochondria, whose mass may be reduced by a half in hypoxia [60]. In addition, the uncertainty about measurement of mitochondrial superoxide radical and H2O2 formation in vivo [88] hampers studies on the role of mitochondrial ROS in hypoxic oxidative damage, redox signaling, and HIF-1 stabilization.

The duration and severity of hypoxic stress differentially activate the responses discussed throughout and lead to substantial phenotypic variations amongst tissues and cell models, which are not consistently and definitely known. Certainly, understanding whether a hierarchy among hypoxia response mechanisms exists and which are the precise timing and conditions of each mechanism to activate, will improve our knowledge of the biochemical mechanisms underlying hypoxia in cells, which eventually may contribute to define therapeutic targets in hypoxia-associated diseases. To this aim it might be worth investigating the hypoxia-induced structural organization of both the respiratory chain enzymes in supramolecular complexes and the assembly of the ATP synthase to form oligomers affecting ROS production [65] and inner mitochondrial membrane structure [89], respectively.

7.9.2 Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability

DR WisePS WardJES ShayJR CrossJJ Gruber, UM Sachdeva, et al.
Proc Nat Acad Sci Oct 27, 2011; 108(49):19611–19616

Citrate is a critical metabolite required to support both mitochondrial bioenergetics and cytosolic macromolecular synthesis. When cells proliferate under normoxic conditions, glucose provides the acetyl-CoA that condenses with oxaloacetate to support citrate production. Tricarboxylic acid (TCA) cycle anaplerosis is maintained primarily by glutamine. Here we report that some hypoxic cells are able to maintain cell proliferation despite a profound reduction in glucose-dependent citrate production. In these hypoxic cells, glutamine becomes a major source of citrate. Glutamine-derived α-ketoglutarate is reductively carboxylated by the NADPH-linked mitochondrial isocitrate dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate. The increased IDH2-dependent carboxylation of glutamine-derived α-ketoglutarate in hypoxia is associated with a concomitant increased synthesis of 2-hydroxyglutarate (2HG) in cells with wild-type IDH1 and IDH2. When either starved of glutamine or rendered IDH2-deficient by RNAi, hypoxic cells are unable to proliferate. The reductive carboxylation of glutamine is part of the metabolic reprogramming associated with hypoxia-inducible factor 1 (HIF1), as constitutive activation of HIF1 recapitulates the preferential reductive metabolism of glutamine-derived α-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions.

Citrate plays a critical role at the center of cancer cell metabolism. It provides the cell with a source of carbon for fatty acid and cholesterol synthesis (1). The breakdown of citrate by ATP-citrate lyase is a primary source of acetyl-CoA for protein acetylation (2). Metabolism of cytosolic citrate by aconitase and IDH1 can also provide the cell with a source of NADPH for redox regulation and anabolic synthesis. Mammalian cells depend on the catabolism of glucose and glutamine to fuel proliferation (3). In cancer cells cultured at atmospheric oxygen tension (21% O2), glucose and glutamine have both been shown to contribute to the cellular citrate pool, with glutamine providing the major source of the four-carbon molecule oxaloacetate and glucose providing the major source of the two-carbon molecule acetyl-CoA (45). The condensation of oxaloacetate and acetyl-CoA via citrate synthase generates the 6 carbon citrate molecule. However, both the conversion of glucose-derived pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH) and the conversion of glutamine to oxaloacetate through the TCA cycle depend on NAD+, which can be compromised under hypoxic conditions. This raises the question of how cells that can proliferate in hypoxia continue to synthesize the citrate required for macromolecular synthesis.

This question is particularly important given that many cancers and stem/progenitor cells can continue proliferating in the setting of limited oxygen availability (67). Louis Pasteur first highlighted the impact of hypoxia on nutrient metabolism based on his observation that hypoxic yeast cells preferred to convert glucose into lactic acid rather than burning it in an oxidative fashion. The molecular basis for this shift in mammalian cells has been linked to the activity of the transcription factor HIF1 (810). Stabilization of the labile HIF1α subunit occurs in hypoxia. It can also occur in normoxia through several mechanisms including loss of the von Hippel-Lindau tumor suppressor (VHL), a common occurrence in renal carcinoma (11). Although hypoxia and/or HIF1α stabilization is a common feature of multiple cancers, to date the source of citrate in the setting of hypoxia or HIF activation has not been determined.

Here, we study the sources of hypoxic citrate synthesis in a glioblastoma cell line that proliferates in profound hypoxia (0.5% O2). Glucose uptake and conversion to lactic acid increased in hypoxia. However, glucose conversion into citrate dramatically declined. Glutamine consumption remained constant in hypoxia, and hypoxic cells were addicted to the use of glutamine in hypoxia as a source of α-ketoglutarate. Glutamine provided the major carbon source for citrate synthesis during hypoxia. However, the TCA cycle-dependent conversion of glutamine into citric acid was significantly suppressed. In contrast, there was a relative increase in glutamine-dependent citrate production in hypoxia that resulted from carboxylation of α-ketoglutarate. This reductive synthesis required the presence of mitochondrial isocitrate dehydrogenase 2 (IDH2). In confirmation of the reverse flux through IDH2, the increased reductive metabolism of glutamine-derived α-ketoglutarate in hypoxia was associated with increased synthesis of 2HG. Finally, constitutive HIF1α-expressing cells also demonstrated significant reductive-carboxylation-dependent synthesis of citrate in normoxia and a relative defect in the oxidative conversion of glutamine into citrate. Collectively, the data demonstrate that mitochondrial glutamine metabolism can be rerouted through IDH2-dependent citrate synthesis in support of hypoxic cell growth.

Some Cancer Cells Can Proliferate at 0.5% O2 Despite a Sharp Decline in Glucose-Dependent Citrate Synthesis.

At 21% O2, cancer cells have been shown to synthesize citrate by condensing glucose-derived acetyl-CoA with glutamine-derived oxaloacetate through the activity of the canonical TCA cycle enzyme citrate synthase (4). In contrast, less is known regarding the synthesis of citrate by cells that can continue proliferating in hypoxia. The glioblastoma cell line SF188 is able to proliferate at 0.5% O2 (Fig. 1A), a level of hypoxia that is sufficient to stabilize HIF1α (Fig. 1B) and predicted to limit respiration (1213). Consistent with previous observations in hypoxic cells, we found that SF188 cells demonstrated increased lactate production when incubated in hypoxia (Fig. 1C), and the ratio of lactate produced to glucose consumed increased demonstrating an increase in the rate of anaerobic glycolysis. When glucose-derived carbon in the form of pyruvate is converted to lactate, it is diverted away from subsequent metabolism that can contribute to citrate production. However, we observed that SF188 cells incubated in hypoxia maintain their intracellular citrate to ∼75% of the level maintained under normoxia (Fig. 1D). This prompted an investigation of how proliferating cells maintain citrate production under hypoxia.

SF188 glioblastoma cells proliferate at 0.5% O2 despite a profound reduction in glucose-dependent citrate synthesis.

SF188 glioblastoma cells proliferate at 0.5% O2 despite a profound reduction in glucose-dependent citrate synthesis.


Fig. 1. SF188 glioblastoma cells proliferate at 0.5% O2 despite a profound reduction in glucose-dependent citrate synthesis. (A) SF188 cells were plated in complete medium equilibrated with 21% O2 (Normoxia) or 0.5% O2 (Hypoxia), total viable cells were counted 24 h and 48 h later (Day 1 and Day 2), and population doublings were calculated. Data are the mean ± SEM of four independent experiments. (B) Western blot demonstrates stabilized HIF1α protein in cells cultured in hypoxia compared with normoxia. (C) Cells were grown in normoxia or hypoxia for 24 h, after which culture medium was collected. Medium glucose and lactate levels were measured and compared with the levels in fresh medium. (D) Cells were cultured for 24 h as in C. Intracellular metabolism was then quenched with 80% MeOH prechilled to −80 °C that was spiked with a 13C-labeled citrate as an internal standard. Metabolites were then extracted, and intracellular citrate levels were analyzed with GC-MS and normalized to cell number. Data for C and D are the mean ± SEM of three independent experiments. (E) Model depicting the pathway for cit+2 production from [U-13C]glucose. Glucose uniformly 13C-labeled will generate pyruvate+3. Pyruvate+3 can be oxidatively decarboxylated by PDH to produce acetyl-CoA+2, which can condense with unlabeled oxaloacetate to produce cit+2. (F) Cells were cultured for 24 h as in C and D, followed by an additional 4 h of culture in glucose-deficient medium supplemented with 10 mM [U-13C]glucose. Intracellular metabolites were then extracted, and 13C-enrichment in cellular citrate was analyzed by GC-MS and normalized to the total citrate pool size. Data are the mean ± SD of three independent cultures from a representative of two independent experiments. *P < 0.05, ***P < 0.001.

Increased glucose uptake and glycolytic metabolism are critical elements of the metabolic response to hypoxia. To evaluate the contributions made by glucose to the citrate pool under normoxia or hypoxia, SF188 cells incubated in normoxia or hypoxia were cultured in medium containing 10 mM [U-13C]glucose. Following a 4-h labeling period, cellular metabolites were extracted and analyzed for isotopic enrichment by gas chromatography-mass spectrometry (GC-MS). In normoxia, the major 13C-enriched citrate species found was citrate enriched with two 13C atoms (cit+2), which can arise from the NAD+-dependent decarboxylation of pyruvate+3 to acetyl-CoA+2 by PDH, followed by the condensation of acetyl-CoA+2 with unenriched oxaloacetate (Fig. 1 E and F). Compared with the accumulation of cit+2, we observed minimal accumulation of cit+3 and cit+5 under normoxia. Cit+3 arises from pyruvate carboxylase (PC)-dependent conversion of pyruvate+3 to oxaloacetate+3, followed by the condensation of oxaloacetate+3 with unenriched acetyl-CoA. Cit+5 arises when PC-generated oxaloacetate+3 condenses with PDH-generated acetyl-CoA+2. The lack of cit+3 and cit+5 accumulation is consistent with PC activity not playing a major role in citrate production in normoxic SF188 cells, as reported (4).

In hypoxic cells, the major citrate species observed was unenriched. Cit+2, cit+3, and cit+5 all constituted minor fractions of the total citrate pool, consistent with glucose carbon not being incorporated into citrate through either PDH or PC-mediated metabolism under hypoxic conditions (Fig. 1F). These data demonstrate that in contrast to normoxic cells, where a large percentage of citrate production depends on glucose-derived carbon, hypoxic cells significantly reduce their rate of citrate production from glucose.

Glutamine Carbon Metabolism Is Required for Viability in Hypoxia.

In addition to glucose, we have previously reported that glutamine can contribute to citrate production during cell growth under normoxic conditions (4). Surprisingly, under hypoxic conditions, we observed that SF188 cells retained their high rate of glutamine consumption (Fig. 2A). Moreover, hypoxic cells cultured in glutamine-deficient medium displayed a significant loss of viability (Fig. 2B). In normoxia, the requirement for glutamine to maintain viability of SF188 cells can be satisfied by α-ketoglutarate, the downstream metabolite of glutamine that is devoid of nitrogenous groups (14). α-ketoglutarate cannot fulfill glutamine’s roles as a nitrogen source for nonessential amino acid synthesis or as an amide donor for nucleotide or hexosamine synthesis, but can be metabolized through the oxidative TCA cycle to regenerate oxaloacetate, and subsequently condense with glucose-derived acetyl-CoA to produce citrate. To test whether the restoration of carbon from glutamine metabolism in the form of α-ketoglutarate could rescue the viability defect of glutamine-starved SF188 cells even under hypoxia, SF188 cells incubated in hypoxia were cultured in glutamine-deficient medium supplemented with a cell-penetrant form of α-ketoglutarate (dimethyl α-ketoglutarate). The addition of dimethyl α-ketoglutarate rescued the defect in cell viability observed upon glutamine withdrawal (Fig. 2B). These data demonstrate that, even under hypoxic conditions, when the ability of glutamine to replenish oxaloacetate through oxidative TCA cycle metabolism is diminished, SF188 cells retain their requirement for glutamine as the carbon backbone for α-ketoglutarate. This result raised the possibility that glutamine could be the carbon source for citrate production through an alternative, nonoxidative, pathway in hypoxia.

Glutamine carbon is required for hypoxic cell viability

Glutamine carbon is required for hypoxic cell viability

Glutamine carbon is required for hypoxic cell viability


Fig. 2. Glutamine carbon is required for hypoxic cell viability and contributes to increased citrate production through reductive carboxylation relative to oxidative metabolism in hypoxia. (A) SF188 cells were cultured for 24 h in complete medium equilibrated with either 21% O2 (Normoxia) or 0.5% O2(Hypoxia). Culture medium was then removed from cells and analyzed for glutamine levels which were compared with the glutamine levels in fresh medium. Data are the mean ± SEM of three independent experiments. (B) The requirement for glutamine to maintain hypoxic cell viability can be satisfied by α-ketoglutarate. Cells were cultured in complete medium equilibrated with 0.5% O2 for 24 h, followed by an additional 48 h at 0.5% O2 in either complete medium (+Gln), glutamine-deficient medium (−Gln), or glutamine-deficient medium supplemented with 7 mM dimethyl α-ketoglutarate (−Gln +αKG). All medium was preconditioned in 0.5% O2. Cell viability was determined by trypan blue dye exclusion. Data are the mean and range from two independent experiments. (C) Model depicting the pathways for cit+4 and cit+5 production from [U-13C]glutamine (glutamine+5). Glutamine+5 is catabolized to α-ketoglutarate+5, which can then contribute to citrate production by two divergent pathways. Oxidative metabolism produces oxaloacetate+4, which can condense with unlabeled acetyl-CoA to produce cit+4. Alternatively, reductive carboxylation produces isocitrate+5, which can isomerize to cit+5. (D) Glutamine contributes to citrate production through increased reductive carboxylation relative to oxidative metabolism in hypoxic proliferating cancer cells. Cells were cultured for 24 h as in A, followed by 4 h of culture in glutamine-deficient medium supplemented with 4 mM [U-13C]glutamine. 13C enrichment in cellular citrate was quantitated with GC-MS. Data are the mean ± SD of three independent cultures from a representative of three independent experiments. **P < 0.01.

Cells Proliferating in Hypoxia Maintain Levels of Additional Metabolites Through Reductive Carboxylation.

Previous work has documented that, in normoxic conditions, SF188 cells use glutamine as the primary anaplerotic substrate, maintaining the pool sizes of TCA cycle intermediates through oxidative metabolism (4). Surprisingly, we found that, when incubated in hypoxia, SF188 cells largely maintained their levels of aspartate (in equilibrium with oxaloacetate), malate, and fumarate (Fig. 3A). To distinguish how glutamine carbon contributes to these metabolites in normoxia and hypoxia, SF188 cells incubated in normoxia or hypoxia were cultured in medium containing 4 mM [U-13C]glutamine. After a 4-h labeling period, metabolites were extracted and the intracellular pools of aspartate, malate, and fumarate were analyzed by GC-MS.

In normoxia, the majority of the enriched intracellular asparatate, malate, and fumarate were the +4 species, which arise through oxidative metabolism of glutamine-derived α-ketoglutarate (Fig. 3 B and C). The +3 species, which can be derived from the citrate generated by the reductive carboxylation of glutamine-derived α-ketoglutarate, constituted a significantly lower percentage of the total aspartate, malate, and fumarate pools. By contrast, in hypoxia, the +3 species constituted a larger percentage of the total aspartate, malate, and fumarate pools than they did in normoxia. These data demonstrate that, in addition to citrate, hypoxic cells preferentially synthesize oxaloacetate, malate, and fumarate through the pathway of reductive carboxylation rather than the oxidative TCA cycle.

IDH2 Is Critical in Hypoxia for Reductive Metabolism of Glutamine and for Cell Proliferation.

We hypothesized that the relative increase in reductive carboxylation we observed in hypoxia could arise from the suppression of α-ketoglutarate oxidation through the TCA cycle. Consistent with this, we found that α-ketoglutarate levels increased in SF188 cells following 24 h in hypoxia (Fig. 4A). Surprisingly, we also found that levels of the closely related metabolite 2-hydroxyglutarate (2HG) increased in hypoxia, concomitant with the increase in α-ketoglutarate under these conditions. 2HG can arise from the noncarboxylating reduction of α-ketoglutarate (Fig. 4B). Recent work has found that specific cancer-associated mutations in the active sites of either IDH1 or IDH2 lead to a 10- to 100-fold enhancement in this activity facilitating 2HG production (1517), but SF188 cells lack IDH1/2 mutations. However, 2HG levels are also substantially elevated in the inborn error of metabolism 2HG aciduria, and the majority of patients with this disease lack IDH1/2 mutations. As 2HG has been demonstrated to arise in these patients from mitochondrial α-ketoglutarate (18), we hypothesized that both the increased reductive carboxylation of glutamine-derived α-ketoglutarate to citrate and the increased 2HG accumulation we observed in hypoxia could arise from increased reductive metabolism by wild-type IDH2 in the mitochondria.

Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2

Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2

Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2


Fig. 4. Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2. (A) α-ketoglutarate and 2HG increase in hypoxia. SF188 cells were cultured in complete medium equilibrated with either 21% O2 (Normoxia) or 0.5% O2 (Hypoxia) for 24 h. Intracellular metabolites were then extracted, cell extracts spiked with a 13C-labeled citrate as an internal standard, and intracellular α-ketoglutarate and 2HG levels were analyzed with GC-MS. Data shown are the mean ± SEM of three independent experiments. (B) Model for reductive metabolism from glutamine-derived α-ketoglutarate. Glutamine+5 is catabolized to α-ketoglutarate+5. Carboxylation of α-ketoglutarate+5 followed by reduction of the carboxylated intermediate (reductive carboxylation) will produce isocitrate+5, which can then isomerize to cit+5. In contrast, reductive activity on α-ketoglutarate+5 that is uncoupled from carboxylation will produce 2HG+5. (C) IDH2 is required for reductive metabolism of glutamine-derived α-ketoglutarate in hypoxia. SF188 cells transfected with a siRNA against IDH2 (siIDH2) or nontargeting negative control (siCTRL) were cultured for 2 d in complete medium equilibrated with 0.5% O2. (Upper) Cells were then cultured at 0.5% O2 for an additional 4 h in glutamine-deficient medium supplemented with 4 mM [U-13C]glutamine. 13C enrichment in intracellular citrate and 2HG was determined and normalized to the relevant metabolite total pool size. (Lower) Cells transfected and cultured in parallel at 0.5% O2 were counted by hemacytometer (excluding nonviable cells with trypan blue staining) or harvested for protein to assess IDH2 expression by Western blot. Data shown for GC-MS and cell counts are the mean ± SD of three independent cultures from a representative experiment. **P < 0.01, ***P < 0.001.

In an experiment to test this hypothesis, SF188 cells were transfected with either siRNA directed against mitochondrial IDH2 (siIDH2) or nontargeting control, incubated in hypoxia for 2 d, and then cultured for another 4 h in hypoxia in media containing 4 mM [U-13C]glutamine. After the labeling period, metabolites were extracted and analyzed by GC-MS (Fig. 4C). Hypoxic SF188 cells transfected with siIDH2 displayed a decreased contribution of cit+5 to the total citrate pool, supporting an important role for IDH2 in the reductive carboxylation of glutamine-derived α-ketoglutarate in hypoxic conditions. The contribution of cit+4 to the total citrate pool did not decrease with siIDH2 treatment, consistent with IDH2 knockdown specifically affecting the pathway of reductive carboxylation and not other fundamental TCA cycle-regulating processes. In confirmation of reverse flux occurring through IDH2, the contribution of 2HG+5 to the total 2HG pool decreased in siIDH2-treated cells. Supporting the importance of citrate production by IDH2-mediated reductive carboxylation for hypoxic cell proliferation, siIDH2-transfected SF188 cells displayed a defect in cellular accumulation in hypoxia. Decreased expression of IDH2 protein following siIDH2 transfection was confirmed by Western blot. Collectively, these data point to the importance of mitochondrial IDH2 for the increase in reductive carboxylation flux of glutamine-derived α-ketoglutarate to maintain citrate levels in hypoxia, and to the importance of this reductive pathway for hypoxic cell proliferation.

Reprogramming of Metabolism by HIF1 in the Absence of Hypoxia Is Sufficient to Induce Increased Citrate Synthesis by Reductive Carboxylation Relative to Oxidative Metabolism.

The relative increase in the reductive metabolism of glutamine-derived α-ketoglutarate at 0.5% O2 may be explained by the decreased ability to carry out oxidative NAD+-dependent reactions as respiration is inhibited (1213). However, a shift to preferential reductive glutamine metabolism could also result from the active reprogramming of cellular metabolism by HIF1 (810), which inhibits the generation of mitochondrial acetyl-CoA necessary for the synthesis of citrate by oxidative glucose and glutamine metabolism (Fig. 5A). To better understand the role of HIF1 in reductive glutamine metabolism, we used VHL-deficient RCC4 cells, which display constitutive expression of HIF1α under normoxia (Fig. 5B). RCC4 cells expressing either a nontargeting control shRNA (shCTRL) or an shRNA directed at HIF1α (shHIF1α) were incubated in normoxia and cultured in medium with 4 mM [U-13C]glutamine. Following a 4-h labeling period, metabolites were extracted and the cellular citrate pool was analyzed by GC-MS. In shCTRL cells, which have constitutive HIF1α expression despite incubation in normoxia, the majority of the total citrate pool was constituted by the cit+5 species, with low levels of all other species including cit+4 (Fig. 5C). By contrast, in HIF1α-deficient cells the contribution of cit+5 to the total citrate pool was greatly decreased, whereas the contribution of cit+4 to the total citrate pool increased and was the most abundant citrate species. These data demonstrate that the relative enhancement of the reductive carboxylation pathway for citrate synthesis can be recapitulated by constitutive HIF1 activation in normoxia.

Reprogramming of metabolism by HIF1 in the absence of hypoxia

Reprogramming of metabolism by HIF1 in the absence of hypoxia


Reprogramming of metabolism by HIF1 in the absence of hypoxia is sufficient to induce reductive carboxylation of glutamine-derived α-ketoglutarate.

Fig. 5. Reprogramming of metabolism by HIF1 in the absence of hypoxia is sufficient to induce reductive carboxylation of glutamine-derived α-ketoglutarate. (A) Model depicting how HIF1 signaling’s inhibition of pyruvate dehydrogenase (PDH) activity and promotion of lactate dehydrogenase-A (LDH-A) activity can block the generation of mitochondrial acetyl-CoA from glucose-derived pyruvate, thereby favoring citrate synthesis from reductive carboxylation of glutamine-derived α-ketoglutarate. (B) Western blot demonstrating HIF1α protein in RCC4 VHL−/− cells in normoxia with a nontargeting shRNA (shCTRL), and the decrease in HIF1α protein in RCC4 VHL−/− cells stably expressing HIF1α shRNA (shHIF1α). (C) HIF1-induced reprogramming of glutamine metabolism. Cells from B at 21% O2 were cultured for 4 h in glutamine-deficient medium supplemented with 4 mM [U-13C]glutamine. Intracellular metabolites were then extracted, and 13C enrichment in cellular citrate was determined by GC-MS. Data shown are the mean ± SD of three independent cultures from a representative of three independent experiments. ***P < 0.001.

Compared with glucose metabolism, much less is known regarding how glutamine metabolism is altered under hypoxia. It has also remained unclear how hypoxic cells can maintain the citrate production necessary for macromolecular biosynthesis. In this report, we demonstrate that in contrast to cells at 21% O2, where citrate is predominantly synthesized through oxidative metabolism of both glucose and glutamine, reductive carboxylation of glutamine carbon becomes the major pathway of citrate synthesis in cells that can effectively proliferate at 0.5% O2. Moreover, we show that in these hypoxic cells, reductive carboxylation of glutamine-derived α-ketoglutarate is dependent on mitochondrial IDH2. Although others have previously suggested the existence of reductive carboxylation in cancer cells (1920), these studies failed to demonstrate the intracellular localization or specific IDH isoform responsible for the reductive carboxylation flux. Recently, we identified IDH2 as an isoform that contributes to reductive carboxylation in cancer cells incubated at 21% O2 (16), but remaining unclear were the physiological importance and regulation of this pathway relative to oxidative metabolism, as well as the conditions where this reductive pathway might be advantageous for proliferating cells.

Here we report that IDH2-mediated reductive carboxylation of glutamine-derived α-ketoglutarate to citrate is an important feature of cells proliferating in hypoxia. Moreover, the reliance on reductive glutamine metabolism can be recapitulated in normoxia by constitutive HIF1 activation in cells with loss of VHL. The mitochondrial NADPH/NADP+ ratio required to fuel the reductive reaction through IDH2 can arise from the increased NADH/NAD+ ratio existing in the mitochondria under hypoxic conditions (2122), with the transfer of electrons from NADH to NADP+ to generate NADPH occurring through the activity of the mitochondrial transhydrogenase (23). Our data do not exclude a complementary role for cytosolic IDH1 in impacting reductive glutamine metabolism, potentially through its oxidative function in an IDH2/IDH1 shuttle that transfers high energy electrons in the form of NADPH from mitochondria to cytosol (1624).

In further support of the increased mitochondrial reductive glutamine metabolism that we observe in hypoxia, we report here that incubation in hypoxia can lead to elevated 2HG levels in cells lacking IDH1/2 mutations. 2HG production from glutamine-derived α-ketoglutarate significantly decreased with knockdown of IDH2, supporting the conclusion that 2HG is produced in hypoxia by enhanced reverse flux of α-ketoglutarate through IDH2 in a truncated, noncarboxylating reductive reaction. However, other mechanisms may also contribute to 2HG elevation in hypoxia. These include diminished oxidative activity and/or enhanced reductive activity of the 2HG dehydrogenase, a mitochondrial enzyme that normally functions to oxidize 2HG back to α-ketoglutarate (25). The level of 2HG elevation we observe in hypoxic cells is associated with a concomitant increase in α-ketoglutarate, and is modest relative to that observed in cancers with IDH1/2 gain-of-function mutations. Nonetheless, 2HG elevation resulting from hypoxia in cells with wild-type IDH1/2 may hold promise as a cellular or serum biomarker for tissues undergoing chronic hypoxia and/or excessive glutamine metabolism.

The IDH2-dependent reductive carboxylation pathway that we propose in this report allows for continued citrate production from glutamine carbon when hypoxia and/or HIF1 activation prevents glucose carbon from contributing to citrate synthesis. Moreover, as opposed to continued oxidative TCA cycle functioning in hypoxia which can increase reactive oxygen species (ROS), reductive carboxylation of α-ketoglutarate in the mitochondria may serve as an electron sink that decreases the generation of ROS. HIF1 activity is not limited to the setting of hypoxia, as a common feature of several cancers is the normoxic stabilization of HIF1α through loss of the VHL tumor suppressor or other mechanisms. We demonstrate here that altered glutamine metabolism through a mitochondrial reductive pathway is a central aspect of hypoxic proliferating cell metabolism and HIF1-induced metabolic reprogramming. These findings are relevant for the understanding of numerous constitutive HIF1-expressing malignancies, as well as for populations, such as stem progenitor cells, which frequently proliferate in hypoxic conditions.

7.9.3 Hypoxia-Inducible Factors in Physiology and Medicine

Gregg L. Semenza
Cell. 2012 Feb 3; 148(3): 399–408.

Oxygen homeostasis represents an organizing principle for understanding metazoan evolution, development, physiology, and pathobiology. The hypoxia-inducible factors (HIFs) are transcriptional activators that function as master regulators of oxygen homeostasis in all metazoan species. Rapid progress is being made in elucidating homeostatic roles of HIFs in many physiological systems, determining pathological consequences of HIF dysregulation in chronic diseases, and investigating potential targeting of HIFs for therapeutic purposes. Oxygen homeostasis represents an organizing principle for understanding metazoan evolution, development, physiology, and pathobiology. The hypoxia-inducible factors (HIFs) are transcriptional activators that function as master regulators of oxygen homeostasis in all metazoan species. Rapid progress is being made in elucidating homeostatic roles of HIFs in many physiological systems, determining pathological consequences of HIF dysregulation in chronic diseases, and investigating potential targeting of HIFs for therapeutic purposes.


Oxygen is central to biology because of its utilization in the process of respiration. O2 serves as the final electron acceptor in oxidative phosphorylation, which carries with it the risk of generating reactive oxygen species (ROS) that react with cellular macromolecules and alter their biochemical or physical properties, resulting in cell dysfunction or death. As a consequence, metazoan organisms have evolved elaborate cellular metabolic and systemic physiological systems that are designed to maintain oxygen homeostasis. This review will focus on the role of hypoxia-inducible factors (HIFs) as master regulators of oxygen homeostasis and, in particular, on recent advances in understanding their roles in physiology and medicine. Due to space limitations and the remarkably pleiotropic effects of HIFs, the description of such roles will be illustrative rather than comprehensive.

O2 and Evolution, Part 1

Accumulation of O2 in Earth’s atmosphere starting ~2.5 billion years ago led to evolution of the extraordinarily efficient system of oxidative phosphorylation that transfers chemical energy stored in carbon bonds of organic molecules to the high-energy phosphate bond in ATP, which is used to power physicochemical reactions in living cells. Energy produced by mitochondrial respiration is sufficient to power the development and maintenance of multicellular organisms, which could not be sustained by energy produced by glycolysis alone (Lane and Martin, 2010). The modest dimensions of primitive metazoan species were such that O2 could diffuse from the atmosphere to all of the organism’s thousand cells, as is the case for the worm Caenorhabditis elegans. To escape the constraints placed on organismal growth by diffusion, systems designed to conduct air to cells deep within the body evolved and were sufficient for O2delivery to organisms with hundreds of thousands of cells, such as the fly Drosophila melanogaster. The final leap in body scale occurred in vertebrates and was associated with the evolution of complex respiratory, circulatory, and nervous systems designed to efficiently capture and distribute O2 to hundreds of millions of millions of cells in the case of the adult Homo sapiens.

Hypoxia-Inducible Factors

Hypoxia-inducible factor 1 (HIF-1) is expressed by all extant metazoan species analyzed (Loenarz et al., 2011). HIF-1 consists of HIF-1α and HIF-1β subunits, which each contain basic helix-loop-helix-PAS (bHLH-PAS) domains (Wang et al., 1995) that mediate heterodimerization and DNA binding (Jiang et al., 1996a). HIF-1β heterodimerizes with other bHLH-PAS proteins and is present in excess, such that HIF-1α protein levels determine HIF-1 transcriptional activity (Semenza et al., 1996).

Under well-oxygenated conditions, HIF-1α is bound by the von Hippel-Lindau (VHL) protein, which recruits an ubiquitin ligase that targets HIF-1α for proteasomal degradation (Kaelin and Ratcliffe, 2008). VHL binding is dependent upon hydroxylation of a specific proline residue in HIF-1α by the prolyl hydroxylase PHD2, which uses O2 as a substrate such that its activity is inhibited under hypoxic conditions (Epstein et al., 2001). In the reaction, one oxygen atom is inserted into the prolyl residue and the other atom is inserted into the co-substrate α-ketoglutarate, splitting it into CO2 and succinate (Kaelin and Ratcliffe, 2008). Factor inhibiting HIF-1 (FIH-1) represses HIF-1α transactivation function (Mahon et al., 2001) by hydroxylating an asparaginyl residue, using O2 and α-ketoglutarate as substrates, thereby blocking the association of HIF-1α with the p300 coactivator protein (Lando et al., 2002). Dimethyloxalylglycine (DMOG), a competitive antagonist of α-ketoglutarate, inhibits the hydroxylases and induces HIF-1-dependent transcription (Epstein et al., 2001). HIF-1 activity is also induced by iron chelators (such as desferrioxamine) and cobalt chloride, which inhibit hydroxylases by displacing Fe(II) from the catalytic center (Epstein et al., 2001).

Studies in cultured cells (Jiang et al., 1996b) and isolated, perfused, and ventilated lung preparations (Yu et al., 1998) revealed an exponential increase in HIF-1α levels at O2 concentrations less than 6% (~40 mm Hg), which is not explained by known biochemical properties of the hydroxylases. In most adult tissues, O2concentrations are in the range of 3-5% and any decrease occurs along the steep portion of the dose-response curve, allowing a graded response to hypoxia. Analyses of cultured human cells have revealed that expression of hundreds of genes was increased in response to hypoxia in a HIF-1-dependent manner (as determined by RNA interference) with direct binding of HIF-1 to the gene (as determined by chromatin immunoprecipitation [ChIP] assays); in addition, the expression of hundreds of genes was decreased in response to hypoxia in a HIF-1-dependent manner but binding of HIF-1 to these genes was not detected (Mole et al., 2009), indicating that HIF-dependent repression occurs via indirect mechanisms, which include HIF-1-dependent expression of transcriptional repressors (Yun et al., 2002) and microRNAs (Kulshreshtha et al., 2007). ChIP-seq studies have revealed that only 40% of HIF-1 binding sites are located within 2.5 kb of the transcription start site (Schödel et al., 2011).

