Archive for the ‘Pyruvate Kinase’ Category

Agios Pharmaceuticals target the metabolism of cancer cells for making drugs that essentially try to repair cancer cells

Reporter: Aviva Lev-Ari, PhD, RN

A small biotech behind a groundbreaking approach to tackling cancer just got its first drug approved



Cancer Metabolism


Metabolic Immuno-Oncology




The VOICE of Larry H. Bernstein, MD, FCAP

Cancer cells didn’t need as much oxygen to metabolize sugar as normal cells. 

Not correct. Cancer cells metabolize glucose by aerobic glycolysis (4 ATP) with an impaired mitochondrial oxygen utilization (36 ATP). 

There is a reverse Warburg effect in which the underlying stromal cell carries out crosstalk with the epithelial cell. 

There is also a 3rd dimension. Cells undergo a series of adaptive changes tied to proteostasis. This involves the sulfur amino acid cysteine and disulfide bonds, which is involved with protein oligomerization in the ER, and also signaling in the mitochondria with mDNA and the nucleus. 

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Cholesterol metabolism in pancreatic cancer

Larry H. Bernstein, MD, FCAP, Curator




New Pancreatic Treatment Shows Promise


Study demonstrates how controlling cholesterol metabolism in pancreatic cancer cells reduces metastasis. [NIH].   http://www.genengnews.com/Media/images/GENHighlight/thumb_May4_2016_NIH_PancreaticCancerCells8616346835.jpg

Scientists say they have shown how controlling cholesterol metabolism in pancreatic cancer cells reduces metastasis, pointing to a potential new treatment using drugs previously developed for atherosclerosis.

“We show for the first time that if you control the cholesterol metabolism you could reduce pancreatic cancer spread to other organs,” said Ji-Xin Cheng, Ph.D., a professor in Purdue University’s Weldon School of Biomedical Engineering and Department of Chemistry. “We chose pancreatic cancer to test this approach because it is the most aggressive disease of all the cancers.”

Dr. Cheng had previously led a team of researchers discovering a link between prostate cancer’s aggressiveness and the accumulation of a compound produced when cholesterol is metabolized in cells, findings that could bring new diagnostic and treatment methods. The new study involved researchers at the Purdue Center for Cancer Research and School of Biomedical Engineering, the Indiana University Simon Cancer Center and School of Medicine, and Purdue’s Department of Biological Sciences, Department of Comparative Pathobiology, and Department of Biochemistry.

The findings, detailed in a paper (“Abrogating Cholesterol Esterification Suppresses Growth and Metastasis of Pancreatic Cancer”) just published in Oncogene, suggest that a class of drugs previously developed to treat atherosclerosis could be repurposed for treatment of pancreatic cancer and other forms of cancer. Atherosclerosis is the buildup of fats, cholesterol, and other substances in arteries, restricting blood flow.

The researchers found accumulations of the compound cholesteryl ester in human pancreatic cancer specimens and cell lines, demonstrating a link between cholesterol esterification and metastasis. Excess quantities of cholesterol result in cholesteryl ester being stored in lipid droplets within cancer cells.

“The results of this study demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification,” said Jingwu Xie, Ph.D., the Jonathan and Jennifer Simmons Professor at the Indiana University School of Medicine and a researcher at the Indiana University Melvin and Bren Simon Cancer Center.

The paper’s lead author is Purdue postdoctoral fellow Junjie Li, Ph.D. Purdue researchers have developed an analytical tool, Raman spectromicroscopy, that allows compositional analysis of single lipid droplets in living cells.

“We identified an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines,” Dr. Li said. “Depletion of cholesterol esterification significantly reduced pancreatic tumor growth and metastasis in mice.”

Findings show that drugs like avasimibe, previously developed for treatment of atherosclerosis, reduced the accumulation of cholesteryl ester. Pancreatic cancer usually kills within a few months of diagnosis. It is hoped the potential new treatment might extend life of these patients for a year, Cheng said.

The accumulation of cholesteryl ester is controlled by an enzyme called acyl-coenzyme A acyltransferase-1 (ACAT-1), and findings have correlated a higher expression of the enzyme with a poor survival rate for patients. The researchers analyzed tissue samples from pancreatic cancer patients and then tested the drug treatment in a type of laboratory mice referred to as an orthotopic mouse model, developed at the IU School of Medicine. Specimens of human pancreatic tissues were obtained from the Simon Cancer Center Solid Tissue Bank.

Imaging showed a decrease of the number of lipid droplets, and Raman spectral analysis verified a significant reduction of cholesteryl ester in the lipid droplets, suggesting that avasimibe acted by blocking cholesterol esterification. The drug did not induce weight loss, and there was no apparent organ toxicity in the liver, kidney, lung and spleen, Dr. Cheng said.

Findings also showed that blocking storage of cholesteryl ester causes cancer cells to die, specifically due to damage to the endoplasmic reticulum, a workhorse of protein and lipid synthesis.

“By using avasimibe, a potent inhibitor of ACAT-1, we found that pancreatic cancer cells were much more sensitive to ACAT-1 inhibition than normal cells,” added Dr. Cheng.

Additional research confirmed that the anticancer effect of avasimibe is specific to ACAT-1 inhibition. The researchers performed various biochemical assays and “genetic ablation” to confirm the drug’s anticancer effect.

“The results showed that avasimibe treatment for four weeks remarkably suppressed tumor size and largely reduced tumor growth rate,” said paper co-author Timothy Ratliff, the Robert Wallace Miller Director of Purdue’s Center for Cancer Research. “Metastatic lesions in lymph nodes and distant organs also were assessed at the end of the study. A much higher number of metastatic lesions in lymph nodes were detected in the control group than the avasimibe-treated group.”

Each mouse in the control group showed at least one metastatic lesion in the liver. In contrast, only three mice in the avasimibe-treated group showed single lesion in liver.


Abrogating cholesterol esterification suppresses growth and metastasis of pancreatic cancer

J Li1, D Gu2, S S-Y Lee1, B Song1, S Bandyopadhyay3, S Chen4, S F Konieczny3,5, T L Ratliff5,6, X Liu5,7, J Xie2 and J-X Cheng1,5
ncogene 2 May 2016;                                         http://dx.doi.org:/10.1038/onc.2016.168

Cancer cells are known to execute reprogramed metabolism of glucose, amino acids and lipids. Here, we report a significant role of cholesterol metabolism in cancer metastasis. By using label-free Raman spectromicroscopy, we found an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines, mediated by acyl-CoA cholesterol acyltransferase-1 (ACAT-1) enzyme. Expression of ACAT-1 showed a correlation with poor patient survival. Abrogation of cholesterol esterification, either by an ACAT-1 inhibitor or by shRNA knockdown, significantly suppressed tumor growth and metastasis in an orthotopic mouse model of pancreatic cancer. Mechanically, ACAT-1 inhibition increased intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum stress and caused apoptosis. Collectively, our results demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification.

Metastasis is the major cause of cancer-related mortality. Though localized tumors can often be treated by surgery or other therapies, treatment options for metastatic diseases are limited. Cancer metastasis has been revealed to be a multiple step process, including cancer cell migration, local invasion, intravasation, circulation through blood and lymph vessels, extravasation, survival and colonization in distant organs.1, 2, 3Mediators identified in these processes have provided the basis for the development of therapies to target metastasis. Current therapeutic strategies for treating metastatic tumors mainly focus on targeting the adhesive molecules and extracellular proteases.4However, these therapeutics have not been proven to be effective in clinical trials, partially owing to the various escape mechanisms used by the metastatic cancer cells.2, 5, 6 Thus, an unmet need exists to develop new therapeutic strategies for treating metastatic cancers.

Recent advances in cancer metabolism have unveiled many potential therapeutic targets for cancer treatment. Metabolic reprogramming, a strategy used by cancer cells to adapt to the rapid proliferation, is being recognized as a new hallmark of cancer.7 Substantial studies have found increased glycolysis, glutaminolysis, nucleotide and lipid synthesis in cancer cells.7, 8, 9,10 Considering that altered metabolic pathways only happen in cancer cells but not in normal cells, targeting these pathways may provide cancer-specific treatments. A number of inhibitors of metabolic enzymes, such as glycolysis inhibitors, are under clinical trials as targeted cancer therapeutics.11

Of various metabolic pathways, lipid metabolism has been suggested to have an important role in cancer cell migration, invasion and metastasis.12 A recent study reported that surrounding adipocytes provide energy source for ovarian cancer cells to promote its rapid growth and metastasis.13 Blocking lipidde novo synthesis pathway has been shown to suppress tumor regrowth and metastasis after anti-angiogenesis treatment withdrawal.14 In parallel, lipolysis by the enzyme monoacylglycerol lipase was shown to regulate the fatty acid network, which promotes cancer cell migration, invasion and growth.15

Cholesterol, a critical component of the plasma membrane, is also implied to be correlated to cancer metastasis.16 It has been shown that prostate cancer bone metastases contain a high level of cholesterol.17 Modulation of cholesterol level in plasma membrane was shown to regulate the capability of cell migration.18, 19Moreover, cholesterol-enriched lipid rafts were shown to have an essential role in cancer cell adhesion and migration.20 Mammalian cells obtain cholesterol either from de novo synthesis or from the uptake of low-density lipoprotein (LDL).21 Inside cells, excess free cholesterol is esterified and stored as cholesteryl ester (CE) in lipid droplets (LDs), which is mediated by acyl-CoA cholesterol acyltransferase (ACAT).22 Increased CE level has been reported in breast cancer,23 leukemia,24 glioma25 and prostate cancer.26Despite these advances, the role of cholesterol esterification in cancer progression, especially in cancer metastasis, is not well understood.

In this article, we report a link between cholesterol esterification and metastasis in pancreatic cancer. Using stimulated Raman scattering (SRS) microscopy and Raman spectroscopy to map LDs stored inside single cells and analyze the composition of individual LDs, we identified an aberrant accumulation of CE in human pancreatic cancer specimens and cell lines. Abrogation of cholesterol esterification, either by inhibiting ACAT-1 enzyme activity or by shRNA knockdown of ACAT-1 expression, significantly reduced pancreatic tumor growth and metastasis in an orthotopic mouse model. Mechanistically, inhibition of cholesterol esterification disturbed cholesterol homeostasis by increasing intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum (ER) stress and eventually led to apoptosis.

In this study, we revealed a link between CE accumulation and pancreatic cancer metastasis. Accumulation of CE via ACAT-1 provides a mechanism to keep high metabolic activity and avoid toxicity from excess free cholesterol. Previously, CE has been reported in breast cancer,23 leukemia,24 glioma25 and prostate cancer.26 Inhibition of cholesterol esterification was shown to suppress tumor growth or cancer cell proliferation.24, 25, 26 Here, we demonstrate that inhibition of cholesterol esterification can be used to treat metastatic pancreatic cancer.

Cholesterol is an essential lipid having important roles in membrane construction, hormone production and signaling.21Aberrant cholesterol metabolism is known to be associated with cardiovascular diseases and cancers.35, 36 Statins, inhibitors of HMG-CoA reductase, have been explored as potential therapies for pancreatic cancer.37 However, statins were not associated with a reduced risk of pancreatic cancer in clinical trials.38 One possible reason is that HMG-CoA reductase is also required for downstream protein prenylation, a critical process for protein activation.39Thus, the effect of statin is not just inhibiting cholesterol synthesis, but also other pathways which may render toxicity to normal cells. This non-specific toxicity is a possible reason for the limited anti-cancer outcome of statin in clinical trials.

Our study identified cholesterol esterification as a novel target for suppression of pancreatic cancer proliferation and metastasis. Inhibitors of ACAT-1 are expected to have great value as cancer-targeting therapeutics, as CE accumulation only occurs in cancer tissues or cell lines. Our animal studies with avasimibe treatment showed no adverse effect to the animals at a dosage of 15mg/kg. More importantly, modulation of cholesterol esterification suppressed not only tumor growth but also tumor metastasis. These results are expected to stimulate further biological studies to fully appreciate the role of cholesterol metabolism in cancer initiation and progression. As CE accumulation happens in several types of aggressive cancer, blocking cholesterol esterification could be pursued as a therapeutic strategy for other types of cancers. By combining with existing chemotherapies, such as gemcitabine, we believe this metabolic treatment possesses high possibilities to extend patients’ survival time by retarding cancer progression and metastasis.

The molecular mechanism that links CE accumulation to cancer aggressiveness needs further studies. One possible mechanism is that cholesterol esterification keeps signaling pathways active by maintaining a low free cholesterol environment. One of the possible targets is the caveolin-1 signaling pathway. Caveolin-1, a regulator of cellular cholesterol homeostasis, is considered as a marker for pancreatic cancer progression.11 Particularly, a promoting role of caveolin-1 in pancreatic cancer metastasis has been reported.40 Our preliminary studies showed ACAT-1 inhibition reduced the expression level of SREBP1, caveolin-1 and phosphorylated ERK1/2 (unpublished data). The effect on caveolin-1 is probably mediated by SREBP1, which senses the intracellular cholesterol homeostasis.41 Meanwhile, caveolin-1 may have an important role in mediating the action of SREBP1 on MAPK pathways,42, 43 which are known to have essential roles in cancer cell metastasis.44 Therefore, it is possible that increased free cholesterol level induced by ACAT-1 inhibition inactivates SREBP1, leading to downregulation of caveolin-1/MAPK pathway, which contributes to the reduced cancer aggressiveness.