In vertebrates, HIF-2α is a HIF-1α paralog that is also regulated by prolyl and asparaginyl hydroxylation and dimerizes with HIF-1β, but is expressed in a cell-restricted manner and plays important roles in erythropoiesis, vascularization, and pulmonary development, as described below. In D. melanogaster, the gene encoding the HIF-1α ortholog is designated similar and its paralog is designated trachealess because inactivating mutations result in defective development of the tracheal tubes (Wilk et al., 1996). In contrast, C. elegans has only a single HIF-1α homolog (Epstein et al., 2001). Thus, in both invertebrates and vertebrates, evolution of specialized systems for O2 delivery was associated with the appearance of a HIF-1α paralog.

O2 and Metabolism

The regulation of metabolism is a principal and primordial function of HIF-1. Under hypoxic conditions, HIF-1 mediates a transition from oxidative to glycolytic metabolism through its regulation of: PDK1, encoding pyruvate dehydrogenase (PDH) kinase 1, which phosphorylates and inactivates PDH, thereby inhibiting the conversion of pyruvate to acetyl coenzyme A for entry into the tricarboxylic acid cycle (Kim et al., 2006Papandreou et al., 2006); LDHA, encoding lactate dehydrogenase A, which converts pyruvate to lactate (Semenza et al. 1996); and BNIP3 (Zhang et al. 2008) and BNIP3L (Bellot et al., 2009), which mediate selective mitochondrial autophagy (Figure 1). HIF-1 also mediates a subunit switch in cytochrome coxidase that improves the efficiency of electron transfer under hypoxic conditions (Fukuda et al., 2007). An analogous subunit switch is also observed in Saccharomyces cerevisiae, although it is mediated by a completely different mechanism (yeast lack HIF-1), suggesting that it may represent a fundamental response of eukaryotic cells to hypoxia.

Regulation of Glucose Metabolism nihms-350382-f0001

Regulation of Glucose Metabolism nihms-350382-f0001

Regulation of Glucose Metabolism

Figure 1
Regulation of Glucose Metabolism

It is conventional wisdom that cells switch to glycolysis when O2 becomes limiting for mitochondrial ATP production. Yet, HIF-1α-null mouse embryo fibroblasts, which do not down-regulate respiration under hypoxic conditions, have higher ATP levels at 1% O2 than wild-type cells at 20% O2, demonstrating that under these conditions O2 is not limiting for ATP production (Zhang et al., 2008). However, the HIF-1α-null cells die under prolonged hypoxic conditions due to ROS toxicity (Kim et al. 2006Zhang et al., 2008). These studies have led to a paradigm shift with regard to our understanding of the regulation of cellular metabolism (Semenza, 2011): the purpose of this switch is to prevent excess mitochondrial generation of ROS that would otherwise occur due to the reduced efficiency of electron transfer under hypoxic conditions (Chandel et al., 1998). This may be particularly important in stem cells, in which avoidance of DNA damage is critical (Suda et al., 2011).

Role of HIFs in Development

Much of mammalian embryogenesis occurs at O2 concentrations of 1-5% and O2 functions as a morphogen (through HIFs) in many developmental systems (Dunwoodie, 2009). Mice that are homozygous for a null allele at the locus encoding HIF-1α die by embryonic day 10.5 with cardiac malformations, vascular defects, and impaired erythropoiesis, indicating that all three components of the circulatory system are dependent upon HIF-1 for normal development (Iyer et al., 1998Yoon et al., 2011). Depending on the genetic background, mice lacking HIF-2α: die by embryonic day 12.5 with vascular defects (Peng et al., 2000) or bradycardia due to deficient catecholamine production (Tian et al., 1998); die as neonates due to impaired lung maturation (Compernolle et al., 2002); or die several months after birth due to ROS-mediated multi-organ failure (Scortegagna et al., 2003). Thus, while vertebrate evolution was associated with concomitant appearance of the circulatory system and HIF-2α, both HIF-1 and HIF-2 have important roles in circulatory system development. Conditional knockout of HIF-1α in specific cell types has demonstrated important roles in chondrogenesis (Schipani et al., 2001), adipogenesis (Yun et al., 2002), B-lymphocyte development (Kojima et al., 2002), osteogenesis (Wang et al., 2007), hematopoiesis (Takubo et al., 2010), T-lymphocyte differentiation (Dang et al., 2011), and innate immunity (Zinkernagel et al., 2007). While knockout mouse experiments point to the adverse effects of HIF-1 loss-of-function on development, it is also possible that increased HIF-1 activity, induced by hypoxia in embryonic tissues as a result of abnormalities in placental blood flow, may also dysregulate development and result in congenital malformations. For example, HIF-1α has been shown to interact with, and stimulate the transcriptional activity of, Notch, which plays a key role in many developmental pathways (Gustafsson et al., 2005).

Translational Prospects

Drug discovery programs have been initiated at many pharmaceutical and biotech companies to develop prolyl hydroxylase inhibitors (PHIs) that, as described above for DMOG, induce HIF activity for treatment of disorders in which HIF mediates protective physiological responses. Local and/or short term induction of HIF activity by PHIs, gene therapy, or other means are likely to be useful novel therapies for many of the diseases described above. In the case of ischemic cardiovascular disease, local therapy is needed to provide homing signals for the recruitment of BMDACs. Chronic systemic use of PHIs must be approached with great caution: individuals with genetic mutations that constitutively activate the HIF pathway (described below) have increased incidence of cardiovascular disease and mortality (Yoon et al., 2011). On the other hand, the profound inhibition of HIF activity and vascular responses to ischemia that are associated with aging suggest that systemic replacement therapy might be contemplated as a preventive measure for subjects in whom impaired HIF responses to hypoxia can be documented. In C. elegans, VHL loss-of-function increases lifespan in a HIF-1-dependent manner (Mehta et al., 2009), providing further evidence for a mutually antagonistic relationship between HIF-1 and aging.


Cancers contain hypoxic regions as a result of high rates of cell proliferation coupled with the formation of vasculature that is structurally and functionally abnormal. Increased HIF-1α and/or HIF-2α levels in diagnostic tumor biopsies are associated with increased risk of mortality in cancers of the bladder, brain, breast, colon, cervix, endometrium, head/neck, lung, ovary, pancreas, prostate, rectum, and stomach; these results are complemented by experimental studies, which demonstrate that genetic manipulations that increase HIF-1α expression result in increased tumor growth, whereas loss of HIF activity results in decreased tumor growth (Semenza, 2010). HIFs are also activated by genetic alterations, most notably, VHL loss of function in clear cell renal carcinoma (Majmunder et al., 2010). HIFs activate transcription of genes that play key roles in critical aspects of cancer biology, including stem cell maintenance (Wang et al., 2011), cell immortalization, epithelial-mesenchymal transition (Mak et al., 2010), genetic instability (Huang et al., 2007), vascularization (Liao and Johnson, 2007), glucose metabolism (Luo et al., 2011), pH regulation (Swietach et al., 2007), immune evasion (Lukashev et al., 2007), invasion and metastasis (Chan and Giaccia, 2007), and radiation resistance (Moeller et al., 2007). Given the extensive validation of HIF-1 as a potential therapeutic target, drugs that inhibit HIF-1 have been identified and shown to have anti-cancer effects in xenograft models (Table 1Semenza, 2010).

Table 1  Drugs that Inhibit HIF-1

Process Inhibited Drug Class Prototype
HIF-1 α synthesis Cardiac glycosidemTOR inhibitorMicrotubule targeting agent

Topoisomerase I inhibitor



HIF-1 α protein stability HDAC inhibitorHSP90 inhibitorCalcineurin inhibitor

Guanylate cyclase activator



Heterodimerization Antimicrobial agent Acriflavine
DNA binding AnthracyclineQuinoxaline antibiotic DoxorubicinEchinomycin
Transactivation Proteasome inhibitorAntifungal agent BortezomibAmphotericin B
Signal transduction BCR-ABL inhibitorCyclooxygenase inhibitorEGFR inhibitor

HER2 inhibitor

ImatinibIbuprofenErlotinib, Gefitinib


Over 100 women die every day of breast cancer in the U.S. The mean PO2 is 10 mm Hg in breast cancer as compared to > 60 mm Hg in normal breast tissue and cancers with PO2 < 10 mm Hg are associated with increased risk of metastasis and patient mortality (Vaupel et al., 2004). Increased HIF-1α protein levels, as identified by immunohistochemical analysis of tumor biopsies, are associated with increased risk of metastasis and/or patient mortality in unselected breast cancer patients and in lymph node-positive, lymph node-negative, HER2+, or estrogen receptor+ subpopulations (Semenza, 2011). Metastasis is responsible for > 90% of breast cancer mortality. The requirement for HIF-1 in breast cancer metastasis has been demonstrated for both autochthonous tumors in transgenic mice (Liao et al., 2007) and orthotopic transplants in immunodeficient mice (Zhang et al., 2011Wong et al., 2011). Primary tumors direct the recruitment of bone marrow-derived cells to the lungs and other sites of metastasis (Kaplan et al., 2005). In breast cancer, hypoxia induces the expression of lysyl oxidase (LOX), a secreted protein that remodels collagen at sites of metastatic niche formation (Erler et al., 2009). In addition to LOX, breast cancers also express LOX-like proteins 2 and 4. LOX, LOXL2, and LOXL4 are all HIF-1-regulated genes and HIF-1 inhibition blocks metastatic niche formation regardless of which LOX/LOXL protein is expressed, whereas available LOX inhibitors are not effective against all LOXL proteins (Wong et al., 2011), again illustrating the role of HIF-1 as a master regulator that controls the expression of multiple genes involved in a single (patho)physiological process.

Translational Prospects

Small molecule inhibitors of HIF activity that have anti-cancer effects in mouse models have been identified (Table 1). Inhibition of HIF impairs both vascular and metabolic adaptations to hypoxia, which may decrease O2 delivery and increase O2 utilization. These drugs are likely to be useful (as components of multidrug regimens) in the treatment of a subset of cancer patients in whom high HIF activity is driving progression. As with all novel cancer therapeutics, successful translation will require the development of methods for identifying the appropriate patient cohort. Effects of combination drug therapy also need to be considered. VEGF receptor tyrosine kinase inhibitors, which induce tumor hypoxia by blocking vascularization, have been reported to increase metastasis in mouse models (Ebos et al., 2009), which may be mediated by HIF-1; if so, combined use of HIF-1 inhibitors with these drugs may prevent unintended counter-therapeutic effects.

HIF inhibitors may also be useful in the treatment of other diseases in which dysregulated HIF activity is pathogenic. Proof of principle has been established in mouse models of ocular neovascularization, a major cause of blindness in the developed world, in which systemic or intraocular injection of the HIF-1 inhibitor digoxin is therapeutic (Yoshida et al., 2010). Systemic administration of HIF inhibitors for cancer therapy would be contraindicated in patients who also have ischemic cardiovascular disease, in which HIF activity is protective. The analysis of SNPs at the HIF1A locus described above suggests that the population may include HIF hypo-responders, who are at increased risk of severe ischemic cardiovascular disease. It is also possible that HIF hyper-responders, such as individuals with hereditary erythrocytosis, are at increased risk of particularly aggressive cancer.

O2 and Evolution, Part 2

When lowlanders sojourn to high altitude, hypobaric hypoxia induces erythropoiesis, which is a relatively ineffective response because the problem is not insufficient red cells, but rather insufficient ambient O2. Chronic erythrocytosis increases the risk of heart attack, stroke, and fetal loss during pregnancy. Many high-altitude Tibetans maintain the same hemoglobin concentration as lowlanders and yet, despite severe hypoxemia, they also maintain aerobic metabolism. The basis for this remarkable evolutionary adaptation appears to have involved the selection of genetic variants at multiple loci encoding components of the oxygen sensing system, particularly HIF-2α (Beall et al., 2010Simonson et al., 2010Yi et al., 2010). Given that hereditary erythrocytosis is associated with modest HIF-2α gain-of-function, the Tibetan genotype associated with absence of an erythrocytotic response to hypoxia may encode reduced HIF-2α activity along with other alterations that increase metabolic efficiency. Delineating the molecular mechanisms underlying these metabolic adaptations may lead to novel therapies for ischemic disorders, illustrating the importance of oxygen homeostasis as a nexus where evolution, biology, and medicine converge.

7.9.4 Hypoxia-inducible factor 1. Regulator of mitochondrial metabolism and mediator of ischemic preconditioning

Semenza GL1.
Biochim Biophys Acta. 2011 Jul; 1813(7):1263-8.

Hypoxia-inducible factor 1 (HIF-1) mediates adaptive responses to reduced oxygen availability by regulating gene expression. A critical cell-autonomous adaptive response to chronic hypoxia controlled by HIF-1 is reduced mitochondrial mass and/or metabolism. Exposure of HIF-1-deficient fibroblasts to chronic hypoxia results in cell death due to excessive levels of reactive oxygen species (ROS). HIF-1 reduces ROS production under hypoxic conditions by multiple mechanisms including: a subunit switch in cytochrome c oxidase from the COX4-1 to COX4-2 regulatory subunit that increases the efficiency of complex IV; induction of pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; induction of BNIP3, which triggers mitochondrial selective autophagy; and induction of microRNA-210, which blocks assembly of Fe/S clusters that are required for oxidative phosphorylation. HIF-1 is also required for ischemic preconditioning and this effect may be due in part to its induction of CD73, the enzyme that produces adenosine. HIF-1-dependent regulation of mitochondrial metabolism may also contribute to the protective effects of ischemic preconditioning.

The story of life on Earth is a tale of oxygen production and utilization. Approximately 3 billion years ago, primitive single-celled organisms evolved the capacity for photosynthesis, a biochemical process in which photons of solar energy are captured by chlorophyll and used to power the reaction of CO2 and H2O to form glucose and O2. The subsequent rise in the atmospheric O2 concentration over the next billion years set the stage for the ascendance of organisms with the capacity for respiration, a process that consumes glucose and O2 and generates CO2, H2O, and energy in the form of ATP. Some of these single-celled organisms eventually took up residence within the cytoplasm of other cells and devoted all of their effort to energy production as mitochondria. Compared to the conversion of glucose to lactate by glycolysis, the complete oxidation of glucose by respiration provided such a large increase in energy production that it made possible the evolution of multicellular organisms. Among metazoan organisms, the progressive increase in body size during evolution was accompanied by progressively more complex anatomic structures that function to ensure the adequate delivery of O2 to all cells, ultimately resulting in the sophisticated circulatory and respiratory systems of vertebrates.

All metazoan cells can sense and respond to reduced O2 availability (hypoxia). Adaptive responses to hypoxia can be cell autonomous, such as the alterations in mitochondrial metabolism that are described below, or non-cell-autonomous, such as changes in tissue vascularization (reviewed in ref. 1). Primary responses to hypoxia need to be distinguished from secondary responses to sequelae of hypoxia, such as the adaptive responses to ATP depletion that are mediated by AMP kinase (reviewed in ref 2). In contrast, recent data suggest that O2 and redox homeostasis are inextricably linked and that changes in oxygenation are inevitably associated with changes in the levels of reactive oxygen species (ROS), as will be discussed below.

HIF-1 Regulates Oxygen Homeostasis in All Metazoan Species

A key regulator of the developmental and physiological networks required for the maintenance of O2homeostasis is hypoxia-inducible factor 1 (HIF-1). HIF-1 is a heterodimeric transcription factor that is composed of an O2-regulated HIF-1α subunit and a constitutively expressed HIF-1β subunit [3,4]. HIF-1 regulates the expression of hundreds of genes through several major mechanisms. First, HIF-1 binds directly to hypoxia response elements, which are cis-acting DNA sequences located within target genes [5]. The binding of HIF-1 results in the recruitment of co-activator proteins that activate gene transcription (Fig. 1A). Only rarely does HIF-1 binding result in transcriptional repression [6]. Instead, HIF-1 represses gene expression by indirect mechanisms, which are described below. Second, among the genes activated by HIF-1 are many that encode transcription factors [7], which when synthesized can bind to and regulate (either positively or negatively) secondary batteries of target genes (Fig. 1B). Third, another group of HIF-1 target genes encode members of the Jumonji domain family of histone demethylases [8,9], which regulate gene expression by modifying chromatin structure (Fig. 1C). Fourth, HIF-1 can activate the transcription of genes encoding microRNAs [10], which bind to specific mRNA molecules and either block their translation or mediate their degradation (Fig. 1D). Fifth, the isolated HIF-1α subunit can bind to other transcription factors [11,12] and inhibit (Fig. 1E) or potentiate (Fig. 1F) their activity.

Mechanisms by which HIF-1 regulates gene expression. nihms232046f1

Mechanisms by which HIF-1 regulates gene expression. nihms232046f1

Mechanisms by which HIF-1 regulates gene expression.


Fig. 1 Mechanisms by which HIF-1 regulates gene expression. (A) Top: HIF-1 binds directly to target genes at a cis-acting hypoxia response element (HRE) and recruits coactivator proteins such as p300 to increase gene transcription.