Besides the caveolin-1/MAPK signaling, other possibilities include the potential alteration of the membrane composition, such as lipid rafts, by ACAT-1 inhibition. Lipid rafts are known to provide platforms for multiple cellular signaling pathways.20 Thus, modulation of cholesterol metabolism is likely to have more profound effects via other signaling pathways. Future studies are needed to fully elucidate the molecular mechanism.


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A Reconstructed View of Personalized Medicine

Author: Larry H. Bernstein, MD, FCAP


There has always been Personalized Medicine if you consider the time a physician spends with a patient, which has dwindled. But the current recognition of personalized medicine refers to breakthrough advances in technological innovation in diagnostics and treatment that differentiates subclasses within diagnoses that are amenable to relapse eluding therapies.  There are just a few highlights to consider:

  1. We live in a world with other living beings that are adapting to a changing environmental stresses.
  2. Nutritional resources that have been available and made plentiful over generations are not abundant in some climates.
  3. Despite the huge impact that genomics has had on biological progress over the last century, there is a huge contribution not to be overlooked in epigenetics, metabolomics, and pathways analysis.

A Reconstructed View of Personalized Medicine

There has been much interest in ‘junk DNA’, non-coding areas of our DNA are far from being without function. DNA has two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), and the pyrimidines (cytosine [C], thymine [T], and  no uracil [U]),  while RNA contains only A, G, C, and U (no T).  The Watson-Crick proposal set the path of molecular biology for decades into the 21st century, culminating in the Human Genome Project.

There is no uncertainty about the importance of “Junk DNA”.  It is both an evolutionary remnant, and it has a role in cell regulation.  Further, the role of histones in their relationship the oligonucleotide sequences is not understood.  We now have a large output of research on noncoding RNA, including siRNA, miRNA, and others with roles other than transcription. This requires major revision of our model of cell regulatory processes.  The classic model is solely transcriptional.

  • DNA-> RNA-> Amino Acid in a protein.

Redrawn we have

  • DNA-> RNA-> DNA and
  • DNA->RNA-> protein-> DNA.

Neverthess, there were unrelated discoveries that took on huge importance.  For example, since the 1920s, the work of Warburg and Meyerhoff, followed by that of Krebs, Kaplan, Chance, and others built a solid foundation in the knowledge of enzymes, coenzymes, adenine and pyridine nucleotides, and metabolic pathways, not to mention the importance of Fe3+, Cu2+, Zn2+, and other metal cofactors.  Of huge importance was the work of Jacob, Monod and Changeux, and the effects of cooperativity in allosteric systems and of repulsion in tertiary structure of proteins related to hydrophobic and hydrophilic interactions, which involves the effect of one ligand on the binding or catalysis of another,  demonstrated by the end-product inhibition of the enzyme, L-threonine deaminase (Changeux 1961), L-isoleucine, which differs sterically from the reactant, L-threonine whereby the former could inhibit the enzyme without competing with the latter. The current view based on a variety of measurements (e.g., NMR, FRET, and single molecule studies) is a ‘‘dynamic’’ proposal by Cooper and Dryden (1984) that the distribution around the average structure changes in allostery affects the subsequent (binding) affinity at a distant site.

What else do we have to consider?  The measurement of free radicals has increased awareness of radical-induced impairment of the oxidative/antioxidative balance, essential for an understanding of disease progression.  Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). The unifying factor in determining toxicity and carcinogenicity for all these metals is the generation of reactive oxygen and nitrogen species. Various studies have confirmed that metals activate signaling pathways and the carcinogenic effect of metals has been related to activation of mainly redox sensitive transcription factors, involving NF-kappaB, AP-1 and p53.

I have provided mechanisms explanatory for regulation of the cell that go beyond the classic model of metabolic pathways associated with the cytoplasm, mitochondria, endoplasmic reticulum, and lysosome, such as, the cell death pathways, expressed in apoptosis and repair.  Nevertheless, there is still a missing part of this discussion that considers the time and space interactions of the cell, cellular cytoskeleton and extracellular and intracellular substrate interactions in the immediate environment.

There is heterogeneity among cancer cells of expected identical type, which would be consistent with differences in phenotypic expression, aligned with epigenetics.  There is also heterogeneity in the immediate interstices between cancer cells.  Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. In the case of breast cancer, there is interaction with estrogen , and we refer to an androgen-unresponsive prostate cancer.

Finally,  the interaction between enzyme and substrates may be conditionally unidirectional in defining the activity within the cell.  The activity of the cell is dynamically interacting and at high rates of activity.  In a study of the pyruvate kinase (PK) reaction the catalytic activity of the PK reaction was reversed to the thermodynamically unfavorable direction in a muscle preparation by a specific inhibitor. Experiments found that in there were differences in the active form of pyruvate kinase that were clearly related to the environmental condition of the assay – glycolitic or glyconeogenic. The conformational changes indicated by differential regulatory response were used to present a dynamic conformational model functioning at the active site of the enzyme. In the model, the interaction of the enzyme active site with its substrates is described concluding that induced increase in the vibrational energy levels of the active site decreases the energetic barrier for substrate induced changes at the site. Another example is the inhibition of H4 lactate dehydrogenase, but not the M4, by high concentrations of pyruvate. An investigation of the inhibition revealed that a covalent bond was formed between the nicotinamide ring of the NAD+ and the enol form of pyruvate.  The isoenzymes of isocitrate dehydrogenase, IDH1 and IDH2 mutations occur in gliomas and in acute myeloid leukemias with normal karyotype. IDH1 and IDH2 mutations are remarkably specific to codons that encode conserved functionally important arginines in the active site of each enzyme. In this case, there is steric hindrance by Asp279 where the isocitrate substrate normally forms hydrogen bonds with Ser94.

Personalized medicine has been largely viewed from a lens of genomics.  But genomics is only the reading frame.  The living activities of cell processes are dynamic and occur at rapid rates.  We have to keep in mind that personalized in reference to genotype is not complete without reconciliation of phenotype, which is the reference to expressed differences in outcomes.


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Is the Warburg effect an effect of deregulated space occupancy of methylome?

Larry H. Bernstein and Radoslav Bozov, co-curation




It would appear that pyruvate is directly used by cancer cell machinery for sustaining genome independence, and that CRISP-Cas9 system is essentially a modified CAD protein for making small bases.

13C-labeled biochemical probes for the study of cancer metabolism with dynamic nuclear polarization-enhanced magnetic resonance imaging

Lucia Salamanca-Cardona and Kayvan R. Keshari

Cancer & Metabolism 2015; 3:9          http://dx.doi.org:/10.1186/s40170-015-0136-2

In recent years, advances in metabolic imaging have become dependable tools for the diagnosis and treatment assessment in cancer. Dynamic nuclear polarization (DNP) has recently emerged as a promising technology in hyperpolarized (HP) magnetic resonance imaging (MRI) and has reached clinical relevance with the successful visualization of [1-13C] pyruvate as a molecular imaging probe in human prostate cancer. This review focuses on introducing representative compounds relevant to metabolism that are characteristic of cancer tissue: aerobic glycolysis and pyruvate metabolism, glutamine addiction and glutamine/glutamate metabolism, and the redox state and ascorbate/dehydroascorbate metabolism. In addition, a brief introduction of probes that can be used to trace necrosis, pH changes, and other pathways relevant to cancer is presented to demonstrate the potential that HP MRI has to revolutionize the use of molecular imaging for diagnosis and assessment of treatments in cancer.


Since the hallmark discovery of the Warburg effect in cancer cells in the 1920s, it has been widely accepted that the metabolic properties of cancer cells are vastly different from those of normal cells [1]. Starting from the observation that many cancerous (neoplastic) cells have higher rates of glucose utilization and lactate production, the development of tools and methods to correlate specific cellular metabolic processes to different types of cancer cells has received increased research focus [2, 3]. Several imaging techniques are currently in use for this purpose, including radiography, scintigraphy, positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance (MR) [4, 5].

For more than 30 years, MR has been a revolutionary diagnostic tool, used in a wide range of settings from the central nervous system to cardiomyopathies and cancers. MR imaging (MRI) can outline molecular and cellular processes with high spatial resolution. Typically, MRI of body tissues is achieved via contrast visualization of the protons (1H) of water, which are present in high abundance in living systems. This can be extended to MR spectroscopy (MRS), which can further differentiate between less abundant, carbon-bearing, biological metabolites in vivo utilizing 1Hs of these compounds [6, 7]. However, despite its usefulness in imaging whole body tissues, 1H MRS has low spectral resolution and poor sensitivity for these less abundant metabolites. In addition,13C MRS is increasingly difficult, in comparison to 1H MRS, in that both the gyromagnetic ratio (approximately 25 % of 1H) and natural abundance (1.1 % of 1H) are significantly lower, making the detection of carbon-bearing compounds difficult [8, 9]. The low spectral resolution of 1H MRS for metabolites can be addressed by using 13C-enriched compounds, and with this direct 13C MRS, metabolic processes can be traced, utilizing enriched tags on specific carbons in a given metabolite [10]. While enrichment of molecules in 13C can also moderately address the sensitivity limitation of MRS, recent work in hyperpolarization (HP) provides a means of dramatically increasing sensitivity and enhancing signals, well beyond that of the equilibrium state obtained via MRS. [11, 12]. The focus of this review will be the introduction of this approach in the setting of cancer metabolism, delineating probes of interest, which have been applied to study metabolic processes in vivo.

Obtaining a hyperpolarized probe

In MR, a desired target is placed in a magnetic field where the nuclear spins of molecules are aligned with or against the direction of the magnetic field. The nuclear spins have thus different energies, and an MR signal is detected upon relaxation of nuclear spins of higher energy. At thermal equilibrium, the number of spins aligned with the magnetic field nearly equals the number of spins opposing the direction of the magnetic field. Thus, at thermal equilibrium, spin polarization is in the order of >0.0005 % resulting in a limited signal. Signal increases on the order of 100,000-fold can be achieved by hyperpolarizing the system via the redistribution of the spin population levels found at equilibrium [10, 13]. There are several techniques that have been used to achieve hyperpolarization of various nuclei: spin-exchange optical pumping of 3He and 129Xe, parahydrogen-induced polarization (PHIP), and dissolution dynamic nuclear polarization (DNP) [11,14, 15]. Both PHIP and DNP techniques can polarize biologically relevant nuclei like 13C and 15N, although there is a wider range of molecules that can be targeted for hyperpolarization using dissolution DNP [14, 1618].

The goal of DNP is the transfer of polarization from highly polarized unpaired electron spins to the nuclear spins of a desired target compound. This is achieved by applying an external magnetic field to a free-radical agent in order to polarize electron spins, followed by saturating the electron spin resonance via microwave irradiation in order to obtain polarization transfer. The free-radical agent is generally a stable organic compound that is compatible with aqueous buffers, which are used as solvent in order to obtain a homogeneous distribution of the radical [13]. Nearly 100 % of the electrons on the free-radical agent are polarized when the free-radical/solvent mixture is subjected to high magnetic fields (≥3.3 T) followed by rapid freezing to 1 K using liquid helium in order to obtain a sample frozen to an amorphous state, which is necessary for retention and transfer of polarization [18]. For biological applications, after transfer of electron spin polarization to the nuclei of interest has occurred, the preparation must exist in solution, which can be achieved utilizing a dissolution process in which the solid sample is rapidly melted via injection of a hot solvent, typically a biologically compatible buffer, into the frozen sample [13]. The dissolution process results in a liquid sample at room temperature, while still preserving the enhanced polarization obtained by the microwave irradiation of the frozen sample [8]. Additionally, the use of chelating agents (e.g., EDTA) with the solvent to eliminate trace metals and more recently the use of gadolinium (Gd) chelates with the DNP sample have been used to further enhance and retain polarization in the liquid sample, albeit with caution over potential toxic effects when applied in vivo and the potential for loss of hyperpolarization due to T 1 shortening [11, 19, 20]. More in-depth exploration of the technical aspects of probe development has been previously reviewed [8, 11].

Considerations in probe selection and current research

The usefulness of a molecule for hyperpolarized MRS is dependent on the polarization lifetime of the nucleus of interest, and this property is determined by the spin-lattice relaxation constant (T1) [21]. Dipolar coupling, the magnetic field range, and molecular size can also affect the T1 of a given nucleus. In general, high magnetic fields and large molecular weights decrease the T1. Dipole-dipole coupling of 13C with 1H is common in biologically relevant molecules, and it shortens relaxation times; therefore, carbon atoms directly bound to 1H are generally not useful as probes for HP. For example, all carbons present in glucose (an important substrate in cancer cells) have relaxation times shorter than 2 s [22]. On the other hand, carbonyl carbons of biologically relevant molecules generally have T1’s above 20 s even at high magnetic fields like [1-13C] pyruvic acid, which has relaxation times of 67, 48, and 44 s at 3, 11.7, and 14.1 T, respectively [2325]. Even carbons that are less oxidized than carbonyls, like the hemi-ketal in [2-13C] fructose have T1’s one order of magnitude higher than glucose carbons. Short spin-lattice relaxation times can sometimes be increased by deuterium enrichment of the sample. With this technique, protons that are directly bound to carbons are exchanged for deuterium atoms which results in the reduction of dipole-dipole relaxation, further preserving the hyperpolarized state [26]. This has resulted in increased T1’s of 13C nuclei in molecules such as glucose (T1 increased from 2 s to 10–14 s), providing the possibility of utilizing them in future metabolic studies [2729]. Despite the effect of deuterium enrichment, research efforts have largely focused on developing carbonyl-bearing molecules as molecular imaging probes. The focus of this review is to introduce representative compounds relevant to metabolism that are characteristic of cancer tissue and have been applied in the work of multiple groups: aerobic glycolysis, glutamine addiction, and the redox state.