HIF-1α and HIF-1β are present in all metazoan species, including the simple roundworm Caenorhabitis elegans [13], which consists of ~103 cells and has no specialized systems for O2 delivery. The fruit flyDrosophila melanogaster evolved tracheal tubes, which conduct air into the interior of the body from which it diffuses to surrounding cells. In vertebrates, the development of the circulatory and respiratory systems was accompanied by the appearance of HIF-2α, which is also O2-regulated and heterodimerizes with HIF-1β [14] but is only expressed in a restricted number of cell types [15], whereas HIF-1α and HIF-1β are expressed in all human and mouse tissues [16]. In Drosophila, the ubiquitiously expressed HIF-1α ortholog is designatedSimilar [17] and the paralogous gene that is expressed specifically in tracheal tubes is designated Trachealess[18].

HIF-1 Activity is Regulated by Oxygen

In the presence of O2, HIF-1α and HIF-2α are subjected to hydroxylation by prolyl-4-hydroxylase domain proteins (PHDs) that use O2 and α-ketoglutarate as substrates and generate CO2 and succinate as by-products [19]. Prolyl hydroxylation is required for binding of the von Hipple-Lindau protein, which recruits a ubiquitin-protein ligase that targets HIF-1α and HIF-2α for proteasomal degradation (Fig. 2). Under hypoxic conditions, the rate of hydroxylation declines and the non-hydroxylated proteins accumulate. HIF-1α transactivation domain function is also O2-regulated [20,21]. Factor inhibiting HIF-1 (FIH-1) represses transactivation domain function [22] by hydroxylating asparagine residue 803 in HIF-1α, thereby blocking the binding of the co-activators p300 and CBP [23].

Negative regulation of HIF-1 activity by oxygen nihms232046f2

Negative regulation of HIF-1 activity by oxygen nihms232046f2

Negative regulation of HIF-1 activity by oxygen


Fig. 2 Negative regulation of HIF-1 activity by oxygen. Top: In the presence of O2: prolyl hydroxylation of HIF-1a leads to binding of the von Hippel-Lindau protein (VHL), which recruits a ubiquitin protein-ligase that targets HIF-1a for proteasomal degradation;

When cells are acutely exposed to hypoxic conditions, the generation of ROS at complex III of the mitochondrial electron transport chain (ETC) increases and is required for the induction of HIF-1α protein levels [24]. More than a decade after these observations were first made, the precise mechanism by which hypoxia increases ROS generation and by which ROS induces HIF-1α accumulation remain unknown. However, the prolyl and asparaginyl hydroxylases contain Fe2+ in their active site and oxidation to Fe3+would block their catalytic activity. Since O2 is a substrate for the hydroxylation reaction, anoxia also results in a loss of enzyme activity. However, the concentration at which O2 becomes limiting for prolyl or asparaginyl hydroxylase activity in vivo is not known.

HIF-1 Regulates the Balance Between Oxidative and Glycolytic Metabolism

All metazoan organisms depend on mitochondrial respiration as the primary mechanism for generating sufficient amounts of ATP to maintain cellular and systemic homeostasis. Respiration, in turn, is dependent on an adequate supply of O2 to serve as the final electron acceptor in the ETC. In this process, electrons are transferred from complex I (or complex II) to complex III, then to complex IV, and finally to O2, which is reduced to water. This orderly transfer of electrons generates a proton gradient across the inner mitochondrial membrane that is used to drive the synthesis of ATP. At each step of this process, some electrons combine with O2 prematurely, resulting in the production of superoxide anion, which is reduced to hydrogen peroxide through the activity of mitochondrial superoxide dismutase. The efficiency of electron transport appears to be optimized to the physiological range of O2 concentrations, such that ATP is produced without the production of excess superoxide, hydrogen peroxide, and other ROS at levels that would result in the increased oxidation of cellular macromolecules and subsequent cellular dysfunction or death. In contrast, when O2levels are acutely increased or decreased, an imbalance between O2 and electron flow occurs, which results in increased ROS production.

MEFs require HIF-1 activity to make two critical metabolic adaptations to chronic hypoxia. First, HIF-1 activates the gene encoding pyruvate dehydrogenase (PDH) kinase 1 (PDK1), which phosphorylates and inactivates the catalytic subunit of PDH, the enzyme that converts pyruvate to acetyl coenzyme A (AcCoA) for entry into the mitochondrial tricarboxylic acid (TCA) cycle [25]. Second, HIF-1 activates the gene encoding BNIP3, a member of the Bcl-2 family of mitochondrial proteins, which triggers selective mitochondrial autophagy [26]. Interference with the induction of either of these proteins in hypoxic cells results in increased ROS production and increased cell death. Overexpression of either PDK1 or BNIP3 rescues HIF-1α-null MEFs. By shunting pyruvate away from the mitochondria, PDK1 decreases flux through the ETC and thereby counteracts the reduced efficiency of electron transport under hypoxic conditions, which would otherwise increase ROS production. PDK1 functions cooperatively with the product of another HIF-1 target gene, LDHA [27], which converts pyruvate to lactate, thereby further reducing available substrate for the PDH reaction.

PDK1 effectively reduces flux through the TCA cycle and thereby reduces flux through the ETC in cells that primarily utilize glucose as a substrate for oxidative phosphorylation. However, PDK1 is predicted to have little effect on ROS generation in cells that utilize fatty acid oxidation as their source of AcCoA. Hence another strategy to reduce ROS generation under hypoxic conditions is selective mitochondrial autophagy [26]. MEFs reduce their mitochondrial mass and O2 consumption by >50% after only two days at 1% O2. BNIP3 competes with Beclin-1 for binding to Bcl-2, thereby freeing Beclin-1 to activate autophagy. Using short hairpin RNAs to knockdown expression of BNIP3, Beclin-1, or Atg5 (another component of the autophagy machinery) phenocopied HIF-1α-null cells by preventing hypoxia-induced reductions in mitochondrial mass and O2 consumption as a result of failure to induce autophagy [26]. HIF-1-regulated expression of BNIP3L also contributes to hypoxia-induced autophagy [28]. Remarkably, mice heterozygous for the HIF-1α KO allele have a significantly increased ratio of mitochondrial:nuclear DNA in their lungs (even though this is the organ that is exposed to the highest O2 concentrations), indicating that HIF-1 regulates mitochondrial mass under physiological conditions in vivo [26]. In contrast to the selective mitochondrial autophagy that is induced in response to hypoxia as described above, autophagy (of unspecified cellular components) induced by anoxia does not require HIF-1, BNIP3, or BNIP3L, but is instead regulated by AMP kinase [29].

The multiplicity of HIF-1-mediated mechanisms identified so far by which cells regulate mitochondrial metabolism in response to changes in cellular O2 concentration (Fig. 3) suggests that this is a critical adaptive response to hypoxia. The fundamental nature of this physiological response is underscored by the fact that yeast also switch COX4 subunits in an O2-dependent manner but do so by an entirely different molecular mechanism [33], since yeast do not have a HIF-1α homologue. Thus, it appears that by convergent evolution both unicellular and multicellular eukaryotes possess mechanisms by which they modulate mitochondrial metabolism to maintain redox homeostasis despite changes in O2 availability. Indeed, it is the balance between energy, oxygen, and redox homeostasis that represents the key to life with oxygen.

Regulation of mitochondrial metabolism by HIF-1  nihms232046f3

Regulation of mitochondrial metabolism by HIF-1 nihms232046f3

Regulation of mitochondrial metabolism by HIF-1α


Fig. 3 Regulation of mitochondrial metabolism by HIF-1α. Acute hypoxia leads to increased mitochondrial generation of reactive oxygen species (ROS). Decreased O2 and increased ROS levels lead to decreased HIF-1α hydroxylation (see Fig. 2) and increased HIF-1-dependent 


7.9.5 Regulation of cancer cell metabolism by hypoxia-inducible factor 1

Semenza GL1.
Semin Cancer Biol. 2009 Feb; 19(1):12-6.

The Warburg Effect: The Re-discovery of the Importance of Aerobic Glycolysis in Tumor Cells

The induction of hypoxia-inducible factor 1 (HIF-1) activity, either as a result of intratumoral hypoxia or loss-of-function mutations in the VHL gene, leads to a dramatic reprogramming of cancer cell metabolism involving increased glucose transport into the cell, increased conversion of glucose to pyruvate, and a concomitant decrease in mitochondrial metabolism and mitochondrial mass. Blocking these adaptive metabolic responses to hypoxia leads to cell death due to toxic levels of reactive oxygen species. Targeting HIF-1 or metabolic enzymes encoded by HIF-1 target genes may represent a novel therapeutic approach to cancer.



7.9.6 Coming up for air. HIF-1 and mitochondrial oxygen consumption

Simon MC1.
Cell Metab. 2006 Mar;3(3):150-1.

Hypoxic cells induce glycolytic enzymes; this HIF-1-mediated metabolic adaptation increases glucose flux to pyruvate and produces glycolytic ATP. Two papers in this issue of Cell Metabolism (Kim et al., 2006; Papandreou et al., 2006) demonstrate that HIF-1 also influences mitochondrial function, suppressing both the TCA cycle and respiration by inducing pyruvate dehydrogenase kinase 1 (PDK1). PDK1 regulation in hypoxic cells promotes cell survival.

Comment on

Oxygen deprivation (hypoxia) occurs in tissues when O2 supply via the cardiovascular system fails to meet the demand of O2-consuming cells. Hypoxia occurs naturally in physiological settings (e.g., embryonic development and exercising muscle), as well as in pathophysiological conditions (e.g., myocardial infarction, inflammation, and solid tumor formation). For over a century, it has been appreciated that O2-deprived cells exhibit increased conversion of glucose to lactate (the “Pasteur effect”). Activation of the Pasteur effect during hypoxia in mammalian cells is facilitated by HIF-1, which mediates the upregulation of glycolytic enzymes that support an increase in glycolytic ATP production as mitochondria become starved for O2, the substrate for oxidative phosphorylation (Seagroves et al., 2001). Thus, mitochondrial respiration passively decreases due to O2 depletion in hypoxic tissues. However, reports by Kim et al. (2006) and Papandreou et al. (2006) in this issue of Cell Metabolism demonstrate that this critical metabolic adaptation is more complex and includes an active suppression of mitochondrial pyruvate catabolism and O2consumption by HIF-1.

Mitochondrial oxidative phosphorylation is regulated by multiple mechanisms, including substrate availability. Major substrates include O2 (the terminal electron acceptor) and pyruvate (the primary carbon source). Pyruvate, as the end product of glycolysis, is converted to acetyl-CoA by the pyruvate dehydrogenase enzymatic complex and enters the tricarboxylic acid (TCA) cycle. Pyruvate conversion into acetyl-CoA is irreversible; this therefore represents an important regulatory point in cellular energy metabolism. Pyruvate dehydrogenase kinase (PDK) inhibits pyruvate dehydrogenase activity by phosphorylating its E1 subunit (Sugden and Holness, 2003). In the manuscripts by Kim et al. (2006) and Papandreou et al. (2006), the authors find that PDK1 is a HIF-1 target gene that actively regulates mitochondrial respiration by limiting pyruvate entry into the TCA cycle. By excluding pyruvate from mitochondrial metabolism, hypoxic cells accumulate pyruvate, which is then converted into lactate via lactate dehydrogenase (LDH), another HIF-1-regulated enzyme. Lactate in turn is released into the extracellular space, regenerating NAD+ for continued glycolysis by O2-starved cells (see Figure 1). This HIF-1-dependent block to mitochondrial O2 consumption promotes cell survival, especially when O2 deprivation is severe and prolonged.




Figure 1. Multiple hypoxia-induced cellular metabolic changes are regulated by HIF-1

By stimulating the expression of glucose transporters and glycolytic enzymes, HIF-1 promotes glycolysis to generate increased levels of pyruvate. In addition, HIF-1 promotes pyruvate reduction to lactate by activating lactate dehydrogenase (LDH). Pyruvate reduction to lactate regenerates NAD+, which permits continued glycolysis and ATP production by hypoxic cells. Furthermore, HIF-1 induces pyruvate dehydrogenase kinase 1 (PDK1), which inhibits pyruvate dehydrogenase and blocks conversion of pyruvate to acetyl CoA, resulting in decreased flux through the tricarboxylic acid (TCA) cycle. Decreased TCA cycle activity results in attenuation of oxidative phosphorylation and excessive mitochondrial reactive oxygen species (ROS) production. Because hypoxic cells already exhibit increased ROS, which have been shown to promote HIF-1 accumulation, the induction of PDK1 prevents the persistence of potentially harmful ROS levels.

Papandreou et al. demonstrate that hypoxic regulation of PDK has important implications for antitumor therapies. Recent interest has focused on cytotoxins that target hypoxic cells in tumor microenvironments, such as the drug tirapazamine (TPZ). Because intracellular O2 concentrations are decreased by mitochondrial O2 consumption, HIF-1 could protect tumor cells from TPZ-mediated cell death by maintaining intracellular O2 levels. Indeed, Papandreou et al. show that HIF-1-deficient cells grown at 2% O2 exhibit increased sensitivity to TPZ relative to wild-type cells, presumably due to higher rates of mitochondrial O2 consumption. HIF-1 inhibition in hypoxic tumor cells should have multiple therapeutic benefits, but the use of HIF-1 inhibitors in conjunction with other treatments has to be carefully evaluated for the most effective combination and sequence of drug delivery. One result of HIF-1 inhibition would be a relative decrease in intracellular O2 levels, making hypoxic cytotoxins such as TPZ more potent antitumor agents. Because PDK expression has been detected in multiple human tumor samples and appears to be induced by hypoxia (Koukourakis et al., 2005), small molecule inhibitors of HIF-1 combined with TPZ represent an attractive therapeutic approach for future clinical studies.

Hypoxic regulation of PDK1 has other important implications for cell survival during O2 depletion. Because the TCA cycle is coupled to electron transport, Kim et al. suggest that induction of the pyruvate dehydrogenase complex by PDK1 attenuates not only mitochondrial respiration but also the production of mitochondrial reactive oxygen species (ROS) in hypoxic cells. ROS are a byproduct of electron transfer to O2, and cells cultured at 1 to 5% O2 generate increased mitochondrial ROS relative to those cultured at 21% O2 (Chandel et al., 1998 and Guzy et al., 2005). In fact, hypoxia-induced mitochondrial ROS have also been shown to be necessary for the stabilization of HIF-1 in hypoxic cells (Brunelle et al., 2005Guzy et al., 2005 and Mansfield et al., 2005). However, the persistence of ROS could ultimately be lethal to tissues during chronic O2 deprivation, and PDK1 induction by HIF-1 should promote cell viability during long-term hypoxia. Kim et al. present evidence that HIF-1-deficient cells exhibit increased apoptosis after 72 hr of culture at 0.5% O2 compared to wild-type cells and that cell survival is rescued by enforced expression of exogenous PDK1. Furthermore, PDK1 reduces ROS production by the HIF-1 null cells. These findings support a novel prosurvival dimension of cellular hypoxic adaptation where PDK1 inhibits the TCA cycle, mitochondrial respiration, and chronic ROS production.

The HIF-1-mediated block to mitochondrial O2 consumption via PDK1 regulation also has implications for O2-sensing pathways by hypoxic cells. One school of thought suggests that perturbing mitochondrial O2consumption increases intracellular O2 concentrations and suppresses HIF-1 induction by promoting the activity of HIF prolyl hydroxylases, the O2-dependent enzymes that regulate HIF-1 stability (Hagen et al., 2003 and Doege et al., 2005). This model suggests that mitochondria function as “O2 sinks.” Although Papandreou et al. demonstrate that increased mitochondrial respiration due to PDK1 depletion results in decreased intracellular O2 levels (based on pimonidazole staining), these changes failed to reduce HIF-1 levels in hypoxic cells. Another model for hypoxic activation of HIF-1 describes a critical role for mitochondrial ROS in prolyl hydroxylase inhibition and HIF-1 stabilization in O2-starved cells (Brunelle et al., 2005Guzy et al., 2005 and Mansfield et al., 2005) (see Figure 1). The mitochondrial “O2 sink” hypothesis can account for some observations in the literature but fails to explain the inhibition of HIF-1 stabilization by ROS scavengers (Chandel et al., 1998Brunelle et al., 2005Guzy et al., 2005 and Sanjuán-Pla et al., 2005). While the relationship between HIF-1 stability, mitochondrial metabolism, ROS, and intracellular O2 redistribution will continue to be debated for some time, these most recent findings shed new light on findings by Louis Pasteur over a century ago.