Pyruvate and aerobic glycolysis

Of particular interest to cancer metabolism is the increased conversion of glucose to lactate as a result of modulated aerobic glycolysis. This process, also known as the Warburg effect, is characteristic of many tumors with altered metabolism where pyruvate generated from glucose metabolism via glycolysis is preferentially converted to lactate by lactate dehydrogenase (LDH) as opposed to entering the tricarboxylic acid cycle [1]. With this phenotype, cancer cells show a preference for lactate fermentation even in the presence of oxygen, thus bypassing oxidative respiration for ATP generation. Because of this, pyruvate has been the preferred probe for HP MRS research since it is an intermediate metabolite in pathways characteristic of aberrant metabolism in cancer cells, including increased lactate production as a result of aerobic glycolysis where detection of HP pyruvate-derived lactate can be used as a marker for cancer and response to treatment [30, 31] as well as an intermediate in amino acid metabolism (e.g., interconversion to alanine via transamination with glutamate) (Fig. 1). In addition, as mentioned before, carbonyl carbons in pyruvate have long relaxation times where even the methyl carbon can have T1’s above 50 s after deuterium enrichment [32]. The interconversion of pyruvate to lactate has been exploited for MRI by using [1-13C] pyruvate and detecting the accumulation of increased lactate in cancerous tissue as compared to surrounding benign tissue. Increased conversion of pyruvate to lactate and alanine has been demonstrated to precede MYC-driven tumorigenesis by using HP [1-13C] pyruvate in murine models [33]. Furthermore, in the same study, a decrease in the flux of alanine was observed at the tumor stage while a decrease in lactate conversion was indicative of tumor regression [33]. In transgenic adenocarcinoma of mouse prostate (TRAMP) models, in vivo studies using HP [1-13C] pyruvate demonstrated that hyperpolarized pyruvate and its metabolic products can be used non-invasively and with high specificity to obtain a profile of the histologic grade of prostate cancers [34]. In vivo imaging following hyperpolarized pyruvate has also been used to evaluate the role of glutaminase and LDH in human lymphoma models [35] as well as to elucidate metabolism of pyruvate in breast cancer [36] and renal cell carcinoma with treatment [30, 37].
Flux of hyperpolarized [1-13C] pyruvate to [1-13C] lactate in prostate regions. a MR image from patient with prostate cancer showing regions of cancerous tissue and surrounding normal tissue. bd Localized dynamic hyperpolarized [1-13C]pyruvate and [1-13C]lactate spectral from voxels overlapping the contralateral region of prostate (turquoise), a region of prostate cancer (yellow), and a vessel outside the prostate (green). Adapted with permission from ref. [43]

Early work that utilized HP pyruvate to assess the response of tumors to treatment was conducted in mice xenografted with EL-4 lymphoma cells and treated with etoposide, a topoisomerase inhibitor that causes rapid cell death [38, 39]. In this study, tumor necrosis was correlated to a decrease in the flux of hyperpolarized lactate which was suggested to be due to a decrease in NAD+ and NADH in the intracellular pool as well as loss of LDH function. More recently, HP [1-13C] pyruvate has been used as a biomarker to evaluate early response to radiation therapy in glioma tumors by observing a decrease in hyperpolarized lactate suggested to be a result of changes in tumor perfusion which can be detected between 24 and 96 h following treatment [40, 41]. HP [1-13C] pyruvate has also been used to detect early response to temozolomide (TMZ) treatment on human glioblastoma rat models [42]. The study successfully showed for the first time detection of response to TMZ therapy 1 day after TMZ administration. The continued reports on using HP pyruvate as an imaging probe for assessing treatment response indicate the potential of the compound to become a standard in the field. Moreover, these studies demonstrate the usefulness of HP [1-13C] pyruvate as a tool for early assessment of therapy response, which can improve treatment selection at the clinical level. Pyruvate has also been validated as a metabolic imaging marker for use in humans [43]. In a two-phase study, patients with biopsy-proven prostate cancer of various histological grades were injected with HP [1-13C] pyruvate. In the first phase, a maximum dose level was determined to establish pharmacological safety of the HP probe while still injecting enough pyruvate to allow visualization. This addressed one of the major challenges faced in translating HP MRI to clinical applications: the potential toxicity of compounds that must be injected into patients. In the second phase, metabolism of pyruvate was visualized in real time and differences in the ratio of [1-13C] lactate to [1-13C] pyruvate between identified cancerous regions and normal tissue regions were successfully observed (Fig. 1ad). [1-13C] lactate in regions that did not contain tumor was not detected, confirming previous biopsy and preclinical studies that demonstrated low flux of [1-13C] pyruvate to lactate and low concentrations of lactate in benign prostate tissues [44, 45]. Preliminary results indicated the possibility of detecting previously unobserved cancerous regions by HP [1-13C] pyruvate, later confirmed to be Gleason 4+3 cancer by biopsy, though further investigation into the relationship between grade and metabolism in prostate cancer patients is needed. While there are challenges associated with translation to clinical use for HP [1-13C] pyruvate, the first in human study demonstrated the feasibility of hyperpolarization technology as a safe diagnostic tool and provides the potential for utilizing this approach in preclinical models with direct translation to the clinic.

Glutamine metabolism

Glutamine is an amino acid that plays an important cellular role as nitrogen donor in the form of an amide group for purine and pyrimidine biosynthesis, leaving a glutamate molecule in the process although glutamine can also be converted to glutamate by glutaminase in a reaction independent of nucleotide biosynthesis. Glutamate is the primary nitrogen donor for the biosynthesis of non-essential amino acids. Transaminases catalyze the transfer of the amine group from glutamate to α-ketoacids to synthesize alanine, aspartate (precursor for asparagine), serine (precursor for glycine and cysteine), ornithine (precursor for arginine), and proline which is derived from the glutamate carbon backbone. Glutamine is considered a non-essential amino acid as it can be recycled from glutamate and ammonia in a reaction catalyzed by glutamine synthetase; however, some cancer cells show increase consumption of glutamine and are unable to grow in the absence of exogenous glutamine [46, 47]. This metabolic characteristic of cells to require exogenous glutamine for growth has been termed “glutamine addiction” and has generated extensive research interest as an indicator of development of cancerous tissues [48]. In particular to the field of HP MRI, the conversion rate of glutamine to glutamate (Fig. 2) was explored in hepatocellular carcinoma (HCC) using a [5-13C] glutamine probe (Fig. 2) [49]. Using the ratio between [5-13C] glutamine and [5-13C] glutamate, it was demonstrated that HCC cells convert glutamine at a higher rate than normal cells supporting the notion of glutamine addiction. One important aspect of this study was the choice of [5-13C] glutamine as a probe as opposed to [1-13C] glutamine, which has a longer T1 (16.1 vs. 24.6 s at 9.4 T) [49, 50]. [5-13C] glutamine was selected because the chemical shift change obtained from [1-13C] in glutamine and glutamate is far too small, which could prevent proper identification and quantification of the peaks. This highlights the importance of understanding not only the target compound to be hyperpolarized but also the metabolic products to be detected and their resulting spectra in MR. This is further emphasized with studies that demonstrate the usefulness of [1-13C] glutamine as a source for [1-13C] glutamate in order to follow the metabolism of α-ketoglutarate to observe the metabolic state of the TCA cycle in transformed cells [51]. Furthermore, [1-13C] α-ketoglutarate has been hyperpolarized and used to visualize other metabolic events involving [1-13C] glutamate such as mutations in IDH1 expression in glioma tumors and pathways dependent on hypoxia-inducible factor (HIF) [5153]. More recently, [5-13C] glutamine has been used to visualize the metabolism of liver cancer in vivo and in vitro, as well as the treatment response of prostate cancer cells in vitro [54]. Based on the promise of glutamine as a biomarker for cancer diagnosis and treatment response, extending the spin-lattice relaxation time of the [5-13C] glutamine has been researched and successfully accomplished. The facile synthesis of [5-13C-4-2H2] glutamine has been reported, and its study showed that by relying on the effect of deuterium enrichment to lessen dipolar coupling effects, the T1 of [5-13C] glutamine could be increased from approximately 15 to 30 s [55]. Visualization of real-time conversion of glutamine to glutamate in SF188 cells was achieved using this probe, demonstrating the promise of [5-13C-4-2H2] glutamine as a probe for molecular imaging of metabolic events in real time. Further investigation of this probe applied to in vivo preclinical models will lay the foundation for its clinical translational potential in the future.
Metabolism of [5-13C] glutamine to [5-13C] glutamate. a Time-dependent spectral data following conversion of [5-13C] glutamine to [5-13C] glutamate. The signals are from 13C-enriched [5-13C]glutamate at 181.5 ppm and [5-13C]glutamine at 178.5 ppm and from natural abundance 13C label in [1-13C]glutamate at 175.2 ppm and [1-13C]glutamine at 174.7 ppm. b Plot of the ratio of the signal intensities of [5-13C]glutamate/[5-13C]glutamine showing the ratio in hepatoma cells (shaded circle), cell lysate (square), and control (triangle). These results demonstrated that hepatoma cancer cells convert glutamine to glutamine at a higher rate than normal cells. Adapted with permission from ref. [49]

Dehydroascorbate as a redox sensor

Reactive oxygen species (ROS) like the hydroxyl radical, superoxide, and hydrogen peroxide have been shown to cause DNA damage and can lead to mutations that transform normal cells into cancerous cells [56]. The reduction/oxidation (redox) state, which is dependent on the balance between oxidizing equivalents like ROS and reducing cofactors, can provide insight into the physiological condition of the cell with respect to potential cancer transformations. Furthermore, the presence of ROS in tissue has been implicated to be a factor in developing resistance to radiation therapies [57]. During oxidative stress (i.e., when there is an increase in ROS), redox homeostasis is maintained by the action of antioxidant compounds, such as ascorbate (or vitamin C, VitC), which can scavenge for ROS and reduce the compounds to rid the cells of damaging agents [58]. In this process, ascorbate that is available to cells in high concentrations can be oxidized to dehydroascorbate (DHA) while reducing ROS. DHA can then be transported into the cell where DHA is reduced back to ascorbate resulting in a process of recycling ascorbate and DHA (Fig. 3) [59]. In this sense, the ratio of DHA to ascorbate can be used as a molecular marker to investigate the redox state and thus the physiological state of tissues. Additionally, conversion of DHA to ascorbate can be enzymatically catalyzed in an NADPH-dependent manner or via oxidation of glutathione (GSH) to glutathione sulfide (GSSG); thus, visualization of ascorbate/DHA metabolism offers a method for probing in vivo metabolism of NADPH as well as determination of GSSG to GSH ratio, both of which have been implicated to be indicators of oxidative stress in the cells, particularly for neurodegenerative, cardiovascular, and cancer diseases [6062]. Hyperpolarized [1-13C] DHA was successfully used in murine models to detect increased reducing capacity in prostate cancer with the purpose of developing a non-invasive, early diagnostic tool for improving selection of treatment therapies [62, 63]. DHA demonstrates a relatively long T1 at clinically relevant field strengths (>50 s at 3 T) and adequate chemical shift separation between it and its metabolic product ascorbate (δ = 3.8 ppm). Increased reduction of HP [1-13C] DHA to ascorbate was observed in tumor tissue compared to normal tissue as well as other metabolic organs (Fig. 3). This was additionally demonstrated in lymphoma cells, further supporting the potential for using DHA as a probe in living systems [64]. A following study validated these results, and the correlation between increased DHA reduction and glutathione was established in vivo, thus showing the utility of [1-13C] DHA as a molecular imaging probe to detect events that go beyond the direct metabolism of DHA [63]. Notwithstanding the potential of HP DHA as a diagnostic probe, the toxicity of DHA remains to be validated. Earlier studies on mammalian cells showed DHA toxicity starting at 10 mM, while a study carried on rats demonstrated neurological effects of DHA starting at injections of 50 mg/kg [65, 66]. However, as outlined above, successful use of DHA injections in rats and mice for hyperpolarization has been demonstrated without reported side effects on the animals. More research is needed to determine the parameters regarding the toxicity of DHA in larger animal models using pure formulations to assess its potential for clinical trials. Further work in DHA could demonstrate its applicability for the study of ROS and redox changes in model systems.
Determination of redox state by imaging of HP [1-13C] ascorbate (VitC) and [1-13C] dehydroascorbate (DHA). Oxidative stress caused by ROS (1.) can be alleviated by oxidation of ascorbate to DHA (2.), and recycling of DHA to ascorbate can occur indirectly with oxidation of glutathione (3.) or directly with oxidation of NADH (4.). The ratio of [ascorbate] to [DHA] has been successfully used in mice models as a biomarker to determine pH in vivo. Adapted with permission from ref. [62]