Selected reading

Brunelle et al., 2005

J.K. Brunelle, E.L. Bell, N.M. Quesada, K. Vercauteren, V. Tiranti, M. Zeviani, R.C. Scarpulla, N.S. Chandel

Cell Metab., 1 (2005), pp. 409–414

Article  PDF (324 K) View Record in Scopus Citing articles (357)

Chandel et al., 1998

N.S. Chandel, E. Maltepe, E. Goldwasser, C.E. Mathieu, M.C. Simon, P.T. Schumacker

Proc. Natl. Acad. Sci. USA, 95 (1998), pp. 11715–11720

View Record in Scopus Full Text via CrossRef Citing articles (973)

Doege et al., 2005Doege, S. Heine, I. Jensen, W. Jelkmann, E. Metzen

Blood, 106 (2005), pp. 2311–2317

View Record in Scopus Full Text via CrossRef Citing articles (84)

Guzy et al., 2005

R.D. Guzy, B. Hoyos, E. Robin, H. Chen, L. Liu, K.D. Mansfield, M.C. Simon, U. Hammerling, P.T. Schumacker

Cell Metab., 1 (2005), pp. 401–408

Article  PDF (510 K) View Record in Scopus Citing articles (593)

Hagen et al., 2003

Hagen, C.T. Taylor, F. Lam, S. Moncada

Science, 302 (2003), pp. 1975–1978

View Record in Scopus Full Text via CrossRef Citing articles (450)

7.9.7 HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption

Papandreou I1Cairns RAFontana LLim ALDenko NC.
Cell Metab. 2006 Mar; 3(3):187-97.

The HIF-1 transcription factor drives hypoxic gene expression changes that are thought to be adaptive for cells exposed to a reduced-oxygen environment. For example, HIF-1 induces the expression of glycolytic genes. It is presumed that increased glycolysis is necessary to produce energy when low oxygen will not support oxidative phosphorylation at the mitochondria. However, we find that while HIF-1 stimulates glycolysis, it also actively represses mitochondrial function and oxygen consumption by inducing pyruvate dehydrogenase kinase 1 (PDK1). PDK1 phosphorylates and inhibits pyruvate dehydrogenase from using pyruvate to fuel the mitochondrial TCA cycle. This causes a drop in mitochondrial oxygen consumption and results in a relative increase in intracellular oxygen tension. We show by genetic means that HIF-1-dependent block to oxygen utilization results in increased oxygen availability, decreased cell death when total oxygen is limiting, and reduced cell death in response to the hypoxic cytotoxin tirapazamine.

Comment in

Tissue hypoxia results when supply of oxygen from the bloodstream does not meet demand from the cells in the tissue. Such a supply-demand mismatch can occur in physiologic conditions such as the exercising muscle, in the pathologic condition such as the ischemic heart, or in the tumor microenvironment (Hockel and Vaupel, 2001 and Semenza, 2004). In either the physiologic circumstance or pathologic conditions, there is a molecular response from the cell in which a program of gene expression changes is initiated by the hypoxia-inducible factor-1 (HIF-1) transcription factor. This program of gene expression changes is thought to help the cells adapt to the stressful environment. For example, HIF-1-dependent expression of erythropoietin and angiogenic compounds results in increased blood vessel formation for delivery of a richer supply of oxygenated blood to the hypoxic tissue. Additionally, HIF-1 induction of glycolytic enzymes allows for production of energy when the mitochondria are starved of oxygen as a substrate for oxidative phosphorylation. We now find that this metabolic adaptation is more complex, with HIF-1 not only regulating the supply of oxygen from the bloodstream, but also actively regulating the oxygen demand of the tissue by reducing the activity of the major cellular consumer of oxygen, the mitochondria.

Perhaps the best-studied example of chronic hypoxia is the hypoxia associated with the tumor microenvironment (Brown and Giaccia, 1998). The tumor suffers from poor oxygen supply through a chaotic jumble of blood vessels that are unable to adequately perfuse the tumor cells. The oxygen tension within the tumor is also a function of the demand within the tissue, with oxygen consumption influencing the extent of tumor hypoxia (Gulledge and Dewhirst, 1996 and Papandreou et al., 2005b). The net result is that a large fraction of the tumor cells are hypoxic. Oxygen tensions within the tumor range from near normal at the capillary wall, to near zero in the perinecrotic regions. This perfusion-limited hypoxia is a potent microenvironmental stress during tumor evolution (Graeber et al., 1996 and Hockel and Vaupel, 2001) and an important variable capable of predicting for poor patient outcome. (Brizel et al., 1996Cairns and Hill, 2004Hockel et al., 1996 and Nordsmark and Overgaard, 2004).

The HIF-1 transcription factor was first identified based on its ability to activate the erythropoetin gene in response to hypoxia (Wang and Semenza, 1993). Since then, it is has been shown to be activated by hypoxia in many cells and tissues, where it can induce hypoxia-responsive target genes such as VEGF and Glut1 (Airley et al., 2001 and Kimura et al., 2004). The connection between HIF-regulation and human cancer was directly linked when it was discovered that the VHL tumor suppressor gene was part of the molecular complex responsible for the oxic degradation of HIF-1α (Maxwell et al., 1999). In normoxia, a family of prolyl hydroxylase enzymes uses molecular oxygen as a substrate and modifies HIF-1α and HIF2α by hydroxylation of prolines 564 and 402 (Bruick and McKnight, 2001 and Epstein et al., 2001). VHL then recognizes the modified HIF-α proteins, acts as an E3-type of ubiquitin ligase, and along with elongins B and C is responsible for the polyubiquitination of HIF-αs and their proteosomal degradation (Bruick and McKnight, 2001Chan et al., 2002Ivan et al., 2001 and Jaakkola et al., 2001). Mutations in VHL lead to constitutive HIF-1 gene expression, and predispose humans to cancer. The ability to recognize modified HIF-αs is at least partly responsible for VHL activity as a tumor suppressor, as introduction of nondegradable HIF-2α is capable of overcoming the growth–inhibitory activity of wild-type (wt) VHL in renal cancer cells (Kondo et al., 2003).

Mitochondrial function can be regulated by PDK1 expression. Mitochondrial oxidative phosphorylation (OXPHOS) is regulated by several mechanisms, including substrate availability (Brown, 1992). The major substrates for OXPHOS are oxygen, which is the terminal electron acceptor, and pyruvate, which is the primary carbon source. Pyruvate is the end product of glycolysis and is converted to acetyl-CoA through the activity of the pyruvate dehydrogenase complex of enzymes. The acetyl-CoA then directly enters the TCA cycle at citrate synthase where it is combined with oxaloacetate to generate citrate. In metazoans, the conversion of pyruvate to acetyl-CoA is irreversible and therefore represents a critical regulatory point in cellular energy metabolism. Pyruvate dehydrogenase is regulated by three known mechanisms: it is inhibited by acetyl-CoA and NADH, it is stimulated by reduced energy in the cell, and it is inhibited by regulatory phosphorylation of its E1 subunit by pyruvate dehydrogenase kinase (PDK) (Holness and Sugden, 2003 and Sugden and Holness, 2003). There are four members of the PDK family in vertebrates, each with specific tissue distributions (Roche et al., 2001). PDK expression has been observed in human tumor biopsies (Koukourakis et al., 2005), and we have reported that PDK3 is hypoxia-inducible in some cell types (Denko et al., 2003). In this manuscript, we find that PDK1 is also a hypoxia-responsive protein that actively regulates the function of the mitochondria under hypoxic conditions by reducing pyruvate entry into the TCA cycle. By excluding pyruvate from mitochondrial consumption, PDK1 induction may increase the conversion of pyruvate to lactate, which is in turn shunted to the extracellular space, regenerating NAD for continued glycolysis.

Identification of HIF-dependent mitochondrial proteins through genomic and bioinformatics approaches

In order to help elucidate the role of HIF-1α in regulating metabolism, we undertook a genomic search for genes that were regulated by HIF-1 in tumor cells exposed to hypoxia in vitro. We used genetically matched human RCC4 cells that had lost VHL during tumorigenesis and displayed constitutive HIF-1 activity, and a cell line engineered to re-express VHL to establish hypoxia-dependent HIF activation. These cells were treated with 18 hr of stringent hypoxia (<0.01% oxygen), and microarray analysis performed. Using a strict 2.5-fold elevation as our cutoff, we identified 173 genes that were regulated by hypoxia and/or VHL status (Table S1 in the Supplemental Data available with this article online). We used the pattern of expression in these experiments to identify putative HIF-regulated genes—ones that were constitutively elevated in the parent RCC4s independent of hypoxia, downregulated in the RCC4VHL cells under normoxia, and elevated in response to hypoxia. Of the 173 hypoxia and VHL-regulated genes, 74 fit the putative HIF-1 target pattern. The open reading frames of these genes were run through a pair of bioinformatics engines in order to predict subcellular localization, and 10 proteins scored as mitochondrial on at least one engine. The genes, fold induction, and mitochondrial scores are listed in Table 1.

HIF-1 downregulates mitochondrial oxygen consumption

Having identified several putative HIF-1 responsive gene products that had the potential to regulate mitochondrial function, we then directly measured mitochondrial oxygen consumption in cells exposed to long-term hypoxia. While other groups have studied mitochondrial function under acute hypoxia (Chandel et al., 1997), this is one of the first descriptions of mitochondrial function after long-term hypoxia where there have been extensive hypoxia-induced gene expression changes. Figure 1A is an example of the primary oxygen trace from a Clark electrode showing a drop in oxygen concentration in cell suspensions of primary fibroblasts taken from normoxic and hypoxic cultures. The slope of the curve is a direct measure of the total cellular oxygen consumption rate. Exposure of either primary human or immortalized mouse fibroblasts to 24 hr of hypoxia resulted in a reduction of this rate by approximately 50% (Figures 1A and 1B). In these experiments, the oxygen consumption can be stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide 3-chloro phenylhydrazone) and was completely inhibited by 2 mM potassium cyanide. We determined that the change in total cellular oxygen consumption was due to changes in mitochondrial activity by the use of the cell-permeable poison of mitochondrial complex 3, Antimycin A. Figure 1C shows that the difference in the normoxic and hypoxic oxygen consumption in murine fibroblasts is entirely due to the Antimycin-sensitive mitochondrial consumption. The kinetics with which mitochondrial function slows in hypoxic tumor cells also suggests that it is due to gene expression changes because it takes over 6 hr to achieve maximal reduction, and the reversal of this repression requires at least another 6 hr of reoxygenation (Figure 1D). These effects are not likely due to proliferation or toxicity of the treatments as these conditions are not growth inhibitory or toxic to the cells (Papandreou et al., 2005a).

Since we had predicted from the gene expression data that the mitochondrial oxygen consumption changes were due to HIF-1-mediated expression changes, we tested several genetically matched systems to determine what role HIF-1 played in the process (Figure 2). We first tested the cell lines that had been used for microarray analysis and found that the parental RCC4 cells had reduced mitochondrial oxygen consumption when compared to the VHL-reintroduced cells. Oxygen consumption in the parental cells was insensitive to hypoxia, while it was reduced by hypoxia in the wild-type VHL-transfected cell lines. Interestingly, stable introduction of a tumor-derived mutant VHL (Y98H) that cannot degrade HIF was also unable to restore oxygen consumption. These results indicate that increased expression of HIF-1 is sufficient to reduce oxygen consumption (Figure 2A). We also investigated whether HIF-1 induction was required for the observed reduction in oxygen consumption in hypoxia using two genetically matched systems. We measured normoxic and hypoxic oxygen consumption in murine fibroblasts derived from wild-type or HIF-1α null embryos (Figure 2B) and from human RKO tumor cells and RKO cells constitutively expressing ShRNAs directed against the HIF-1α gene (Figures 2C and 4C). Neither of the HIF-deficient cell systems was able to reduce oxygen consumption in response to hypoxia. These data from the HIF-overexpressing RCC cells and the HIF-deficient cells indicate that HIF-1 is both necessary and sufficient for reducing mitochondrial oxygen consumption in hypoxia.

HIF-dependent mitochondrial changes are functional, not structural

Because addition of CCCP could increase oxygen consumption even in the hypoxia-treated cells, we hypothesized that the hypoxic inhibition was a regulated activity, not a structural change in the mitochondria in response to hypoxic stress. We confirmed this interpretation by examining several additional mitochondrial characteristics in hypoxic cells such as mitochondrial morphology, quantity, and membrane potential. We examined morphology by visual inspection of both the transiently transfected mitochondrially localized DsRed protein and the endogenous mitochondrial protein cytochrome C. Both markers were indistinguishable in the parental RCC4 and the RCC4VHL cells (Figure 3A). Likewise, we measured the mitochondrial membrane potential with the functional dye rhodamine 123 and found that it was identical in the matched RCC4 cells and the matched HIF wt and knockout (KO) cells when cultured in normoxia or hypoxia (Figure 3B). Finally, we determined that the quantity of mitochondria per cell was not altered in response to HIF or hypoxia by showing that the amount of the mitochondrial marker protein HSP60 was identical in the RCC4 and HIF cell lines (Figure 3C)

PDK1 is a HIF-1 inducible target protein

After examination of the list of putative HIF-regulated mitochondrial target genes, we hypothesized that PDK1 could mediate the functional changes that we observed in hypoxia. We therefore investigated PDK1 protein expression in response to HIF and hypoxia in the genetically matched cell systems. Figure 4A shows that in the RCC4 cells PDK1 and the HIF-target gene BNip3 (Greijer et al., 2005 and Papandreou et al., 2005a) were both induced by hypoxia in a VHL-dependent manner, with the expression of PDK1 inversely matching the oxygen consumption measured in Figure 1 above. Likewise, the HIF wt MEFs show oxygen-dependent induction of PDK1 and BNip3, while the HIF KO MEFs did not show any expression of either of these proteins under any oxygen conditions (Figure 4B). Finally, the parental RKO cells were able to induce PDK1 and the HIF target gene BNip3L in response to hypoxia, while the HIF-depleted ShRNA RKO cells could not induce either protein (Figure 4C). Therefore, in all three cell types, the HIF-1-dependent regulation of oxygen consumption seen in Figure 2, corresponds to the HIF-1-dependent induction of PDK1 seen in Figure 4.

In order to determine if PDK1 was a direct HIF-1 target gene, we analyzed the genomic sequence flanking the 5′ end of the gene for possible HIF-1 binding sites based on the consensus core HRE element (A/G)CGTG (Caro, 2001). Several such sites exist within the first 400 bases upstream, so we generated reporter constructs by fusing the genomic sequence from −400 to +30 of the start site of transcription to the firefly luciferase gene. In transfection experiments, the chimeric construct showed significant induction by either cotransfection with a constitutively active HIF proline mutant (P402A/P564G) (Chan et al., 2002) or exposure of the transfected cells to 0.5% oxygen (Figure 4D). Most noteworthy, when the reporter gene was transfected into the HIF-1α null cells, it did not show induction when the cells were cultured in hypoxia, but it did show induction when cotransfected with expression HIF-1α plasmid. We then generated deletions down to the first 36 bases upstream of transcription and found that even this short sequence was responsive to HIF-1 (Figure 4D). Analysis of this small fragment showed only one consensus HRE site located in an inverted orientation in the 5′ untranslated region. We synthesized and cloned a mutant promoter fragment in which the core element ACGTG was replaced with AAAAG, and this construct lost over 90% of its hypoxic induction. These experiments suggest that it is this HRE within the proximal 5′ UTR that HIF-1 uses to transactivate the endogenous PDK1 gene in response to hypoxia.

PDK1 is responsible for the HIF-dependent mitochondrial oxygen consumption changes

In order to directly test if PDK1 was the HIF-1 target gene responsible for the hypoxic reduction in mitochondrial oxygen consumption, we generated RKO cell lines with either knockdown or overexpression of PDK1 and measured the oxygen consumption in these derivatives. The PDK1 ShRNA stable knockdown line was generated as a pool of clones cotransfected with pSUPER ShPDK1 and pTK-hygro resistance gene. After selection for growth in hygromycin, the cells were tested by Western blot for the level of PDK1 protein expression. We found that normoxic PDK1 is reduced by 75%, however, there was measurable expression of PDK1 in these cells in response to hypoxia (Figure 5A). When we measured the corresponding oxygen consumption in these cells, we found a change commensurate with the level of PDK1. The knockdown cells show elevated baseline oxygen consumption, and partial reduction in this activity in response to hypoxia. Therefore, reduction of PDK1 expression by genetic means increased mitochondrial oxygen consumption in both normoxic and hypoxic conditions. Interestingly, these cells still induced HIF-1α (Figure 5A) and HIF-1 target genes such as BNip3L in response to hypoxia (data not shown), suggesting that altered PDK1 levels do not alter HIF-1α function.