Other metabolic imaging probes

While the three probes discussed earlier are the most well studied in metabolic events that are characteristic of cancer cells in general, other molecules have been evaluated in their potential to be used as biomarkers. Hyperpolarized bicarbonate (H13CO3) has been successfully used to determine the pH in extracellular matrix of lymphoma tumors in mice, and a correlation between acidic environments and cancer was established [67]. The relaxation times for bicarbonate compounds at 3 T are between 34 and 50 s, which is enough time to detect the rapid conversion of H13CO3 and 13CO2 catalyzed by carbonic anhydrase [23]. The attractive feature of this probe is based on how ubiquitous acidic extracellular environments are to a wide variety of diseases; thus, HP bicarbonate has the potential for clinical translation beyond cancer research, though extensive work will be necessary to generate a preparation which will result in an adequate dose for the clinic [68, 69]. More recently, the potential of α-ketoisocaproate (KIC) as a molecular probe for in vivo detection of branched chain amino acid transaminase (BCAT) has been explored. BCAT catalyzes the conversion of KIC to leucine, and its expression has been suggested to correlate to genetic characterization of certain tumors. In a pilot study, HP α-keto-[1-13C]-isocaproate was shown to have a T1 of 100 s so its metabolism can be sensitively traced for over a minute after injection [70]. In the same study, metabolism of HP [1-13C] KIC to [1-13C] leucine by BCAT was observed in murine lymphoma tumor tissue but was absent in rat mammary adenocarcinoma with a correlation between BCAT expression and [1-13C] leucine signal detection [70]. Additionally, in the same models, [1-13C] pyruvate conversion to [1-13C] lactate and [1-13C] alanine was detected in both types of tumors. These findings show the promise of using [1-13C] KIC as a discriminative probe in addition to pyruvate in order to diagnose different types of cancer [71, 72]. Furthermore, the correlation between BCAT expression and [1-13C] leucine detection was also shown in rat brain tissue, confirming the usefulness of HP [1-13C] KIC in assessing BCAT activity in vivo [73]. Choline is another compound that has been evaluated as a molecular imaging probe since elevated choline and choline-derived metabolites have been correlated by 1H-MRS imaging to cancer in the brain, breast, colon, cervix, and prostate [7476]. Despite its potential as a global marker for cancer because of the long T1 relaxation times that can be achieved with deuterium and 15N enrichment [77, 78], HP applications of 13C enriched choline are limited due to the small change in chemical shifts of choline and choline-derived metabolites as well as its potential toxicity [16, 79, 80]. It has been shown that choline toxicity occurs at doses of 53 mg/kg in mice, although a recent study successfully detected HP 13C choline in vivo without adverse effects in rats at doses of 50 mg/kg by using atropine to prevent cholinergic intoxication [81, 82] though metabolic products have been difficult to visualize in vivo. As mentioned earlier, the usefulness of glucose as a probe is limited due to the short relaxation times of all the carbons present in the molecule and although the T1’s can be increased through deuterium enrichment, the lifetime of the probe remains a hurdle for clinical applications [27, 28]. Thus, fructose (a pentose analog of glucose) has been successfully used as an alternative to probe glycolytic pathways [83] in TRAMP models where differences in HP [2-13C] fructose uptake and metabolism was visualized in tumor regions compared to surrounding normal tissues. Like choline, the limiting factor in the usefulness of [2-13C] fructose for in vivo studies is in small chemical shifts between the metabolite and its phosphorylated product. Finally, tumor necrosis can be used as a measure of treatment response, particularly early necrosis. HP [1,4-,13C] malate has been visualized in lymphoma mice models after injection of HP [1,4-13C] fumarate [84]. In normal cells, fumarate has a slow rate of transport into the mitochondria; however, in necrotic cells where the mitochondrial membrane is degraded, fumarase has access to the HP fumarate and its ubiquitous cofactor, water, thus facilitating rapid conversion to malate. Preliminary studies have shown the potential for its use in animal models though further work is required to determine the necessary density of necrotic cells for detection and the timings required for adequate visualization in patients.


The application of hyperpolarized 13C imaging has been extensively investigated in preclinical models, and the successful demonstration of HP [1-13C] pyruvate in patients with prostate cancer has validated the potential of HP MRI as a safe diagnostic and treatment assessment tool. Application of other probes beyond pyruvate is still in its infancy, particularly because of the need to further study the currently developed models under conditions that are relevant to a clinical setting (i.e., lower magnetic fields) as well as to study the necessary parameters (probe toxicity dose limits, safety limits for rapid injection) to withstand the necessary hurdles to translation. Nevertheless, these vast research findings are promising and indicate an eventual translation to humans. Furthermore, there is a large variety of biologically relevant molecules that have the potential to be hyperpolarized (Fig. 4), and molecular imaging of metabolic events in real time using not only one single probe but a combination of relevant probes could become an invaluable tool in elucidating so far undiscovered metabolic and proteomic interactions that play a role in cancer development and treatment. This gives HP MRI the great potential to revolutionize current molecular imaging technologies.
Metabolic pathways with compounds that can be used as molecular imaging probes for HP MRI. A wide variety of metabolic pathways have already been visualized or have the potential to be visualized using hyperpolarization technology that can be applied to different pathological states of the cell including cardiovascular disease and a large variety of cancers. 1. Metabolism of C1 (red dots) in pyruvate. Theasterisks on selected compounds represent enrichment of 13C in the second pass of pyruvate in TCA cycle. 2. Metabolism of C1 (brown dots) in DHA using a pool of NADPH derived from the pentose phosphate pathway. 3. Metabolism of C1 (blue dots) and C5 (green dots) of glutamine. 4. Metabolism of C1 and C4 (purple dots) of fumarate unrelated to TCA metabolites. 5. Metabolism of extracellular bicarbonate (gray dots). MTC1 monocarboxylate transporter 1, MTC4 monocarboxylate transporter 4,System ASC amino acid transporter, GLUTs glucose transporters, DCT dicarboxylate transporter, DHARdehydroascorbate reductase, GR glutathione reductase, GSH glutathione, GSSG glutathione disulfide,LDH lactate dehydrogenase, ALT alanine transaminase, CA carbonic anhydrase, PC pyruvate carboxylase,PDH pyruvate dehydrogenase, CS citrate synthase, GLS glutaminase, GLDH glutamate dehydrogenase,IDH isocitrate dehydrogenase, OGDC oxoglutarate dehydrogenase complex, SCS succinyl CoA synthetase, SQR succinate dehydrogenase, FH fumarate hydratase, MDH malate dehydrogenase, FUMfumarase. Cofactors have been omitted for brevity


ALT:   alanine transaminase;   BCAT:  branched chain amino acid transaminase;   DHA:  dehydroascorbate;   DNP:  dynamic nuclear polarization;   EDTA:  ethylenediaminetetraacetic acid;   GSH:  glutathione;   GSSG:   glutathione sulfide;   HCC:  hepatocellular carcinoma;   HIF:  hypoxia-inducible factor;   HP:  hyperpolarized/hyperpolarization;   IDH:  isocitrate dehydrogenase;   KIC:  ketoisocaproate;   LDH:  lactate dehydrogenase;   MR: magnetic resonance;   MRI:  Magnetic resonance imaging;   MRS:  magnetic resonance spectroscopy;   NAD(H):  nicotinamide adenine dinucleotide;   NADP(H):  nicotinamide adenine dinucleotide phosphate;   PET:  positron emission tomography;   ROS:  reactive oxygen species;   SPECT:  single-photon emission computed tomography;   TRAMP:  transgenic adenocarcinoma of mouse prostate


  1. Warburg O. On the origin of cancer cells. Science. 1956;123:309–14.View ArticlePubMed
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  4. Shie P, Cardarelli R, Brandon D, Erdman W, AbdulRahim N. Meta-analysis: comparison of F-18 fluorodeoxyglucose-positron emission tomography and bone scintigraphy in the detection of bone metastases in patients with breast cancer. Clin Nucl Med. 2008;33:97–101.View ArticlePubMed
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  8. Comment A, Merritt ME. Hyperpolarized magnetic resonance as a sensitive detector of metabolic function. Biochemistry. 2014;53:7333–57.View ArticlePubMed
  9. Rider OJ, Tyler DJ. Clinical implications of cardiac hyperpolarized magnetic resonance imaging. J Cardiov Magn Reson. 2013;15:93.View Article
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sjwilliamspa commented on Is the Warburg effect an effect of deregulated space occupancy of methylome?

Is the Warburg effect an effect of deregulated space occupancy of methylome? Larry H. Bernstein and Radoslav Bozov, …

It would be an interesting figure, although I am not sure anyone has been able to measure it, is the spatial distribution of lactate and pyruvate over the tumor as a function of diffusion distance such as a heat map to see if pyruvate and lactate levels have a gradiant over a solid tumor. I am not sure it would but interesting to see where tumor cells, which undergo Warburg type metabolic phenotype actually exist, if it is a function of angiogenesis or a function of the proliferative capacity of cells in situ.

Response by LHB…

Radoslav Bozov has repeatedly referred to the real problem of space/time in the required experimental view that is intractable, as seen by Erwin Schroedinger.  It is confounded by
the restrictions imposed by research, and to an extent also the dilemma of location and velocity.

I think it is to an extent also inherent in the modern revelations of autophagy and apoptosis that were not part of the view in the mid 20th century.  However, the work of B. Chance led to a substantially better understanding of the hydride transfer in the NAD/NADH.  What is overlooked is the important role cited by NO Kaplan of NADPH/NADP vs NADH/NAD associated with synthetic and, alternatively, catabolic processes in the cell. What role the pyridine nucleotide transhydrogenase would play is anyones guess.   In any case the proliferation of malignant cells is dependent on NADPH.  This would limit the NAD/NADH related reactions. The effect in the cytoplasm is PYR –> LAC, with generation of NAD from NADH.  In addition, the type of isoenzyme favored should be consequential.  For instance, the M-type LDH does not form an abortive ternary complex LDH*NAD+*PYR. In addition, Bernstein, Everse and Grisham showed that in cancer there is an aberrant cytoplasmic MDH.

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P13K delta-gamma anticancer agent

Larry H. Bernstein, MD, FCAP, Curator



RP 6350, Rhizen Pharmaceuticals S.A. and Novartis tieup for Rhizen’s inhaled dual Pl3K-delta gamma inhibitor



(A)           and                         (Al)                  and                (A2)

(S)-2-(l-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one (Compound A1 is RP 6350).




RP 6350, RP6350, RP-6350


mw 415

Rhizen Pharmaceuticals is developing RP-6530, a PI3K delta and gamma dual inhibitor, for the potential oral treatment of cancer and inflammation  In November 2013, a phase I trial in patients with hematologic malignancies was initiated in Italy ]\. In September 2015, a phase I/Ib study was initiated in the US, in patients with relapsed and refractory T-cell lymphoma. At that time, the study was expected to complete in December 2016

PATENTS……..WO 11/055215 ,  WO 12/151525.

  • Antineoplastics; Small molecules
  • Mechanism of Action Phosphatidylinositol 3 kinase delta inhibitors; Phosphatidylinositol 3 kinase gamma inhibitors
  • Phase I Haematological malignancies
  • Preclinical Multiple myeloma


Swaroop K. V. S. Vakkalanka,
COMPANY Rhizen Pharmaceuticals Sa



PI3K delta/gamma inhibitor RP6530 An orally active, highly selective, small molecule inhibitor of the delta and gamma isoforms of phosphoinositide-3 kinase (PI3K) with potential immunomodulating and antineoplastic activities. Upon administration, PI3K delta/gamma inhibitor RP6530 inhibits the PI3K delta and gamma isoforms and prevents the activation of the PI3K/AKT-mediated signaling pathway. This may lead to a reduction in cellular proliferation in PI3K delta/gamma-expressing tumor cells. In addition, this agent modulates inflammatory responses through various mechanisms, including the inhibition of both the release of reactive oxygen species (ROS) from neutrophils and tumor necrosis factor (TNF)-alpha activity. Unlike other isoforms of PI3K, the delta and gamma isoforms are overexpressed primarily in hematologic malignancies and in inflammatory and autoimmune diseases. By selectively targeting these isoforms, PI3K signaling in normal, non-neoplastic cells is minimally impacted or not affected at all, which minimizes the side effect profile for this agent. Check for active clinical trials using this agent. (NCI Thesaurus)

Company Rhizen Pharmaceuticals S.A.
Description Dual phosphoinositide 3-kinase (PI3K) delta and gamma inhibitor
Molecular Target Phosphoinositide 3-kinase (PI3K) delta ; Phosphoinositide 3-kinase (PI3K) gamma
Mechanism of Action Phosphoinositide 3-kinase (PI3K) delta inhibitor; Phosphoinositide 3-kinase (PI3K) gamma inhibitor
Therapeutic Modality Small molecule


Dual PI3Kδ/γ Inhibition By RP6530 Induces Apoptosis and Cytotoxicity In B-Lymphoma Cells
 Swaroop Vakkalanka, PhD*,1, Srikant Viswanadha, Ph.D.*,2, Eugenio Gaudio, PhD*,3, Emanuele Zucca, MD4, Francesco Bertoni, MD5, Elena Bernasconi, B.Sc.*,3, Davide Rossi, MD, Ph.D.*,6, and Anastasios Stathis, MD*,7
 1Rhizen Pharmaceuticals S A, La Chaux-de-Fonds, Switzerland, 2Incozen Therapeutics Pvt. Ltd., Hyderabad, India, 3Lymphoma & Genomics Research Program, IOR-Institute of Oncology Research, Bellinzona, Switzerland, 4IOSI Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, 5Lymphoma Unit, IOSI-Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, 6Italian Multiple Myeloma Network, GIMEMA, Italy, 7Oncology Institute of Southern Switzerland, Bellinzona, Switzerland

RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.

Blood 2013 122:4411; published ahead of print December 6, 2013
Swaroop Vakkalanka, Srikant Viswanadha, Eugenio Gaudio, Emanuele Zucca, Francesco Bertoni, Elena Bernasconi, Davide Rossi, Anastasios Stathis
  • Dual PI3K delta/gamma Inhibition By RP6530 Induces Apoptosis and Cytotoxicity
  • RP6530, a novel, small molecule PI3K delta/gamma
  • Activity and selectivity of RP6530 for PI3K delta and gamma isoforms

Introduction Activation of the PI3K pathway triggers multiple events including cell growth, cell cycle entry, cell survival and motility. While α and β isoforms are ubiquitous in their distribution, expression of δ and γ is restricted to cells of the hematopoietic system. Because these isoforms contribute to the development, maintenance, transformation, and proliferation of immune cells, dual targeting of PI3Kδ and γ represents a promising approach in the treatment of lymphomas. The objective of the experiments was to explore the therapeutic potential of RP6530, a novel, small molecule PI3Kδ/γ inhibitor, in B-cell lymphomas.

Methods Activity and selectivity of RP6530 for PI3Kδ and γ isoforms and subsequent downstream activity was determined in enzyme and cell-based assays. Additionally, RP6530 was tested for potency in viability, apoptosis, and Akt phosphorylation assays using a range of immortalized B-cell lymphoma cell lines (Raji, TOLEDO, KG-1, JEKO, OCI-LY-1, OCI-LY-10, MAVER, and REC-1). Viability was assessed using the colorimetric MTT reagent after incubation of cells for 72 h. Inhibition of pAKT was estimated by Western Blotting and bands were quantified using ImageJ after normalization with Actin. Primary cells from lymphoid tumors [1 chronic lymphocytic leukemia (CLL), 2 diffuse large B-cell lymphomas (DLBCL), 2 mantle cell lymphoma (MCL), 1 splenic marginal zone lymphoma (SMZL), and 1 extranodal MZL (EMZL)] were isolated, incubated with 4 µM RP6530, and analyzed for apoptosis or cytotoxicity by Annexin V/PI staining.

Results RP6530 demonstrated high potency against PI3Kδ (IC50=24.5 nM) and γ (IC50=33.2 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. Cellular potency was confirmed in target-specific assays, namely anti-FcεR1-(EC50=37.8 nM) or fMLP (EC50=39.0 nM) induced CD63 expression in human whole blood basophils, LPS induced CD19+ cell proliferation in human whole blood (EC50=250 nM), and LPS induced CD45R+ cell proliferation in mouse whole blood (EC50=101 nM). RP6530 caused a dose-dependent inhibition (>50% @ 2-7 μM) in growth of immortalized (Raji, TOLEDO, KG-1, JEKO, REC-1) B-cell lymphoma cells. Effect was more pronounced in the DLBCL cell lines, OCI-LY-1 and OCI-LY-10 (>50% inhibition @ 0.1-0.7 μM), and the reduction in viability was accompanied by corresponding inhibition of pAKT with EC50 of 6 & 70 nM respectively. Treatment of patient-derived primary cells with 4 µM RP6530 caused an increase in cell death. Fold-increase in cytotoxicity as evident from PI+ staining was 1.6 for CLL, 1.1 for DLBCL, 1.2 for MCL, 2.2 for SMZL, and 2.3 for EMZL. Cells in early apotosis (Annexin V+/PI-) were not different between the DMSO blank and RP6530 samples.

Conclusions RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.

Disclosures:Vakkalanka:Rhizen Pharmaceuticals, S.A.: Employment, Equity Ownership; Incozen Therapeutics Pvt. Ltd.: Employment, Equity Ownership.Viswanadha:Incozen Therapeutics Pvt. Ltd.: Employment. Bertoni:Rhizen Pharmaceuticals SA: Research Funding.


PI3K Dual Inhibitor (RP-6530)

Therapeutic Area Respiratory , Oncology – Liquid Tumors , Rheumatology Molecule Type Small Molecule
Indication Peripheral T-cell lymphoma (PTCL) , Non-Hodgkins Lymphoma , Asthma , Chronic Obstructive Pulmonary Disease (COPD) , Rheumatoid Arthritis
Development Phase Phase I Rt. of Administration Oral


Rhizen is developing dual PI3K gamma/delta inhibitors for liquid tumors and inflammatory conditions.

Situation Overview

Dual Pl3K inhibition is strongly implicated as an intervention treatment in allergic and non-allergic inflammation of the airways and autoimmune diseases manifested by a reduction in neutrophilia and TNF in response to LPS. Scientific evidence for PI3-kinase involvement in various cellular processes underlying asthma and COPD stems from inhibitor studies and gene-targeting approaches, which makes it a potential target for treatment of respiratory disease. Resistance to conventional therapies such as corticosteroids in several patients has been attributed to an up-regulation of the PI3K pathway; thus, disruption of PI3K signaling provides a novel strategy aimed at counteracting the immuno-inflammatory response. Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the ? and ? isoforms would be expected to attenuate the progression of immune response encountered in most variations of airway inflammation and arthritis.

Mechanism of Action

While alpha and beta isoforms are ubiquitous in their distribution, expression of delta and gamma is restricted to circulating hematogenous cells and endothelial cells. Unlike PI3K-alpha or beta, mice lacking expression of gamma or delta do not show any adverse phenotype indicating that targeting of these specific isoforms would not result in overt toxicity. Dual delta/gamma inhibition is strongly implicated as an intervention strategy in allergic and non-allergic inflammation of the airways and other autoimmune diseases. Scientific evidence for PI3K-delta and gamma involvement in various cellular processes underlying asthma and COPD stems from inhibitor studies and gene-targeting approaches. Also, resistance to conventional therapies such as corticosteroids in several COPD patients has been attributed to an up-regulation of the PI3K delta/gamma pathway. Disruption of PI3K-delta/gamma signalling therefore provides a novel strategy aimed at counteracting the immuno-inflammatory response. Due to the pivotal role played by PI3K-delta and gamma in mediating inflammatory cell functionality such as leukocyte migration and activation, and mast cell degranulation, blocking these isoforms may also be an effective strategy for the treatment of rheumatoid arthritis as well.

Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the delta and gamma isoforms would be expected to attenuate the progression of immune response encountered in airway inflammation and rheumatoid arthritis.




Clinical Trials

Rhizen has identified an orally active Lead Molecule, RP-6530, that has an excellent pre-clinical profile. RP-6530 is currently in non-GLP Tox studies and is expected to enter Clinical Development in H2 2013.

In December 2013, Rhizen announced the start of a Phase I clinical trial. The study entitled A Phase-I, Dose Escalation Study to Evaluate Safety and Efficacy of RP6530, a dual PI3K delta /gamma inhibitor, in patients with Relapsed or Refractory Hematologic Malignancies is designed primarily to establish the safety and tolerability of RP6530. Secondary objectives include clinical efficacy assessment and biomarker response to allow dose determination and potential patient stratification in subsequent expansion studies.


Partners by Region

Rhizen’s pipeline consists of internally discovered (with 100% IP ownership) novel small molecule programs aimed at high value markets of Oncology, Immuno-inflammtion and Metabolic Disorders. Rhizen has been successful in securing critical IP space in these areas and efforts are on for further expansion in to several indications. Rhizen seeks partnerships to unlock the potential of these valuable assets for further development from global pharmaceutical partners. At present global rights on all programs are available and Rhizen is flexible to consider suitable business models for licensing/collaboration.

In 2012, Rhizen announced a joint venture collaboration with TG Therapeutics for global development and commercialization of Rhizen’s Novel Selective PI3K Kinase Inhibitors. The selected lead RP5264 (hereafter, to be developed as TGR-1202) is an orally available, small molecule, PI3K specific inhibitor currently being positioned for the treatment of hematological malignancies.


This scheme provides a synthetic route for the preparation of compound of formula wherein all the variables are as described herein in above

Figure imgf000094_0001

15 14 10 12 12a

April 2015, preclinical data were presented at the 106th AACR Meeting in Philadelphia, PA. RP-6530 had GI50 values of 17,028 and 22,014 nM, respectively
December 2014, data were presented at the 56th ASH Meeting in San Francisco, CA.
December 2013, preclinical data were presented at the 55th ASH Meeting in New Orleans, LA.
June 2013, preclinical data were presented at the 18th Annual EHA Congress in Stockholm, Sweden. RP-6530 inhibited PI3K delta and gamma isoforms with IC50 values of 24.5 and 33.2 nM, respectively.
  • 01 Sep 2015 Phase-I clinical trials in Hematological malignancies (Second-line therapy or greater) in USA (PO) (NCT02567656)
  • 18 Nov 2014 Preclinical trials in Multiple myeloma in Switzerland (PO) prior to November 2014
  • 18 Nov 2014 Early research in Multiple myeloma in Switzerland (PO) prior to November 2014


WO2011055215A2 Nov 3, 2010 May 12, 2011 Incozen Therapeutics Pvt. Ltd. Novel kinase modulators
WO2012151525A1 May 4, 2012 Nov 8, 2012 Rhizen Pharmaceuticals Sa Novel compounds as modulators of protein kinases
WO2013164801A1 May 3, 2013 Nov 7, 2013 Rhizen Pharmaceuticals Sa Process for preparation of optically pure and optionally substituted 2- (1 -hydroxy- alkyl) – chromen – 4 – one derivatives and their use in preparing pharmaceuticals
US20110118257 May 19, 2011 Rhizen Pharmaceuticals Sa Novel kinase modulators
US20120289496 May 4, 2012 Nov 15, 2012 Rhizen Pharmaceuticals Sa Novel compounds as modulators of protein kinases
WO 2011055215



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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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New Insights on the Warburg Effect [2.2]

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



New Insights on the Warburg Effect [2.2]

Defective Mitochondria Transform Normal Cells into Tumors

GEN News Jul 9, 2015

Ninety-one years ago Otto Warburg demonstrated that cancer cells have impaired respiration, which became known as the Warburg Effect. The interest in this and related work was superceded in the last quarter of the twentieth century by work on the genetic code. Now there is renewed interest.

An international research team reports that a specific defect in mitochondria plays a key role in the transition from normal cells to cancerous ones. The scientists disrupted a key component of mitochondria of otherwise normal cells and the cells took on characteristics of malignant cells.

Their study (“Disruption of cytochrome c oxidase function induces the Warburg effect and metabolic reprogramming”) is published Oncogene and was led by members of the lab of Narayan G. Avadhani, Ph.D., the Harriet Ellison Woodward Professor of Biochemistry in the department of biomedical sciences in the school of veterinary medicine at the University of Pennsylvania. Satish Srinivasan, Ph.D., a research investigator in Dr. Avadhani’s lab, was the lead author.

This is consistent with the 1924 observation by Warburg that cancerous cells consumed glucose at a higher rate than normal cells (Meyerhof ratio) and had defects in their grana, the organelles that are now known as mitochondria. He postulated that these defects led to problems in the process by which the cell produces energy. But the process called oxidative phosphorylation was not yet known. Further work in his laboratory was carried out by Hans Krebs and by Albert Szent Gyorgyi elucidating the tricarboxylic acid cycle.  The discovery of the importance of cytochrome c and adenosine triphosphate in oxidative phosphorylation was made in the post World War II period by Fritz Lippman, with an important contribution by Nathan Kaplan. All of the name scientists, except Kaplan, received Nobel Prizes. The last piece of the puzzle became the demonstation of a sequence of hydrogen transfers on the electron transport chain. The researchers above have now shown that mitochondrial defects indeed contributed to the cells becoming cancerous.

“The first part of the Warburg hypothesis has held up solidly in that most proliferating tumors show high dependence on glucose as an energy source and they release large amounts of lactic acid,” said Dr. Avadhani. “But the second part, about the defective mitochondrial function causing cells to be tumorigenic, has been highly contentious.”

To see whether the second part of Warburg’s postulation was correct, the researchers took cell lines from the skeleton, kidney, breast, and esophagus and used RNA molecules to silence the expression of select components of mitochondrial cytochrome oxidase C, or CcO, a critical enzyme involved in oxidative phosphorylation. CcO uses oxygen to make water and set up a transmembrane potential that is used to synthesize ATP, the molecule used for energy by the body’s cells.

The biologists observed that disrupting only a single protein subunit of cytochrome oxidase C led to major changes in the mitochondria and in the cells themselves. “These cells showed all the characteristics of cancer cells,” noted Dr. Avadhani.