PDK1 expression directly regulates cellular oxygen consumption rate


Figure 5. PDK1 expression directly regulates cellular oxygen consumption rate

  1. A)Western blot of RKO cell and ShRNAPDK1RKO cell lysates after exposure to 24 hr of normoxia or 0.5% O2. Blots were probed for HIF 1α, PDK1, and tubulin as a loading control.
  2. B)Oxygen consumption rate in RKO and ShRNAPDK1RKO cells after exposure to 24 hr of normoxia or 0.5% O2.
  3. C)Western blot of RKOiresGUS cell and RKOiresPDK1 cell lysates after exposure to 24 hr of normoxia or 0.5% O2. Blots were probed for HIF 1α, PDK1, and tubulin as a loading control.
  4. D)Oxygen consumption rate in RKOiresGUS and RKOiresPDK1 cells after exposure to 24 hr of normoxia or 0.5% O2.
  5. E)Model describing the interconnected effects of HIF-1 target gene activation on hypoxic cell metabolism. Reduced oxygen conditions causes HIF-1 to coordinately induce the enzymes shown in boxes. HIF-1 activation results in increased glucose transporter expression to increase intracellular glucose flux, induction of glycolytic enzymes increases the conversion of glucose to pyruvate generating energy and NADH, induction of PDK1 decreases mitochondrial utilization of pyruvate and oxygen, and induction of LDH increases the removal of excess pyruvate as lactate and also regenerates NAD+ for increased glycolysis.

For all graphs, the error bars represent the standard error of the mean.

We also determined if overexpression of PDK1 could lead to reduced mitochondrial oxygen consumption. A separate culture of RKO cells was transfected with a PDK1-IRES-puro expression plasmid and selected for resistance to puromycin. The pool of puromycin resistant cells was tested for PDK1 expression by Western blot. These cells showed a modest increase in PDK1 expression under control conditions when compared to the cells transfected with GUS-IRES-puro, with an additional increase in PDK1 protein in response to hypoxia (Figure 5C). The corresponding oxygen consumption measurements showed that the mitochondria is very sensitive to changes in the levels of PDK1, as even this slight increase was able to significantly reduce oxygen consumption in the normoxic PDK1-puro cultures. Further increase in PDK1 levels with hypoxia further reduced oxygen consumption in both cultures (Figure 5D). The model describing the relationship between hypoxia, HIF-1, PDK1, and intermediate metabolism is described inFigure 5E.

Altering oxygen consumption alters intracellular oxygen tension and sensitivity to hypoxia-dependent cell killing

The intracellular concentration of oxygen is a net result of the rate at which oxygen diffuses into the cell and the rate at which it is consumed. We hypothesized that the rate at which oxygen was consumed within the cell would significantly affect its steady-state intracellular concentrations. We tested this hypothesis in vitro using the hypoxic marker drug pimonidazole (Bennewith and Durand, 2004). We plated high density cultures of HIF wild-type and HIF knockout cells and placed these cultures in normoxic, 2% oxygen, and anoxic incubators for overnight treatment. The overnight treatment gives the cells time to adapt to the hypoxic conditions and establish altered oxygen consumption profiles. Pimonidozole was then added for the last 4 hr of the growth of the culture. Pimonidazole binding was detected after fixation of the cells using an FITC labeled anti-pimonidazole antibody and it was quantitated by flow cytometry. The quantity of the bound drug is a direct indication of the oxygen concentration within the cell (Bennewith and Durand, 2004). The histograms in Figure 6A show that the HIF-1 knockout and wild-type cells show similar staining in the cells grown in 0% oxygen. However, the cells treated with 2% oxygen show the consequence of the genetic removal of HIF-1. The HIF-proficient cells showed relatively less pimonidazole binding at 2% when compared to the 0% culture, while the HIF-deficient cells showed identical binding between the cells at 2% and those at 0%. We interpret these results to mean that the HIF-deficient cells have greater oxygen consumption, and this has lowered the intracellular oxygenation from the ambient 2% to close to zero intracellularly. The HIF-proficient cells reduced their oxygen consumption rate so that the rate of diffusion into the cell is greater than the rate of consumption.

Figure 6. HIF-dependent decrease in oxygen consumption raises intracellular oxygen concentration, protects when oxygen is limiting, and decreases sensitivity to tirapazamine in vitro

  1. A)Pimonidazole was used to determine the intracellular oxygen concentration of cells in culture. HIF wt and HIF KO MEFs were grown at high density and exposed to 2% O2or anoxia for 24 hr in glass dishes. For the last 4 hr of treatment, cells were exposed to 60 μg/ml pimonidazole. Pimonidazole binding was quantitated by flow cytometry after binding of an FITC conjugated anti-pimo mAb. Results are representative of two independent experiments.
  2. B)HIF1α reduces oxygen consumption and protects cells when total oxygen is limited. HIF wt and HIF KO cells were plated at high density and sealed in aluminum jigs at <0.02% oxygen. At the indicated times, cells were harvested, and dead cells were quantitated by trypan blue exclusion. Note both cell lines are equally sensitive to anoxia-induced apoptosis, so the death of the HIF null cells indicates that the increased oxygen consumption removed any residual oxygen in the jig and resulted in anoxia-induced death.
  3. C)PDK1 is responsible for HIF-1’s adaptive response when oxygen is limiting. A similar jig experiment was performed to measure survival in the parental RKO, the RKO ShRNAHIF1α, and the RKOShPDK1 cells. Cell death by trypan blue uptake was measured 48 hr after the jigs were sealed.
  4. D)HIF status alters sensitivity to TPZ in vitro. HIF wt and HIF KO MEFs were grown at high density in glass dishes and exposed to 21%, 2%, and <0.01% O2conditions for 18 hr in the presence of varying concentrations of Tirapazamine. After exposure, cells were harvested and replated under normoxia to determine clonogenic viability. Survival is calculated relative to the plating efficiency of cells exposed to 0 μM TPZ for each oxygen concentration.
  5. E)Cell density alters sensitivity to TPZ. HIF wt and HIF KO MEFs were grown at varying cell densities in glass dishes and exposed to 2% O2in the presence of 10 μM TPZ for 18 hr. After the exposure, survival was determined as described in (C).

For all graphs, the error bars represent the standard error of the mean.

HIF-induced PDK1 can reduce the total amount of oxygen consumed per cell. The reduction in the amount of oxygen consumed could be significant if there is a finite amount of oxygen available, as would be the case in the hours following a blood vessel occlusion. The tissue that is fed by the vessel would benefit from being economical with the oxygen that is present. We experimentally modeled such an event using aluminum jigs that could be sealed with defined amounts of cells and oxygen present (Siim et al., 1996). We placed 10 × 106 wild-type or HIF null cells in the sealed jig at 0.02% oxygen, waited for the cells to consume the remaining oxygen, and measured cell viability. We have previously shown that these two cell types are resistant to mild hypoxia and equally sensitive to anoxia-induced apoptosis (Papandreou et al., 2005a). Therefore, any death in this experiment would be the result of the cells consuming the small amount of remaining oxygen and dying in response to anoxia. We found that in sealed jigs, the wild-type cells are more able to adapt to the limited oxygen supply by reducing consumption. The HIF null cells continued to consume oxygen, reached anoxic levels, and started to lose viability within 36 hr (Figure 6B). This is a secondary adaptive effect of HIF1. We confirmed that PDK1 was responsible for this difference by performing a similar experiment using the parental RKO cells, the RKOShRNAHIF1α and the RKOShRNAPDK1 cells. We found similar results in which both the cells with HIF1α knockdown and PDK1 knockdown were sensitive to the long-term effects of being sealed in a jig with a defined amount of oxygen (Figure 6c). Note that the RKOShPDK1 cells are even more sensitive than the RKOShHIF1α cells, presumably because they have higher basal oxygen consumption rates (Figure 5B).

Because HIF-1 can help cells adapt to hypoxia and maintain some intracellular oxygen level, it may also protect tumor cells from killing by the hypoxic cytotoxin tirapazamine (TPZ). TPZ toxicity is very oxygen dependent, especially at oxygen levels between 1%–4% (Koch, 1993). We therefore tested the relative sensitivity of the HIF wt and HIF KO cells to TPZ killing in high density cultures (Figure 6D). We exposed the cells to the indicated concentrations of drug and oxygen concentrations overnight. The cells were then harvested and replated to determine reproductive viability by colony formation. Both cell types were equally resistant to TPZ at 21% oxygen, while both cell types are equally sensitive to TPZ in anoxic conditions where intracellular oxygen levels are equivalent (Figure 6A). The identical sensitivity of both cell types in anoxia indicates that both cell types are equally competent in repairing the TPZ-induced DNA damage that is presumed to be responsible for its toxicity. However, in 2% oxygen cultures, the HIF null cells displayed a significantly greater sensitivity to the drug than the wild-type cells. This suggests that the increased oxygen consumption rate in the HIF-deficient cells is sufficient to lower the intracellular oxygen concentration relative to that in the HIF-proficient cells. The lower oxygen level is significant enough to dramatically sensitize these cells to killing by TPZ.

If the increased sensitivity to TPZ in the HIF ko cells is determined by intracellular oxygen consumption differences, then this effect should also be cell-density dependent. We showed that this is indeed the case in Figure 6E where oxygen and TPZ concentrations were held constant, and increased cell density lead to increased TPZ toxicity. The effect was much more pronounced in the HIF KO cells, although the HIF wt cells showed some increased toxicity in the highest density cultures, consistent with the fact they were still consuming some oxygen, even with HIF present (Figure 1). The in vitro TPZ survival data is therefore consistent with our hypothesis that control of oxygen consumption can regulate intracellular oxygen concentration, and suggests that increased oxygen consumption could sensitize cells to hypoxia-dependent therapy.


The findings presented here show that HIF-1 is actively responsible for regulating energy production in hypoxic cells by an additional, previously unrecognized mechanism. It has been shown that HIF-1 induces the enzymes responsible for glycolysis when it was presumed that low oxygen did not support efficient oxidative phosphorylation (Iyer et al., 1998 and Seagroves et al., 2001). The use of glucose to generate ATP is capable of satisfying the energy requirements of a cell if glucose is in excess (Papandreou et al., 2005a). We now find that at the same time that glycolysis is increasing, mitochondrial respiration is decreasing. However, the decreased respiration is not because there is not enough oxygen present to act as a substrate for oxidative phosphorylation, but because the flow of pyruvate into the TCA cycle has been reduced by the activity of pyruvate dehydrogenase kinase. Other reports have suggested that oxygen utilization is shifted in cells exposed to hypoxia, but these reports have focused on other regulators such as nitric oxide synthase (Hagen et al., 2003). NO can reduce oxygen consumption through direct inhibition of cytochrome oxidase, but this effect seems to be more significant at physiologic oxygen concentrations, not at severe levels seen in the tumor (Palacios-Callender et al., 2004).

7.9.8 HIF-1. upstream and downstream of cancer metabolism

Semenza GL1.
Curr Opin Genet Dev. 2010 Feb; 20(1):51-6

Hypoxia-inducible factor 1 (HIF-1) plays a key role in the reprogramming of cancer metabolism by activating transcription of genes encoding glucose transporters and glycolytic enzymes, which take up glucose and convert it to lactate; pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; and BNIP3, which triggers selective mitochondrial autophagy. The shift from oxidative to glycolytic metabolism allows maintenance of redox homeostasis and cell survival under conditions of prolonged hypoxia. Many metabolic abnormalities in cancer cells increase HIF-1 activity. As a result, a feed-forward mechanism can be activated that drives HIF-1 activation and may promote tumor progression. Hypoxia-inducible factor 1 (HIF-1) plays a key role in the reprogramming of cancer metabolism by activating transcription of genes encoding glucose transporters and glycolytic enzymes, which take up glucose and convert it to lactate; pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; and BNIP3, which triggers selective mitochondrial autophagy. The shift from oxidative to glycolytic metabolism allows maintenance of redox homeostasis and cell survival under conditions of prolonged hypoxia. Many metabolic abnormalities in cancer cells increase HIF-1 activity. As a result, a feed-forward mechanism can be activated that drives HIF-1 activation and may promote tumor progression.

Metastatic cancer is characterized by reprogramming of cellular metabolism leading to increased uptake of glucose for use as both an anabolic and catabolic substrate. Increased glucose uptake is such a reliable feature that it is utilized clinically to detect metastases by positron emission tomography using 18F-fluorodeoxyglucose (FDG-PET) with a sensitivity of ~90% [1]. As with all aspects of cancer biology, the details of metabolic reprogramming differ widely among individual tumors. However, the role of specific signaling pathways and transcription factors in this process is now understood in considerable detail. This review will focus on the involvement of hypoxia-inducible factor 1 (HIF-1) in both mediating metabolic reprogramming and responding to metabolic alterations. The placement of HIF-1 both upstream and downstream of cancer metabolism results in a feed-forward mechanism that may play a major role in the development of the invasive, metastatic, and lethal cancer phenotype.

O2 concentrations are significantly reduced in many human cancers compared to the surrounding normal tissue. The median PO2 in breast cancers is ~10 mm Hg, as compared to ~65 mm Hg in normal breast tissue [2]. Reduced O2 availability induces HIF-1, which regulates the transcription of hundreds of genes [3*,4*] that encode proteins involved in every aspect of cancer biology, including: cell immortalization and stem cell maintenance; genetic instability; glucose and energy metabolism; vascularization; autocrine growth factor signaling; invasion and metastasis; immune evasion; and resistance to chemotherapy and radiation therapy [5].

HIF-1 is a transcription factor that consists of an O2-regulated HIF-1α and a constitutively expressed HIF-1β subunit [6]. In well-oxygenated cells, HIF-1α is hydroxylated on proline residue 402 (Pro-402) and/or Pro-564 by prolyl hydroxylase domain protein 2 (PHD2), which uses O2 and α-ketoglutarate as substrates in a reaction that generates CO2 and succinate as byproducts [7]. Prolyl-hydroxylated HIF-1α is bound by the von Hippel-Lindau tumor suppressor protein (VHL), which recruits an E3-ubiquitin ligase that targets HIF-1α for proteasomal degradation (Figure 1A). Asparagine 803 in the transactivation domain is hydroxylated in well-oxygenated cells by factor inhibiting HIF-1 (FIH-1), which blocks the binding of the coactivators p300 and CBP [7]. Under hypoxic conditions, the prolyl and asparaginyl hydroxylation reactions are inhibited by substrate (O2) deprivation and/or the mitochondrial generation of reactive oxygen species (ROS), which may oxidize Fe(II) present in the catalytic center of the hydroxylases [8].

HIF-1 and metabolism  nihms156580f1

HIF-1 and metabolism nihms156580f1

HIF-1 and metabolism


Figure 1 HIF-1 and metabolism. (A) Regulation of HIF-1α protein synthesis and stability and HIF-1-dependent metabolic reprogramming. The rate of translation of HIF-1α mRNA into protein in cancer cells is dependent upon the activity of the mammalian 

The finding that acute changes in PO2 increase mitochondrial ROS production suggests that cellular respiration is optimized at physiological PO2 to limit ROS generation and that any deviation in PO2 — up or down — results in increased ROS generation. If hypoxia persists, induction of HIF-1 leads to adaptive mechanisms to reduce ROS and re-establish homeostasis, as described below. Prolyl and asparaginyl hydroxylation provide a molecular mechanism by which changes in cellular oxygenation can be transduced to the nucleus as changes in HIF-1 activity. This review will focus on recent advances in our understanding of the role of HIF-1 in controlling glucose and energy metabolism, but it should be appreciated that any increase in HIF-1 activity that leads to changes in cell metabolism will also affect many other critical aspects of cancer biology [5] that will not be addressed here.

HIF-1 target genes involved in glucose and energy metabolism

HIF-1 activates the transcription of SLC2A1 and SLC2A3, which encode the glucose transporters GLUT1 and GLUT3, respectively, as well as HK1 and HK2, which encode hexokinase, the first enzyme of the Embden-Meyerhoff (glycolytic) pathway [9]. Once taken up by GLUT and phosphorylated by HK, FDG cannot be metabolized further; thus, FDG-PET signal is determined by FDG delivery to tissue (i.e. perfusion) and GLUT/HK expression/activity. Unlike FDG, glucose is further metabolized to pyruvate by the action of the glycolytic enzymes, which are all encoded by HIF-1 target genes (Figure 1A). Glycolytic intermediates are also utilized for nucleotide and lipid synthesis [10]. Lactate dehydrogenase A (LDHA), which converts pyruvate to lactate, and monocarboxylate transporter 4 (MCT4), which transports lactate out of the cell (Figure 1B), are also regulated by HIF-1 [9,11]. Remarkably, lactate produced by hypoxic cancer cells can be taken up by non-hypoxic cells and used as a respiratory substrate [12**].