The normal cells that converted to cancerous cells displayed changes in their metabolism, becoming more reliant on glucose by utilization of the glycolytic pathway. They reduced their synthesis of ATP.  Oxidative phosphorylation was reduced in concert with the ATP reduction. The large switch to glycolysis as primary energy source is a less efficient means of making ATP that is common in cancer cells.

The cells lost contact inhibition and gained an increased ability to invade distant tissues, both hallmarks of cancer cells. When they were grown in a 3D medium, which closely mimics the natural environment in which tumors grow in the body, the cells with disrupted mitochondria formed large, long-lived colonies, akin to tumors.

The researchers also silenced cytochrome oxidase C subunits in breast and esophageal cancer cell lines. They found that the cells became even more invasive, according to Dr. Srinivasan. The team then looked at actual tumors from human patients and found that the most oxygen-starved regions, which are common in tumors, contained defective versions of CcO.

“That result alone couldn’t tell us whether that was the cause or effect of tumors, but our cell system clearly says that mitochondrial dysfunction is a driving force in tumorigenesis,” explained Dr. Avadhani.

The researchers observed that disrupting CcO triggered the mitochondria to activate a stress signal to the nucleus, akin to an SOS alerting the cell that something was wrong. Dr. Avadhani and his colleagues had previously seen a similar pathway activated in cells with depleted mitochondrial DNA, which is also linked to cancer.

Building on these findings, Dr. Avadhani and members of his lab will examine whether inhibiting components of this mitochondrial stress signaling pathway might be a strategy for preventing cancer progression.

“We are targeting the signaling pathway, developing a lot of small molecules and antibodies,” said Dr. Avadhani. “Hopefully if you block the signaling the cells will not go into the so called oncogenic mode and instead would simply die.”

In addition, they noted that looking for defects in CcO could be a biomarker for cancer screening.


Who controls the ATP supply in cancer cells? Biochemistry lessons to understand cancer energy metabolism

Rafael Moreno-Sánchez, Alvaro Marín-Hernández, Emma Saavedra, Juan P. Pardo, Stephen J. Ralph, Sara Rodríguez-Enríquez
Intl J Biochem Cell Biol 7 Feb 2014; 50:10-23

The supply of ATP in mammalian and human cells is provided by glycolysis and oxidative phosphorylation (OxPhos). There are no other pathways or processes able to synthesize ATP at sufficient rates to meet the energy demands of cells. Acetate thiokinase or acetyl-CoA synthetase, a ubiquitous enzyme catalyzing the synthesis of ATP and acetate from acetyl-CoA, PPi and AMP, might represent an exception under hypoxia in cancer cells, although the flux through this branch is negligible (≤10%) when compared to the glycolytic flux (Yoshii et al., 2009).

Glycolysis in human cells can be defined as the metabolic process that transforms 1 mol of glucose (or other hexoses) into 2 moles of lactate plus 2 moles of ATP. These stoichiometric values represent a maximum and due to the several reactions branching off glycolysis, they will be usually lower under physiological conditions, closer to 1.3–1.9 for the lactate/glucose ratio (Travis et al., 1971; Jablonska and Bishop, 1975; Suter and Weidemann, 1975; Hanson and Parsons, 1976; Wu and Davis, 1981; Pick-Kober and Schneider, 1984; Sun et al., 2012). OxPhos is the metabolic process that oxidizes several substrates through the Krebs cycle to produce reducing equivalents (NADH, FADH2), which feed the respiratory chain to generate an H+.

Applying basic biochemical principles, this review analyzes data that contrasts with the Warburg hypothesis that glycolysis is the exclusive ATP provider in cancer cells. Although disregarded for many years, there is increasing experimental evidence demonstrating that oxidative phosphorylation (OxPhos) makes a significant contribution to ATP supply in many cancer cell types and under a variety of conditions.

Substrates oxidized by normal mitochondria such as amino acids and fatty acids are also avidly consumed by cancer cells. In this regard, the proposal that cancer cells metabolize glutamine for anabolic purposes without the need for a functional respiratory chain and OxPhos is analyzed considering thermodynamic and kinetic aspects for the reductive carboxylation of 2-oxoglutarate catalyzed by isocitrate dehydrogenase.

In addition, metabolic control analysis (MCA) studies applied to energy metabolism of cancer cells are reevaluated. Regardless of the experimental/environmental conditions and the rate of lactate production, the flux-control of cancer glycolysis is robust in the sense that it involves the same steps:

  • glucose transport,
  • hexokinase,
  • hexosephosphate isomerase, and
  • glycogen degradation,

all at the beginning of the pathway; these steps together with phosphofructokinase 1 also control glycolysis in normal cells.

The respiratory chain complexes exert significantly higher flux-control on OxPhos in cancer cells than in normal cells. Thus, determination of the contribution of each pathway to ATP supply and/or the flux-control distribution of both pathways in cancer cells is necessary in order to identify differences from normal cells which may lead to the design of rational alternative therapies that selectively target cancer energy metabolism.

Fig. 1. Labeling patterns of 13C-glutamate or 13C-glutamine mitochondrial metabolism in cancer cells.

Fig. 2. Survey in PubMed of papers published in the field of tumor mitochondrial metabolism from 1951 to September 2013.


Emerging concepts in bioenergetics and cancer research: Metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bioenergetic therapy

Emilie Obre, Rodrigue Rossignol
Intl J Biochem Cell Biol 2015; 59:167-181

The field of energy metabolism dramatically progressed in the last decade, owing to a large number of cancer studies, as well as fundamental investigations on related transcriptional networks and cellular interactions with the microenvironment. The concept of metabolic flexibility was clarified in studies showing the ability of cancer cells to remodel the biochemical pathways of energy transduction and linked anabolism in response to glucose, glutamine or oxygen deprivation.

A clearer understanding of the large scale bioenergetic impact of C-MYC, MYCN, KRAS and P53 was obtained, along with its modification during the course of tumor development. The metabolic dialog between different types of cancer cells, but also with the stroma, also complexified the understanding of bioenergetics and raised the concepts of metabolic symbiosis and reverse Warburg effect.

Signaling studies revealed the role of respiratory chain derived reactive oxygen species for metabolic remodeling and metastasis development. The discovery of oxidative tumors in human and mice models related to chemoresistance also changed the prevalent view of dysfunctional mitochondria in cancer cells. Likewise, the influence of energy metabolism-derived oncometabolites emerged as a new means of tumor genetic regulation. The knowledge obtained on the multi-site regulation of energy metabolism in tumors was translated to cancer preclinical studies, supported by genetic proof of concept studies targeting LDHA, HK2, PGAM1, or ACLY.

Here, we review those different facets of metabolic remodeling in cancer, from its diversity in physiology and pathology, to the search of the genetic determinants, the microenvironmental regulators and pharmacological modulators.


Pyruvate kinase M2: A key enzyme of the tumor metabolome and its medical relevance

Mazurek, S.
Biomedical Research 2012; 23(SPEC. ISSUE): Pages 133-142

Tumor cells are characterized by an over expression of the glycolytic pyruvate kinase isoenzyme
type M2 (abbreviations: M2-PK or PKM2). In tumor metabolism the quaternary structure of M2-PK (tetramer/dimer ratio) determines whether glucose is used for glycolytic energy regeneration (highly active tetrameric form, Warburg effect) or synthesis of cell building blocks (nearly inactive dimeric form) which are both prerequisites for cells with a high proliferation rate. In tumor cells the nearly inactive dimeric form of M2- PK is predominant due to direct interactions with different oncoproteins. Besides its key functions in tumor metabolism recent studies revealed that M2-PK may also react as protein kinase as well as co activator of transcription factors. Of medical relevance is the quantification of the dimeric form of M2-PK with either an ELISA or point of care rapid test in plasma and stool that is used for follow-up studies during therapy (plasma M2-PK) and colorectal cancer (CRC) screening (fecal M2-PK; mean sensitivity for CRC in 12 independent studies with altogether 704 samples: 80% ± 7%). An intervention in the regulation mechanisms of the expression, activity and tetramer: dimer ratio of M2-PK has significant consequences for the proliferation rate and tumorigenic capacity of the tumor cells, making this enzyme an intensively

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Therapeutic Implications for Targeted Therapy from the Resurgence of Warburg ‘Hypothesis’

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

(Note that each portion of the discussion is followed by a reference)

It is now a time to pause after almost a century of a biological scientific discoveries that have transformed the practice of medicine and impacted the lives of several generations of young minds determined to probe the limits of our knowledge.  In the century that we have entered into the scientific framework of medicine has brought together a difficult to grasp evolution of the emergence of human existence from wars, famine, droughts, storms, infectious diseases, and insect born pestilence with betterment of human lives, only unevenly divided among societal classes that have existed since time immemorial. In this short time span there have emerged several generations of physicians who have benefited from a far better medical education that their forebears could have known. In this expansive volume on cancer, we follow an incomplete and continuing challenge to understand cancer, a disease that has become associated with longer life spans in developed nations.

While there are significant improvements in the diagnosis and treatment of cancers, there is still a personal as well as locality factor in the occurrence of this group of diseases, which has been viewed incorrectly as a “dedifferentiation” of mature tissue types and the emergence of a cell phenotype that is dependent on glucose, reverts to a cancer “stem cell type” (loss of stemness), loses cell to cell adhesion, loses orderly maturation, and metastasizes to distant sites. At the same time, physician and nurses are stressed in the care of patients by balancing their daily lives and maintaining a perspective.

The conceptual challenge of cancer diagnosis and management has seemed insurmountable, but owes much to the post World War I activities of Otto Heinrich Warburg. It was Warburg who made the observation that cancer cells metabolize glucose by fermentation in much the way Pasteur 60 years earlier observed fermentation of yeast cells. This metabolic phenomenon occurs even in the presence of an oxygen supply, which would provide a huge deficit in ATP production compared with respiration. The cancer cell is “addicted to glucose” and produced lactic acid. Warburg was awarded the Nobel Prize in Medicine for this work in 1931.

In the last 15 years there has been a resurgence of work on the Warburg effect that sheds much new light on the process that was not previously possible, with significant therapeutic implications.  In the first place, the metabolic mechanism for the Warburg effect was incomplete even at the beginning of the 21st century.  This has been partly rectified with the enlightening elucidation of genome modifications, cellular metabolic regulation, and signaling pathways.

The following developments have become central to furthering our understanding of malignant transformation.

  1. There is usually an identifiable risk factor, such as, H. pylori, or of a chronic inflammatory state, as in the case of Barrett’s esophagus.
  2. There are certain changes in glucose metabolism that have been unquestionably been found in the evolution of this disease. The changes are associated with major changes in metabolic pathways, miRN signaling, and the metabolism geared to synthesis of cells with an impairment of the cell death cycle. In these changes, mitochondrial function is central to both the impaired respiration and the autophagy geared to the synthesis of cancer cells.

The emergence of this cell prototype is characterized by the following, again related to the Warburg effect:

  1. Cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis
  2. The mitochondrial pyruvate carrier (MPC), composed of the products of the MPC1 and MPC2 genes, modulates fractional pyruvate oxidation. MPC1 is deleted or underexpressed in multiple cancers and correlates with poor prognosis.
  3. Cancer cells tend to express a partially inhibited splice variant of pyruvate kinase (PK-M2), leading to decreased pyruvate production.
  4. The two proteins that mediate pyruvate conversion to lactate and its export, M-type lactate dehydrogenase and the monocarboxylate transporter MCT-4, are commonly upregulated in cancer cells leading to decreased pyruvate oxidation.
  5. The enzymatic step following mitochondrial entry is the conversion of pyruvate to acetyl-CoA by the pyruvate dehydrogenase (PDH) complex. Cancer cells frequently exhibit increased expression of the PDH kinase PDK1, which phosphorylates and inactivates PDH. This PDH regulatory mechanism is required for oncogene induced transformation and reversed in oncogene-induced senescence.
  6. The PDK inhibitor dichloroacetate has shown some clinical efficacy, which correlates with increased pyruvate oxidation. One of the simplest mechanisms to explain decreased mitochondrial pyruvate oxidation in cancer cells, a loss of mitochondrial pyruvate import, has been observed repeatedly over the past 40 years. This process has been impossible to study at a molecular level until recently, however, as the identities of the protein(s) that mediate mitochondrial pyruvate uptake were unknown.
  7. The mitochondrial pyruvate carrier (MPC) as a multimeric complex that is necessary for efficient mitochondrial pyruvate uptake. The MPC contains two distinct proteins, MPC1 and MPC2; the absence of either leads to a loss of mitochondrial pyruvate uptake and utilization in yeast, flies, and mammalian cells.

A Role for the Mitochondrial Pyruvate Carrier as a Repressor of the Warburg Effect and Colon Cancer Cell Growth

John C. Schell, Kristofor A. Olson, Lei Jiang, Amy J. Hawkins, et al.
Molecular Cell Nov 6, 2014; 56: 400–413.

In addition to the above, the following study has therapeutic importance:

Glycolysis has become a target of anticancer strategies. Glucose deprivation is sufficient to induce growth inhibition and cell death in cancer cells. The increased glucose transport in cancer cells has been attributed primarily to the upregulation of glucose transporter 1 (Glut1),  1 of the more than 10 glucose transporters that are responsible for basal glucose transport in almost all cell types. Glut1 has not been targeted until very recently due to the lack of potent and selective inhibitors.

First, Glut1 antibodies were shown to inhibit cancer cell growth. Other Glut1 inhibitors and glucose transport inhibitors, such as fasentin and phloretin, were also shown to be effective in reducing cancer cell growth. A group of inhibitors of glucose transporters has been recently identified with IC50 values lower than 20mmol/L for inhibiting cancer cell growth. However, no animal or detailed mechanism studies have been reported with these inhibitors.

Recently, a small molecule named STF-31 was identified that selectively targets the von Hippel-Lindau (VHL) deficient kidney cancer cells. STF-31 inhibits VHL deficient cancer cells by inhibiting Glut1. It was further shown that daily intraperitoneal injection of a soluble analogue of STF-31 effectively reduced the growth of tumors of VHL-deficient cancer cells grafted on nude mice. On the other hand, STF-31 appears to be an inhibitor with a narrow cell target spectrum.

These investigators recently reported the identification of a group of novel small compounds that inhibit basal glucose transport and reduce cancer cell growth by a glucose deprivation–like mechanism. These compounds target Glut1 and are efficacious in vivo as anticancer agents. A novel representative compound WZB117 not only inhibited cell growth in cancer cell lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal injection of WZB117 resulted in a more than 70% reduction in the size of human lung cancer of A549 cell origin. Mechanism studies showed that WZB117 inhibited glucose transport in human red blood cells (RBC), which express Glut1 as their sole glucose transporter. Cancer cell treatment with WZB117 led to decreases in levels of Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by increase in ATP sensing enzyme AMP-activated protein kinase (AMPK) and declines in cyclin E2 as well as phosphorylated retinoblastoma, resulting in cell-cycle arrest, senescence, and necrosis. Addition of extracellular ATP rescued compound-treated cancer cells, suggesting that the reduction of intracellular ATP plays an important role in the anticancer mechanism of the molecule.

A Small-Molecule Inhibitor of Glucose Transporter 1 Downregulates Glycolysis, Induces Cell-Cycle Arrest, and Inhibits Cancer Cell Growth In Vitro and In Vivo

Yi Liu, Yanyan Cao, Weihe Zhang, Stephen Bergmeier, et al.
Mol Cancer Ther Aug 2012; 11(8): 1672–82

Alterations in cellular metabolism are among the most consistent hallmarks of cancer. These investigators have studied the relationship between increased aerobic lactate production and mitochondrial physiology in tumor cells. To diminish the ability of malignant cells to metabolize pyruvate to lactate, M-type lactate dehydrogenase levels were knocked down by means of LDH-A short hairpin RNAs. Reduction in LDH-A activity resulted in stimulation of mitochondrial respiration and decrease of mitochondrial membrane potential. It also compromised the ability of these tumor cells to proliferate under hypoxia. The tumorigenicity of the LDH-A-deficient cells was severely diminished, and this phenotype was reversed by complementation with the human ortholog LDH-A protein. These results demonstrate that LDH-A plays a key role in tumor maintenance.

The results are consistent with a functional connection between alterations in glucose metabolism and mitochondrial physiology in cancer. The data also reflect that the dependency of tumor cells on glucose metabolism is a liability for these cells under limited-oxygen conditions. Interfering with LDH-A activity as a means of blocking pyruvate to lactate conversion could be exploited therapeutically. Because individuals with complete deficiency of LDH-A do not show any symptoms under ordinary circumstances, the genetic data suggest that inhibition of LDH-A activity may represent a relatively nontoxic approach to interfere with tumor growth.

Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance

Valeria R. Fantin Julie St-Pierre and Philip Leder
Cancer Cell Jun 2006; 9: 425–434.

The widespread clinical use of positron-emission tomography (PET) for the detection of aerobic glycolysis in tumors and recent findings have rekindled interest in Warburg’s theory. Studies on the physiological changes in malignant conversion provided a metabolic signature for the different stages of tumorigenesis; during tumorigenesis, an increase in glucose uptake and lactate production have been detected. The fully transformed state is most dependent on aerobic glycolysis and least dependent on the mitochondrial machinery for ATP synthesis.

Tumors ferment glucose to lactate even in the presence of oxygen (aerobic glycolysis; Warburg effect). The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis and glucose degradation to lactate. The nonoxidative part of the PPP is controlled by transketolase enzyme reactions. We have detected upregulation of a mutated transketolase transcript (TKTL1) in human malignancies, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. Strong TKTL1 protein expression was correlated to invasive colon and urothelial tumors and to poor patients outcome. TKTL1 encodes a transketolase with unusual enzymatic properties, which are likely to be caused by the internal deletion of conserved residues. We propose that TKTL1 upregulation in tumors leads to enhanced, oxygen-independent glucose usage and a lactate based matrix degradation. As inhibition of transketolase enzyme reactions suppresses tumor growth and metastasis, TKTL1 could be the relevant target for novel anti-transketolase cancer therapies. We suggest an individualized cancer therapy based on the determination of metabolic changes in tumors that might enable the targeted inhibition of invasion and metastasis.

Other important links between cancer-causing genes and glucose metabolism have been already identified. Activation of the oncogenic kinase Akt has been shown to stimulate glucose uptake and metabolism in cancer cells and renders these cells susceptible to death in response to glucose withdrawal. Such tumor cells have been shown to be dependent on glucose because the ability to induce fatty acid oxidation in response to glucose deprivation is impaired by activated Akt. In addition, AMP-activated protein kinase (AMPK) has been identified as a link between glucose metabolism and the cell cycle, thereby implicating p53 as an essential component of metabolic cell-cycle control.

Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted

S Langbein, M Zerilli, A zur Hausen, W Staiger, et al.
British Journal of Cancer (2006) 94, 578–585.

The unique metabolic profile of cancer (aerobic glycolysis) might confer apoptosis resistance and be therapeutically targeted. Compared to normal cells, several human cancers have high mitochondrial membrane potential (DJm) and low expression of the K+ channel Kv1.5, both contributing toapoptosis resistance. Dichloroacetate (DCA) inhibits mitochondrial pyruvate dehydrogenase kinase (PDK), shifts metabolism from glycolysis to glucose oxidation, decreases DJm, increases mitochondrial H2O2, and activates Kv channels in all cancer, but not normal, cells; DCA upregulates Kv1.5 by an NFAT1-dependent mechanism. DCA induces apoptosis, decreases proliferation, and inhibits tumor growth, without apparent toxicity. Molecular inhibition of PDK2 by siRNA mimics DCA. The mitochondria-NFAT-Kv axis and PDK are important therapeutic targets in cancer; the orally available DCA is a promising selective anticancer agent.

Cancer progression and its resistance to treatment depend, at least in part, on suppression of apoptosis. Although mitochondria are recognized as regulators of apoptosis, their importance as targets for cancer therapy has not been adequately explored or clinically exploited. In 1930, Warburg suggested that mitochondrial dysfunction in cancer results in a characteristic metabolic phenotype, that is, aerobic glycolysis (Warburg, 1930). Positron emission tomography (PET) imaging has now confirmed that most malignant tumors have increased glucose uptake and metabolism. This bioenergetic feature is a good marker of cancer but has not been therapeutically pursued..

The small molecule DCA is a metabolic modulator that has been used in humans for decades in the treatment of lactic acidosis and inherited mitochondrial diseases. Without affecting normal cells, DCA reverses the metabolic electrical remodeling that we describe in several cancer lines (hyperpolarized mitochondria, activated NFAT1, downregulated Kv1.5), inducing apoptosis and decreasing tumor growth. DCA in the drinking water at clinically relevant doses for up to 3 months prevents and reverses tumor growth in vivo, without apparent toxicity and without affecting hemoglobin, transaminases, or creatinine levels. The ease of delivery, selectivity, and effectiveness  make DCA an attractive candidate for proapoptotic cancer therapy which can be rapidly translated into phase II–III clinical trials.

A Mitochondria-K+ Channel Axis Is Suppressed in Cancer and Its Normalization Promotes Apoptosis and Inhibits Cancer Growth

Sebastien Bonnet, Stephen L. Archer, Joan Allalunis-Turner, et al.

Cancer Cell Jan 2007; 11: 37–51.

Tumor cells, just as other living cells, possess the potential for proliferation, differentiation, cell cycle arrest, and apoptosis. There is a specific metabolic phenotype associated with each of these conditions, characterized by the production of both energy and special substrates necessary for the cells to function in that particular state. Unlike that of normal living cells, the metabolic phenotype of tumor cells supports the proliferative state. Aim: To present the metabolic hypothesis that (1) cell transformation and tumor growth are associated with the activation of metabolic enzymes that increase glucose carbon utilization for nucleic acid synthesis, while enzymes of the lipid and amino acid synthesis pathways are activated in tumor growth inhibition, and (2) phosphorylation and allosteric and transcriptional regulation of intermediary metabolic enzymes and their substrate availability together mediate and sustain cell transformation from one condition to another. Conclusion: Evidence is presented that demonstrates opposite changes in metabolic phenotypes induced by TGF-β, a cell transforming agent, and tumor growth-inhibiting phytochemicals such as genistein and Avemar, or novel synthetic antileukemic drugs such as STI571 (Gleevec).  Intermediary metabolic enzymes that mediate the growth signaling pathways and promote malignant cell transformation may serve as high efficacy nongenetic novel targets for cancer therapies.

A Metabolic Hypothesis of Cell Growth and Death in Pancreatic Cancer

Laszlo G. Boros, Wai-Nang Paul Lee, and Vay Liang W. Go
Pancreas 2002; 24(1):26–33

Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of kidney cancer. Here, we integrated an unbiased genome-wide RNA interference screen for ccRCC survival regulators with an analysis of recurrently overexpressed genes in ccRCC to identify new therapeutic targets in this disease. One of the most potent survival regulators, the monocarboxylate transporter MCT4 (SLC16A3), impaired ccRCC viability in all eight ccRCC lines tested and was the seventh most overexpressed gene in a meta-analysis of five ccRCC expression datasets.

MCT4 silencing impaired secretion of lactate generated through glycolysis and induced cell cycle arrest and apoptosis. Silencing MCT4 resulted in intracellular acidosis, and reduction in intracellular ATP production together with partial reversion of the Warburg effect in ccRCC cell lines. Intra-tumoral heterogeneity in the intensity of MCT4 protein expression was observed in primary ccRCCs.

MCT4 protein expression analysis based on the highest intensity of expression in primary ccRCCs was associated with poorer relapse-free survival, whereas modal intensity correlated with Fuhrman nuclear grade. Consistent with the potential selection of subclones enriched for MCT4 expression during disease progression, MCT4 expression was greater at sites of metastatic disease. These data suggest that MCT4 may serve as a novel metabolic target to reverse the Warburg effect and limit disease progression in ccRCC.

Clear cell carcinoma (ccRCC) is the commonest subtype of renal cell carcinoma, accounting for 80% of cases. These tumors are highly resistant to cytotoxic chemotherapy and until recently, systemic treatment options for advanced ccRCC were limited to cytokine based therapies, such as interleukin-2 and interferon-α. Recently, anti-angiogenic drugs and mTOR inhibitors, all targeting the HIF–VEGF axis which is activated in up to 91% of ccRCCs through loss of the VHL tumor suppressor gene [1], have been shown to be effective in metastatic ccRCC [2–5]. Although these drugs increase overall survival to more than 2 years [6], resistance invariably occurs, making the identification of new molecular targets a major clinical need to improve outcomes in patients with metastatic ccRCC.

Genome-wide RNA interference analysis of renal carcinoma survival regulators identifies MCT4 as a Warburg effect metabolic target

Marco Gerlinger, Claudio R Santos, Bradley Spencer-Dene, et al.
J Pathol 2012; 227: 146–156

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 a 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 with the surrounding normal tissue. The median PO2 in breast cancers is 10 mmHg, as compared with65 mmHg in normal breast tissue. Reduced O2 availability induces HIF-1, which regulates the transcription of hundreds of genes 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.

HIF-1 is a transcription factor that consists of an O2 regulated HIF-1a and a constitutively expressed HIF-1b subunit. In well-oxygenated cells, HIF-1a is hydroxylated on proline residue 402 (Pro-402) and/or Pro-564 by prolyl hydroxylase domain protein 2 (PHD2), which uses O2 and a-ketoglutarate as substrates in a reaction that generates CO2 and succinate as byproducts. Prolylhydroxylated HIF-1a is bound by the von Hippel–Lindau tumor suppressor protein (VHL), which recruits an E3-ubiquitin ligase that targets HIF-1a 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. 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.

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.

HIF-1: upstream and downstream of cancer metabolism

Gregg L Semenza
Current Opinion in Genetics & Development 2010, 20:51–56

This review comes from a themed issue on Genetic and cellular mechanisms of oncogenesis Edited by Tony Hunter and Richard Marais


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-1a 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.