Pyruvate represents a critical metabolic control point, as it can be converted to acetyl coenzyme A (AcCoA) by pyruvate dehydrogenase (PDH) for entry into the tricarboxylic acid (TCA) cycle or it can be converted to lactate by LDHA (Figure 1B). Pyruvate dehydrogenase kinase (PDK), which phosphorylates and inactivates the catalytic domain of PDH, is encoded by four genes and PDK1 is activated by HIF-1 [13,14]. (Further studies are required to determine whether PDK2PDK3, or PDK4 is regulated by HIF-1.) As a result of PDK1 activation, pyruvate is actively shunted away from the mitochondria, which reduces flux through the TCA cycle, thereby reducing delivery of NADH and FADH2 to the electron transport chain. This is a critical adaptive response to hypoxia, because in HIF-1α–null mouse embryo fibroblasts (MEFs), PDK1 expression is not induced by hypoxia and the cells die due to excess ROS production, which can be ameliorated by forced expression of PDK1 [13]. MYC, which is activated in ~40% of human cancers, cooperates with HIF-1 to activate transcription of PDK1, thereby amplifying the hypoxic response [15]. Pharmacological inhibition of HIF-1 or PDK1 activity increases O2 consumption by cancer cells and increases the efficacy of a hypoxia-specific cytotoxin [16].

Hypoxia also induces mitochondrial autophagy in many human cancer cell lines through HIF-1-dependent expression of BNIP3 and a related BH3 domain protein, BNIP3L [19**]. Autocrine signaling through the platelet-derived growth factor receptor in cancer cells increases HIF-1 activity and thereby increases autophagy and cell survival under hypoxic conditions [21]. Autophagy may also occur in a HIF-1-independent manner in response to other physiological stimuli that are associated with hypoxic conditions, such as a decrease in the cellular ATP:AMP ratio, which activates AMP kinase signaling [22].

In clear cell renal carcinoma, VHL loss of function (LoF) results in constitutive HIF-1 activation, which is associated with impaired mitochondrial biogenesis that results from HIF-1-dependent expression of MXI1, which blocks MYC-dependent expression of PGC-1β, a coactivator that is required for mitochondrial biogenesis [23]. Inhibition of wild type MYC activity in renal cell carcinoma contrasts with the synergistic effect of HIF-1 and oncogenic MYC in activating PDK1 transcription [24].

Genetic and metabolic activators of HIF-1

Hypoxia plays a critical role in cancer progression [2,5] but not all cancer cells are hypoxic and a growing number of O2-independent mechanisms have been identified by which HIF-1 is induced [5]. Several mechanisms that are particularly relevant to cancer metabolism are described below.

Activation of mTOR

Alterations in mitochondrial metabolism

NAD+ levels

It is of interest that the NAD+-dependent deacetylase sirtuin 1 (SIRT1) was found to bind to, deacetylate, and increase transcriptional activation by HIF-2α but not HIF-1α [42**]. Another NAD+-dependent enzyme is poly(ADP-ribose) polymerase 1 (PARP1), which was recently shown to bind to HIF-1α and promote transactivation through a mechanism that required the enzymatic activity of PARP1 [43]. Thus, transactivation mediated by both HIF-1α and HIF-2α can be modulated according to NAD+ levels.

Nitric oxide

Increased expression of nitric oxide (NO) synthase isoforms and increased levels of NO have been shown to increase HIF-1α protein stability in human oral squamous cell carcinoma [44]. In prostate cancer, nuclear co-localization of endothelial NO synthase, estrogen receptor β, HIF-1α, and HIF-2α was associated with aggressive disease and the proteins were found to form chromatin complexes on the promoter of TERT gene encoding telomerase [45**]. The NOS2 gene encoding inducible NO synthase is HIF-1 regulated [5], suggesting another possible feed-forward mechanism.

7.9.9 In Vivo HIF-Mediated Reductive Carboxylation

Gameiro PA1Yang JMetelo AMPérez-Carro R, et al.
Cell Metab. 2013 Mar 5; 17(3):372-85.

Hypoxic and VHL-deficient cells use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate. To gain insights into the role of HIF and the molecular mechanisms underlying RC, we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. HIF expression drastically reduced intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate or knocking down PDK-1 and ACLY restored citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action to maintain lipogenesis. Using [1–13C] glutamine, we demonstrated in vivo RC activity in VHL-deficient tumors growing as xenografts in mice. Lastly, HIF rendered VHL-deficient cells sensitive to glutamine deprivation in vitro, and systemic administration of glutaminase inhibitors suppressed the growth of RCC cells as mice xenografts.

Cancer cells undergo fundamental changes in their metabolism to support rapid growth, adapt to limited nutrient resources, and compete for these supplies with surrounding normal cells. One of the metabolic hallmarks of cancer is the activation of glycolysis and lactate production even in the presence of adequate oxygen. This is termed the Warburg effect, and efforts in cancer biology have revealed some of the molecular mechanisms responsible for this phenotype (Cairns et al., 2011). More recently, 13C isotopic studies have elucidated the complementary switch of glutamine metabolism that supports efficient carbon utilization for anabolism and growth (DeBerardinis and Cheng, 2010). Acetyl-CoA is a central biosynthetic precursor for lipid synthesis, being generated from glucose-derived citrate in well-oxygenated cells (Hatzivassiliou et al., 2005). Warburg-like cells, and those exposed to hypoxia, divert glucose to lactate, raising the question of how the tricarboxylic acid (TCA) cycle is supplied with acetyl-CoA to support lipogenesis. We and others demonstrated, using 13C isotopic tracers, that cells under hypoxic conditions or defective mitochondria primarily utilize glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate by isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) (Filipp et al., 2012Metallo et al., 2012;Mullen et al., 2012Wise et al., 2011).

The transcription factors hypoxia inducible factors 1α and 2α (HIF-1α, HIF-2α) have been established as master regulators of the hypoxic program and tumor phenotype (Gordan and Simon, 2007Semenza, 2010). In addition to tumor-associated hypoxia, HIF can be directly activated by cancer-associated mutations. The von Hippel-Lindau (VHL) tumor suppressor is inactivated in the majority of sporadic clear-cell renal carcinomas (RCC), with VHL-deficient RCC cells exhibiting constitutive HIF-1α and/or HIF-2α activity irrespective of oxygen availability (Kim and Kaelin, 2003). Previously, we showed that VHL-deficient cells also relied on RC for lipid synthesis even under normoxia. Moreover, metabolic profiling of two isogenic clones that differ in pVHL expression (WT8 and PRC3) suggested that reintroduction of wild-type VHL can restore glucose utilization for lipogenesis (Metallo et al., 2012). The VHL tumor suppressor protein (pVHL) has been reported to have several functions other than the well-studied targeting of HIF. Specifically, it has been reported that pVHL regulates the large subunit of RNA polymerase (Pol) II (Mikhaylova et al., 2008), p53 (Roe et al., 2006), and the Wnt signaling regulator Jade-1. VHL has also been implicated in regulation of NF-κB signaling, tubulin polymerization, cilia biogenesis, and proper assembly of extracellular fibronectin (Chitalia et al., 2008Kim and Kaelin, 2003Ohh et al., 1998Thoma et al., 2007Yang et al., 2007). Hypoxia inactivates the α-ketoglutarate-dependent HIF prolyl hydroxylases, leading to stabilization of HIF. In addition to this well-established function, oxygen tension regulates a larger family of α-ketoglutarate-dependent cellular oxygenases, leading to posttranslational modification of several substrates, among which are chromatin modifiers (Melvin and Rocha, 2012). It is therefore conceivable that the effect of hypoxia on RC that was reported previously may be mediated by signaling mechanisms independent of the disruption of the pVHL-HIF interaction. Here we (1) demonstrate that HIF is necessary and sufficient for RC, (2) provide insights into the molecular mechanisms that link HIF to RC, (3) detected RC activity in vivo in human VHL-deficient RCC cells growing as tumors in nude mice, (4) provide evidence that the reductive phenotype ofVHL-deficient cells renders them sensitive to glutamine restriction in vitro, and (5) show that inhibition of glutaminase suppresses growth of VHL-deficient cells in nude mice. These observations lay the ground for metabolism-based therapeutic strategies for targeting HIF-driven tumors (such as RCC) and possibly the hypoxic compartment of solid tumors in general.

Functional Interaction between pVHL and HIF Is Necessary to Inhibit RC

Figure 1  HIF Inactivation Is Necessary for Downregulation of Reductive Carboxylation by pVHL

We observed a concurrent regulation in glucose metabolism in the different VHL mutants. Reintroduction of wild-type or type 2C pVHL mutant, which can meditate HIF-α destruction, stimulated glucose oxidation via pyruvate dehydrogenase (PDH), as determined by the degree of 13C-labeled TCA cycle metabolites (M2 enrichment) (Figures 1D and 1E). In contrast, reintroduction of an HIF nonbinding Type 2B pVHL mutant failed to stimulate glucose oxidation, resembling the phenotype observed in VHL-deficient cells (Figures 1D and 1E). Additional evidence for the overall glucose utilization was obtained from the enrichment of M3 isotopomers using [U13-C6]glucose (Figure S1A), which shows a lower contribution of glucose-derived carbons to the TCA cycle in VHL-deficient RCC cells (via pyruvate carboxylase and/or continued TCA cycling).

To test the effect of HIF activation on the overall glutamine incorporation in the TCA cycle, we labeled an isogenic pair of VHL-deficient and VHL-reconstituted UMRC2 cells with [U-13C5]glutamine, which generates M4 fumarate, M4 malate, M4 aspartate, and M4 citrate isotopomers through glutamine oxidation. As seen in Figure S1BVHL-deficient/VHL-positive UMRC2 cells exhibit similar enrichment of M4 fumarate, M4 malate, and M4 asparate (but not citrate) showing that VHL-deficient cells upregulate reductive carboxylation without compromising oxidative metabolism from glutamine. …  Labeled carbon derived from [5-13C1]glutamine can be incorporated into fatty acids exclusively through RC, and the labeled carbon cannot be transferred to palmitate through the oxidative TCA cycle (Figure 1B, red carbons). Tracer incorporation from [5-13C1]glutamine occurs in the one carbon (C1) of acetyl-CoA, which results in labeling of palmitate at M1, M2, M3, M4, M5, M6, M7, and M8 mass isotopomers. In contrast, lipogenic acetyl-CoA molecules originating from [U-13C6]glucose are fully labeled, and the labeled palmitate is represented by M2, M4, M6, M8, M10, M12, M14, and M16 mass isotopomers.

Figure 2 HIF Inactivation Is Necessary for Downregulation of Reductive Lipogenesis by pVHL

To determine the specific contribution from glucose oxidation or glutamine reduction to lipogenic acetyl-CoA, we performed isotopomer spectral analysis (ISA) of palmitate labeling patterns. ISA indicates that wild-type pVHL or pVHL L188V mutant-reconstituted UMRC2 cells relied mainly on glucose oxidation to produce lipogenic acetyl-CoA, while UMRC2 cells reconstituted with a pVHL mutant defective in HIF inactivation (Y112N or Y98N) primarily employed RC. Upon disruption of the pVHL-HIF interaction, glutamine becomes the preferred substrate for lipogenesis, supplying 70%–80% of the lipogenic acetyl-CoA (Figure 2C). This is not a cell-line-specific phenomenon, but it applies to VHL-deficient human RCC cells in general; the same changes are observed in 786-O cells reconstituted with wild-type pVHL or mutant pVHL or infected with vector only as control (Figure S2).

HIF Is Sufficient to Induce RC (reductive carboxylation) from Glutamine in RCC Cells

As shown in Figure 3C, reintroduction of wild-type VHLinto 786-O cells suppressed RC, whereas the expression of the constitutively active HIF-2α mutant was sufficient to stimulate this reaction, restoring the M1 enrichment of TCA cycle metabolites observed in VHL-deficient 786-O cells. Expression of HIF-2α P-A also led to a concomitant decrease in glucose oxidation, corroborating the metabolic alterations observed in glutamine metabolism (Figures 3D and 3E).

Figure 3 Expression of HIF-2α Is Sufficient to Induce Reductive Carboxylation and Lipogenesis from Glutamine in RCC Cells

Expression of HIF-2α P-A in 786-O cells phenocopied the loss-of-VHL with regards to glutamine reduction for lipogenesis (Figure 3G), suggesting that HIF-2α can induce the glutamine-to-lipid pathway in RCC cells per se. Although reintroduction of wild-type VHL restored glucose oxidation in UMRC2 and UMRC3 cells (Figures S3B–S3I), HIF-2α P-A expression did not measurably affect the contribution of each substrate to the TCA cycle or lipid synthesis in these RCC cells (data not shown). UMRC2 and UMRC3 cells endogenously express both HIF-1α and HIF-2α, whereas 786-O cells exclusively express HIF-2α. There is compelling evidence suggesting, at least in RCC cells, that HIF-α isoforms have overlapping—but also distinct—functions and their roles in regulating bioenergetic processes remain an area of active investigation. Overall, HIF-1α has an antiproliferative effect, and its expression in vitro leads to rapid death of RCC cells while HIF-2α promotes tumor growth (Keith et al., 2011Raval et al., 2005).

Metabolic Flux Analysis Shows Net Reversion of the IDH Flux upon HIF Activation

To determine absolute fluxes in RCC cells, we employed 13C metabolic flux analysis (MFA) as previously described (Metallo et al., 2012). Herein, we performed MFA using a combined model of [U-13C6]glucose and [1-13C1]glutamine tracer data sets from the 786-O derived isogenic clones PRC3 (VHL−/ −)/WT8 (VHL+) cells, which show a robust metabolic regulation by reintroduction of pVHL. To this end, we first determined specific glucose/glutamine consumption and lactate/glutamate secretion rates. As expected, PRC3 exhibited increased glucose consumption and lactate production when compared to WT8 counterparts (Figure 4A). While PRC3 exhibited both higher glutamine consumption and glutamate production rates than WT8 (Figure 4A), the net carbon influx was higher in PRC3 cells (Figure 4B). Importantly, the fitted data show that the flux of citrate to α-ketoglutarate was negative in PRC3 cells (Figure 4C). This indicates that the net (forward plus reverse) flux of isocitrate dehydrogenase and aconitase (IDH + ACO) is toward citrate production. The exchange flux was also higher in PRC3 than WT8 cells, whereas the PDH flux was lower in PRC3 cells. In agreement with the tracer data, these MFA results strongly suggest that the reverse IDH + ACO fluxes surpass the forward flux in VHL-deficient cells. The estimated ATP citrate lyase (ACLY) flux was also lower in PRC3 than in WT8 cells. Furthermore, the malate dehydrogenase (MDH) flux was negative, reflecting a net conversion of oxaloacetate into malate in VHL-deficient cells (Figure 4C). This indicates an increased flux through the reductive pathway downstream of IDH, ACO, and ACLY. Additionally, some TCA cycle flux estimates downstream of α-ketoglutarate were not significantly different between PRC and WT8 (Table S1). This shows that VHL-deficient cells maintain glutamine oxidation while upregulating reductive carboxylation (Figure S1B). This finding is in agreement with the higher glutamine uptake observed in VHL-deficient cells. Table S1 shows the metabolic network and complete MFA results. …

Addition of citrate in the medium, in contrast to acetate, led to an increase in the citrate-to-α-ketoglutarate ratio (Figure 5L) and absolute citrate levels (Figure S4H) not only in VHL-deficient but alsoVHL-reconstituted cells. The ability of exogenous citrate, but not acetate, to also affect RC in VHL-reconstituted cells may be explained by compartmentalization differences or by allosteric inhibition of citrate synthase (Lehninger, 2005); that is, the ability of acetate to raise the intracellular levels of citrate may be limited in (VHL-reconstituted) cells that exhibit high endogenous levels of citrate. Whatever the mechanism, the results imply that increasing the pools of intracellular citrate has a direct biochemical effect in cells with regards to their reliance on RC. Finally, we assayed the transcript and protein levels of enzymes involved in the reductive utilization of glutamine and did not observe significant differences between VHL-deficient andVHL-reconstituted UMRC2 cells (Figures S4I and S4J), suggesting that HIF does not promote RC by direct transactivation of these enzymes. The IDH1/IDH2 equilibrium is defined as follows:


Figure 5 Regulation of HIF-Mediated Reductive Carboxylation by Citrate Levels

We sought to investigate whether HIF could affect the driving force of the IDH reaction by also enhancing NADPH production. We did not observe a significant alteration of the NADP+/NADPH ratio between VHL-deficient and VHL-positive cells in the cell lysate (Figure S4I). Yet, we determined the ratio of the free dinucleotides using the measured ratios of suitable oxidized (α-ketoglutarate) and reduced (isocitrate/citrate) metabolites that are linked to the NADP-dependent IDH enzymes. The determined ratios (Figure S4J) are in close agreement with the values initially reported by the Krebs lab (Veech et al., 1969) and showed that HIF-expressing UMRC2 cells exhibit a higher NADP+/NADPH ratio. Collectively, these data strongly suggest that HIF-regulated citrate levels modulate the reductive flux to maintain adequate lipogenesis.

Reductive Carboxylation from Glutamine Is Detectable In Vivo

Figure 6 Evidence for Reductive Carboxylation Activity In Vivo

Loss of VHL Renders RCC Cells Sensitive to Glutamine Deprivation

We hypothesized that VHL deficiency results in cell addiction to glutamine for proliferation. We treated the isogenic clones PRC3 (VHL-deficient cells) and WT8 (VHL-reconstituted cells) with the glutaminase inhibitor 968 (Wang et al., 2010a). VHL-deficient PRC3 cells were more sensitive to treatment with 968, compared to the VHL-reconstituted WT8 cells (Figure 7A). To confirm that this is not only a cell-line-specific phenomenon, we also cultured UMRC2 cells in the presence of 968 or diluent control and showed selective sensitivity of VHL-deficient cells (Figure 7B).

Figure 7 VHL-Deficient Cells and Tumors Are Sensitive to Glutamine Deprivation

(A–E) Cell proliferation is normalized to the corresponding cell type grown in 1 mM glutamine-containing medium. Effect of treatment with glutaminase (GLS) inhibitor 968 in PRC3/WT8 (A) and UMRC2 cells (B). Rescue of GLS inhibition with dimethyl alpha-ketoglutarate (DM-Akg; 4 mM) or acetate (4 mM) in PRC3/WT8 clonal cells (C) and polyclonal 786-O cells (D). Effect of GLS inhibitor BPTES in UMRC2 cells (E). Student’s t test compares VHL-reconstituted cells to control cells in (A), (B), and (E) and DM-Akg or acetate-rescued cells to correspondent control cells treated with 968 only in (C) and (D) (asterisk in parenthesis indicates comparison between VHL-reconstituted to control cells). Error bars represent SEM.

(F) GLS inhibitor BPTES suppresses growth of human UMRC3 RCC cells as xenografts in nu/nu mice. When the tumors reached 100mm3, injections with BPTES or vehicle control were carried out daily for 14 days (n = 12). BPTES treatment decreases tumor size and mass (see insert). Student’s t test compares control to BPTES-treated mice (F). Error bars represent SEM.

(G) Diagram showing the regulation of reductive carboxylation by HIF.


In summary, our findings show that HIF is necessary and sufficient to promote RC from glutamine. By inhibiting glucose oxidation in the TCA cycle and reducing citrate levels, HIF shifts the IDH reaction toward RC to support citrate production and lipogenesis (Figure 7G). The reductive flux is active in vivo, fuels tumor growth, and can potentially be targeted pharmacologically. Understanding the significance of reductive glutamine metabolism in tumors may lead to metabolism-based therapeutic strategies.

Along with others, we reported that hypoxia and loss of VHL engage cells in reductive carboxylation (RC) from glutamine to support citrate and lipid synthesis (Filipp et al., 2012Metallo et al., 2012Wise et al., 2011). Wise et al. (2011) suggested that inactivation of HIF in VHL-deficient cells leads to reduction of RC. These observations raise the hypothesis that HIF, which is induced by hypoxia and is constitutively active inVHL-deficient cells, mediates RC. In our current work, we provide mechanistic insights that link HIF to RC. First, we demonstrate that polyclonal reconstitution of VHL in several human VHL-deficient RCC cell lines inhibits RC and restores glucose oxidation. Second, the VHL mutational analysis demonstrates that the ability of pVHL to mitigate reductive lipogenesis is mediated by HIF and is not the outcome of previously reported, HIF-independent pVHL function(s). Third, to prove our hypothesis we showed that constitutive expression of a VHL-independent HIF mutant is sufficient to phenocopy the reductive phenotype observed in VHL-deficient cells. In addition, we showed that RC is not a mere in vitro phenomenon, but it can be detected in vivo in human tumors growing as mouse xenografts. Lastly, treatment of VHL-deficient human xenografts with glutaminase inhibitors led to suppression of their growth as tumors.

7.9.10 Evaluation of HIF-1 inhibitors as anticancer agents

Semenza GL1.
Drug Discov Today. 2007 Oct; 12(19-20):853-9

Hypoxia-inducible factor 1 (HIF-1) regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, genetic instability, vascularization, metabolic reprogramming, autocrine growth factor signaling, invasion/metastasis, and treatment failure. In animal models, HIF-1 overexpression is associated with increased tumor growth, vascularization, and metastasis, whereas HIF-1 loss-of-function has the opposite effect, thus validating HIF-1 as a target. In further support of this conclusion, immunohistochemical detection of HIF-1α overexpression in biopsy sections is a prognostic factor in many cancers. A growing number of novel anticancer agents have been shown to inhibit HIF-1 through a variety of molecular mechanisms. Determining which combination of drugs to administer to any given patient remains a major obstacle to improving cancer treatment outcomes.

Aurelian Udristioiu


Aurelian Udristioiu

Lab Director at Emergency County Hospital Targu Jiu

Mechanisms that control T cell metabolic reprogramming are now coming to light, and many of the same oncogenes importance in cancer metabolism are also crucial to drive T cell metabolic transformations, most notably Myc, hypoxia inducible factor (HIF)1a, estrogen-related receptor (ERR) a, and the mTOR pathway.
The proto-oncogenic transcription factor, Myc, is known to promote transcription of genes for the cell cycle, as well as aerobic glycolysis and glutamine metabolism. Recently, Myc has been shown to play an essential role in inducing the expression of glycolytic and glutamine metabolism genes in the initial hours of T cell activation. In a similar fashion, the transcription factor (HIF)1a can up-regulate glycolytic genes to allow cancer cells to survive under hypoxic conditions

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Writer and Curator: Larry H. Bernstein, MD, FCAP 

7.8  Sirtuins

7.8.1 Function and regulation of the mitochondrial Sirtuin isoform Sirt5 in Mammalia

7.8.2 Substrates and Regulation Mechanisms for the Human Mitochondrial Sirtuins- Sirt3 and Sirt5

7.8.3 The mTORC1 Pathway Stimulates Glutamine Metabolism and Cell Proliferation by Repressing SIRT4

7.8.4  Rab1A and small GTPases Activate mTORC1

7.8.5 PI3K.Akt signaling in osteosarcoma

7.8.6 The mTORC1-S6K1 Pathway Regulates Glutamine Metabolism through the eIF4B-Dependent Control of c-Myc Translation

7.8.7 Localization of mouse mitochondrial SIRT proteins

7.8.8 SIRT4 Has Tumor-Suppressive Activity and Regulates the Cellular Metabolic Response to DNA Damage by Inhibiting Mitochondrial Glutamine Metabolism

7.8.9 Mitochondrial sirtuins and metabolic homeostasis

7.8.10 Mitochondrial sirtuins

7.8.11 Sirtuin regulation of mitochondria: energy production, apoptosis, and signaling


7.8.1 Function and regulation of the mitochondrial Sirtuin isoform Sirt5 in Mammalia

Gertz M1Steegborn C.
Biochim Biophys Acta. 2010 Aug; 1804(8):1658-65

Sirtuins are a family of protein deacetylases that catalyze the nicotinamide adenine dinucleotide (NAD(+))-dependent removal of acetyl groups from modified lysine side chains in various proteins. Sirtuins act as metabolic sensors and influence metabolic adaptation but also many other processes such as stress response mechanisms, gene expression, and organismal aging. Mammals have seven Sirtuin isoforms, three of them – Sirt3, Sirt4, and Sirt5 – located to mitochondria, our centers of energy metabolism and apoptosis initiation. In this review, we shortly introduce the mammalian Sirtuin family, with a focus on the mitochondrial isoforms. We then discuss in detail the current knowledge on the mitochondrial isoform Sirt5. Its physiological role in metabolic regulation has recently been confirmed, whereas an additional function in apoptosis regulation remains speculative. We will discuss the biochemical properties of Sirt5 and how they might contribute to its physiological function. Furthermore, we discuss the potential use of Sirt5 as a drug target, structural features of Sirt5 and of an Sirt5/inhibitor complex as well as their differences to other Sirtuins and the current status of modulating Sirt5 activity with pharmacological compounds.

removal of acetyl groups from modified lysine side chain

removal of acetyl groups from modified lysine side chain

removal of acetyl groups from modified lysine side chain

sirtuin structure

sirtuin structure

sirtuin structure

7.8.2 Substrates and Regulation Mechanisms for the Human Mitochondrial Sirtuins- Sirt3 and Sirt5

Schlicker C1Gertz MPapatheodorou PKachholz BBecker CFSteegborn C
J Mol Biol. 2008 Oct 10; 382(3):790-801

The enzymes of the Sirtuin family of nicotinamide-adenine-dinucleotide-dependent protein deacetylases are emerging key players in nuclear and cytosolic signaling, but also in mitochondrial regulation and aging. Mammalian mitochondria contain three Sirtuins, Sirt3, Sirt4, and Sirt5. Only one substrate is known for Sirt3 as well as for Sirt4, and up to now, no target for Sirt5 has been reported. Here, we describe the identification of novel substrates for the human mitochondrial Sirtuin isoforms Sirt3 and Sirt5. We show that Sirt3 can deacetylate and thereby activate a central metabolic regulator in the mitochondrial matrix, glutamate dehydrogenase. Furthermore, Sirt3 deacetylates and activates isocitrate dehydrogenase 2, an enzyme that promotes regeneration of antioxidants and catalyzes a key regulation point of the citric acid cycle. Sirt3 thus can regulate flux and anapleurosis of this central metabolic cycle. We further find that the N- and C-terminal regions of Sirt3 regulate its activity against glutamate dehydrogenase and a peptide substrate, indicating roles for these regions in substrate recognition and Sirtuin regulation. Sirt5, in contrast to Sirt3, deacetylates none of the mitochondrial matrix proteins tested. Instead, it can deacetylate cytochrome c, a protein of the mitochondrial intermembrane space with a central function in oxidative metabolism, as well as apoptosis initiation. Using a mitochondrial import assay, we find that Sirt5 can indeed be translocated into the mitochondrial intermembrane space, but also into the matrix, indicating that localization might contribute to Sirt5 regulation and substrate selection.

Mitochondria are central organelles in cellular energy metabolism, but also in processes such as apoptosis, cellular senescence, and lifespan regulation.1 and 2 Failures in mitochondrial function and regulation contribute to aging-related diseases, such as atherosclerosis3 and Parkinson’s disease,4 likely by increasing cellular levels of reactive oxygen species and the damage they cause.1 Emerging players in metabolic regulation and cellular signaling are members of the Sirtuin family of homologs of “silent information regulator 2” (Sir2), a yeast protein deacetylase.5 and 6 Sir2 was found to be involved in aging processes and lifespan determination in yeast,7 and 8 and its homologs were subsequently identified as lifespan regulators in various higher organisms.89 and 10 Sirtuins form class III of the protein deacetylase superfamily and hydrolyze one nicotinamide adenine dinucleotide (NAD +) as cosubstrate for each lysine residue they deacetylate.11 and 12 The coupling of deacetylation to NAD + was proposed to link changes in cellular energy levels to deacetylation activity,13 and 14 which would indicate Sirtuins as metabolic sensors. Other known regulation mechanisms for Sirtuin activity are the modulation of the expression levels of their genes6 and the autoinhibitory effect of an N-terminal region on the yeast Sirtuin “homologous to SIR2 protein 2” (Hst2).15

The seven mammalian Sirtuin proteins (Sirt1–Sirt7) have various substrate proteins that mediate functions in genetic, cellular, and mitochondrial regulation.5 and 6 The best-studied mammalian Sir2 homolog, Sirt1, was shown to regulate, among others, transcription factor p53, nuclear factor-kappa B, and peroxisome proliferator-activated receptor gamma coactivator-1-alpha.6 Three human Sirtuin proteins are known to be located in the mitochondria, Sirt3, Sirt4, and Sirt5,161718 and 19 although Sirt3 was reported to change its localization to nuclear when coexpressed with Sirt5.20 The recent identification of the first substrates for mitochondrial Sirtuins—acetyl coenzyme A synthetase 221 and 22 and glutamate dehydrogenase (GDH)16—as targets of Sirtuins 3 and 4, respectively, revealed that these Sirtuins control a regulatory network that has implications for energy metabolism and the mechanisms of caloric restriction (CR) and lifespan determination.23 Sirt3 regulates adaptive thermogenesis and decreases mitochondrial membrane potential and reactive oxygen species production, while increasing cellular respiration.24 Furthermore, Sirt3 is down-regulated in several genetically obese mice,24 and variability in the human SIRT3 gene has been linked to survivorship in the elderly. 25 In contrast to the deacetylases Sirt3 and Sirt5, Sirt4 appears to be an ADP ribosyltransferase. 16 Through this activity, Sirt4 inhibits GDH and thereby down-regulates insulin secretion in response to amino acids. 16 For Sirt5, however, there is no report yet on its physiological function or any physiological substrate. It is dominantly expressed in lymphoblasts and heart muscle cells,17 and 26 and its gene contains multiple repetitive elements that might make it a hotspot for chromosomal breaks. 26 Interestingly, the Sirt5 gene has been located to a chromosomal region known for abnormalities associated with malignant diseases. 26

A proteomics study found 277 acetylation sites in 133 mitochondrial proteins;27 many of them should be substrates for the mitochondrial Sirtuins mediating their various functions, but up to now, only one physiological substrate could be identified for Sirt3,21 and 22 and none could be identified for Sirt5. Our understanding of substrate selection by Sirtuins is incomplete, and knowledge of specific Sirtuin targets would be essential for a better understanding of Sirtuin-mediated processes and Sirtuin-targeted therapy. A first study on several Sirtuins showed varying preferences among acetylated peptides.28 Structural and thermodynamic analysis of peptides bound to the Sirtuin Sir2Tm from Thermatoga maritima indicated that positions − 1 and + 2 relative to the acetylation site play a significant role in substrate binding. 29 However, these studies were conducted with nonphysiological Sirtuin/substrate pairs, and other studies indicated little sequence specificity; instead, the yeast Sirtuin Hst2 was described to display contextual and conformational specificity: Hst2 deacetylated acetyl lysine only in the context of a protein, and it preferentially deacetylated within flexible protein regions. 30 Finally, statistical analysis of a proteomics study on acetylated proteins identified preferences at various positions such as + 1, − 2, and − 3, and deacetylation sites appeared to occur preferentially in helical regions. 27 Thus, our present knowledge of Sirtuin substrates and of factors determining Sirtuin specificity is incomplete and insufficient for sequence-based identification of physiological substrates.

Here, we describe the identification of novel targets for the mitochondrial deacetylases Sirt3 and Sirt5. We show that Sirt3 can deacetylate and thereby activate the enzymes GDH and isocitrate dehydrogenase (ICDH) 2—two key metabolic regulators in the mitochondrial matrix. We find that the N- and C-terminal regions of Sirt3 influence its activity against GDH and a peptide substrate, indicating roles in regulation and substrate recognition for these regions. Furthermore, we find that Sirt5 can deacetylate cytochrome c, a protein of the mitochondrial intermembrane space (IMS) with a central function in oxidative metabolism and apoptosis.

The upstream sequence contributes to the target specificity of Sirt3 and Sirt5

Sirtuins have been reported to have little sequence specificity,30 but other studies indicated a sequence preference dominated by positions − 1 and + 2.29 We tested the importance of the amino acid pattern preceding the acetylation site for recognition by the mitochondrial Sirtuins Sirt3 and Sirt5 through a fluorescence assay. First, the fluorogenic and commercially available modified p53-derived tetrapeptide QPK-acetylK, originally developed for Sirt2 assays but also efficiently used by Sirt3, was tested. Even 60 μg of Sirt5 did not lead to any deacetylation signal, whereas 0.35 μg of Sirt3 efficiently deacetylated the peptide (Fig. 1a). We then tested Sirt3 and Sirt5 on a second modified p53-derived tetrapeptide, RHK-acetylK. Sirt3 (0.5 μg) showed a slightly increased activity against this substrate as compared to QPK-acetylK (Fig. 1b); more importantly, 0.5 μg of Sirt5 showed significant activity against this peptide. These results show that the mitochondrial Sirtuins Sirt3 and, especially, Sirt5 indeed recognize the local target sequence, and target positions further upstream of − 1 seem to be involved in substrate recognition. For identification of novel substrates for the mitochondrial Sirtuins and further characterization of their target recognition mechanisms, we then turned to testing full-length proteins, as the downstream sequence and the larger protein context of the deacetylation site might also contribute to substrate selection.