Intratumoral hypoxia The majority of locally advanced solid tumors contain regions of reduced oxygen availability. Intratumoral hypoxia results when cells are located too far from a functional blood vessel for diffusion of adequate amounts of O2 as a result of rapid cancer cell proliferation and the formation of blood vessels that are structurally and functionally abnormal. In the most extreme case, O2 concentrations are below those required for survival, resulting in cell death and establishing a selection for cancer cells in which apoptotic pathways are inactivated, anti-apoptotic pathways are activated, or invasion/metastasis pathways that promote escape from the hypoxic microenvironment are activated. This hypoxic adaptation may arise by alterations in gene expression or by mutations in the genome or both and is associated with reduced patient survival.

Hypoxia-inducible factor 1 (HIF-1) The expression of hundreds of genes is altered in each cell exposed to hypoxia. Many of these genes are regulated by HIF-1. HIF-1 is a heterodimer formed by the association of an O2-regulated HIF1a subunit with a constitutively expressed HIF-1b subunit. The structurally and functionally related HIF-2a protein also dimerizes with HIF-1b and regulates an overlapping battery of target genes. Under nonhypoxic conditions, HIF-1a (as well as HIF-2a) is subject to O2-dependent prolyl hydroxylation and this modification is required for binding of the von Hippel–Lindau tumor suppressor protein (VHL), which also binds to Elongin C and thereby recruits a ubiquitin ligase complex that targets HIF-1a for ubiquitination and proteasomal degradation. Under hypoxic conditions, the rate of hydroxylation and ubiquitination declines, resulting in accumulation of HIF-1a. Immunohistochemical analysis of tumor biopsies has revealed high levels of HIF-1a in hypoxic but viable tumor cells surrounding areas of necrosis.

Genetic alterations in cancer cells increase HIF-1 activity In the majority of clear-cell renal carcinomas, VHL function is lost, resulting in constitutive activation of HIF-1. After re-introduction of functional VHL, renal carcinoma cell lines are no longer tumorigenic, but can be made tumorigenic by expression of HIF2a in which the prolyl residues that are subject to hydroxylation have been mutated. In addition to VHL loss-of-function, many other genetic alterations that inactivate tumor suppressors

Evaluation of HIF-1 inhibitors as anticancer agents

Gregg L. Semenza
Drug Discovery Today Oct 2007; 12(19/20).

Hypoxia-inducible factor-1 (HIF-1), which is present at high levels in human tumors, plays crucial roles in tumor promotion by upregulating its target genes, which are involved in anaerobic energy metabolism, angiogenesis, cell survival, cell invasion, and drug resistance. Therefore, it is apparent that the inhibition of HIF-1 activity may be a strategy for treating cancer. Recently, many efforts to develop new HIF-1-targeting agents have been made by both academic and pharmaceutical industry laboratories. The future success of these efforts will be a new class of HIF-1-targeting anticancer agents, which would improve the prognoses of many cancer patients. This review focuses on the potential of HIF-1 as a target molecule for anticancer therapy, and on possible strategies to inhibit HIF-1 activity. In addition, we introduce YC-1 as a new anti-HIF-1, anticancer agent. Although YC-1 was originally developed as a potential therapeutic agent for thrombosis and hypertension, recent studies demonstrated that YC-1 suppressed HIF-1 activity and vascular endothelial growth factor expression in cancer cells. Moreover, it halted tumor growth in immunodeficient mice without serious toxicity during the treatment period. Thus, we propose that YC-1 is a good lead compound for the development of new anti-HIF-1, anticancer agents.

Although many anticancer regimens have been introduced to date, their survival benefits are negligible, which is the reason that a more innovative treatment is required. Basically, the identification of the specific molecular features of tumor promotion has allowed for rational drug discovery in cancer treatment, and drugs have been screened based upon the modulation of specific molecular targets in tumor cells. Target-based drugs should satisfy the following two conditions.

First, they must act by a described mechanism.

Second, they must reduce tumor growth in vivo, associated with this mechanism.

Many key factors have been found to be involved in the multiple steps of cell growth signal-transduction pathways. Targeting these factors offers a strategy for preventing tumor growth; for example, competitors or antibodies blocking ligand–receptor interaction, and receptor tyrosine kinase inhibitors, downstream pathway inhibitors (i.e., RAS farnesyl transferase inhibitors, mitogen-activated protein kinase and mTOR inhibitors), and cell-cycle arresters (i.e., cyclin-dependent kinase inhibitors) could all be used to inhibit tumor growth.

In addition to the intracellular events, tumor environmental factors should be considered to treat solid tumors. Of these, hypoxia is an important cancer-aggravating factor because it contributes to the progression of a more malignant phenotype, and to the acquisition of resistance to radiotherapy and chemotherapy. Thus, transcription factors that regulate these hypoxic events are good targets for anticancer therapy and in particular HIF-1 is one of most compelling targets. In this paper, we introduce the roles of HIF-1 in tumor promotion and provide a summary of new anticancer strategies designed to inhibit HIF-1 activity.

New anticancer strategies targeting HIF-1

Eun-Jin Yeo, Yang-Sook Chun, Jong-Wan Park
Biochemical Pharmacology 68 (2004) 1061–1069

Classical work in tumor cell metabolism focused on bioenergetics, particularly enhanced glycolysis and suppressed oxidative phosphorylation (the ‘Warburg effect’). But the biosynthetic activities required to create daughter cells are equally important for tumor growth, and recent studies are now bringing these pathways into focus. In this review, we discuss how tumor cells achieve high rates of nucleotide and fatty acid synthesis, how oncogenes and tumor suppressors influence these activities, and how glutamine metabolism enables macromolecular synthesis in proliferating cells.

Otto Warburg’s demonstration that tumor cells rapidly use glucose and convert the majority of it to lactate is still the most fundamental and enduring observation in tumor metabolism. His work, which ushered in an era of study on tumor metabolism focused on the relationship between glycolysis and cellular bioenergetics, has been revisited and expanded by generations of tumor biologists. It is now accepted that a high rate of glucose metabolism, exploited clinically by 18FDGPET scanning, is a metabolic hallmark of rapidly dividing cells, correlates closely with transformation, and accounts for a significant percentage of ATP generated during cell proliferation. A ‘metabolic transformation’ is required for tumorigenesis. Research over the past few years has reinforced this idea, revealing the conservation of metabolic activities among diverse tumor types, and proving that oncogenic mutations can promote metabolic autonomy by driving nutrient uptake to levels that often exceed those required for cell growth and proliferation.

In order to engage in replicative division, a cell must duplicate its genome, proteins, and lipids and assemble the components into daughter cells; in short, it must become a factory for macromolecular biosynthesis. These activities require that cells take up extracellular nutrients like glucose and glutamine and allocate them into metabolic pathways that convert them into biosynthetic precursors (Figure 1). Tumor cells can achieve this phenotype through changes in the expression of enzymes that determine metabolic flux rates, including nutrient transporters and enzymes [8– 10]. Current studies in tumor metabolism are revealing novel mechanisms for metabolic control, establishing which enzyme isoforms facilitate the tumor metabolic phenotype, and suggesting new targets for cancer therapy.

The ongoing challenge in tumor cell metabolism is to understand how individual pathways fit together into the global metabolic phenotype of cell growth. Here we discuss two biosynthetic activities required by proliferating tumor cells: production of ribose-5 phosphate for nucleotide biosynthesis and production of fatty acids for lipid biosynthesis. Nucleotide and lipid biosynthesis share three important characteristics.

  • First, both use glucose as a carbon source.
  • Second, both consume TCA cycle intermediates, imposing the need for a mechanism to replenish the cycle.
  • Third, both require reductive power in the form of NADPH.

In this Essay, we discuss the possible drivers, advantages, and potential liabilities of the altered metabolism of cancer cells (Figure 1, not shown). Although our emphasis on the Warburg effect reflects the focus of the field, we would also like to encourage a broader approach to the study of cancer metabolism that takes into account the contributions of all interconnected small molecule pathways of the cell.

The Tumor Microenvironment Selects for Altered Metabolism One compelling idea to explain the Warburg effect is that the altered metabolism of cancer cells confers a selective advantage for survival and proliferation in the unique tumor microenvironment. As the early tumor expands, it outgrows the diffusion limits of its local blood supply, leading to hypoxia and stabilization of the hypoxia-inducible transcription factor, HIF. HIF initiates a transcriptional program that provides multiple solutions to hypoxic stress (reviewed in Kaelin and Ratcliffe, 2008). Because a decreased dependence on aerobic respiration becomes advantageous, cell metabolism is shifted toward glycolysis by the increased expression of glycolytic enzymes, glucose transporters, and inhibitors of mitochondrial metabolism. In addition, HIF stimulates angiogenesis (the formation of new blood vessels) by upregulating several factors, including most prominently vascular endothelial growth factor (VEGF).

Blood vessels recruited to the tumor microenvironment, however, are disorganized, may not deliver blood effectively, and therefore do not completely alleviate hypoxia (reviewed in Gatenby and Gillies, 2004). The oxygen levels within a tumor vary both spatially and temporally, and the resulting rounds of fluctuating oxygen levels potentially select for tumors that constitutively upregulate glycolysis. Interestingly, with the possible exception of tumors that have lost the von Hippel-Lindau protein (VHL), which normally mediates degradation of HIF, HIF is still coupled to oxygen levels, as evident from the heterogeneity of HIF expression within the tumor microenvironment. Therefore, the Warburg effect—that is, an uncoupling of glycolysis from oxygen levels—cannot be explained solely by upregulation of HIF. Other molecular mechanisms are likely to be important, such as the metabolic changes induced by oncogene activation and tumor suppressor loss.

Oncogene Activation Drives Changes in Metabolism Not only may the tumor microenvironment select for a deranged metabolism, but oncogene status can also drive metabolic changes. Since Warburg’s time, the biochemical study of cancer metabolism has been overshadowed by efforts to identify the mutations that contribute to cancer initiation and progression. Recent work, however, has demonstrated that the key components of the Warburg effect—

  • increased glucose consumption,
  • decreased oxidative phosphorylation, and
  • accompanying lactate production—
  • are also distinguishing features of oncogene activation.

The signaling molecule Ras, a powerful oncogene when mutated, promotes glycolysis (reviewed in Dang and Semenza, 1999; Ramanathan et al., 2005). Akt kinase, a well-characterized downstream effector of insulin signaling, reprises its role in glucose uptake and utilization in the cancer setting (reviewed in Manning and Cantley, 2007), whereas the Myc transcription factor upregulates the expression of various metabolic genes (reviewed in Gordan et al., 2007). The most parsimonious route to tumorigenesis may be activation of key oncogenic nodes that execute a proliferative program, of which metabolism may be one important arm. Moreover, regulation of metabolism is not exclusive to oncogenes.

Cancer Cell Metabolism: Warburg & Beyond

Hsu PP & Sabatini DM
Cell  Sep 5, 2008; 134, 703-705

Tumor cells respond to growth signals by the activation of protein kinases, altered gene expression and significant modifications in substrate flow and redistribution among biosynthetic pathways. This results in a proliferating phenotype with altered cellular function. These transformed cells exhibit unique anabolic characteristics, which includes increased and preferential utilization of glucose through the non-oxidative steps of the pentose cycle for nucleic acid synthesis but limited de novo fatty  acid   synthesis   and   TCA   cycle   glucose   oxidation. This  primarily nonoxidative anabolic profile reflects an undifferentiated highly proliferative aneuploid cell phenotype and serves as a reliable metabolic biomarker to determine cell proliferation rate and the level of cell transformation/differentiation in response to drug treatment.

Novel drugs effective in particular cancers exert their anti-proliferative effects by inducing significant reversions of a few specific non-oxidative anabolic pathways. Here we present evidence that cell transformation of various mechanisms is sustained by a unique disproportional substrate distribution between the two branches of the pentose cycle for nucleic acid synthesis, glycolysis and the TCA cycle for fatty acid synthesis and glucose oxidation. This can be demonstrated by the broad labeling and unique specificity of [1,2-13C2]glucose to trace a large number of metabolites in the metabolome. Stable isotope-based dynamic metabolic profiles (SIDMAP) serve the drug discovery process by providing a powerful new tool that integrates the metabolome into a functional genomics approach to developing new drugs. It can be used in screening kinases and their metabolic targets, which can therefore be more efficiently characterized, speeding up and improving drug testing, approval and labeling processes by saving trial and error type study costs in drug testing.

Metabolic Biomarker and Kinase Drug Target Discovery in Cancer Using Stable Isotope-Based Dynamic Metabolic Profiling (SIDMAP)

László G. Boros, Daniel J. Brackett and George G. Harrigan
Current Cancer Drug Targets, 2003, 3, 447-455 447

Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150 kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, while silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier.

A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila , and Humans

Daniel K. Bricker, Eric B. Taylor, John C. Schell, Thomas Orsak, et al.
Science Express 24 May 2012

Adenosine deaminase acting on RNA (ADAR) enzymes convert adenosine (A) to inosine (I) in double-stranded (ds) RNAs. Since Inosine is read as Guanosine, the biological consequence of ADAR enzyme activity is an A/G conversion within RNA molecules. A-to-I editing events can occur on both coding and non-coding RNAs, including microRNAs (miRNAs), which are small regulatory RNAs of ~20–23 nucleotides that regulate several cell processes by annealing to target mRNAs and inhibiting their translation. Both miRNA precursors and mature miRNAs undergo A-to-I RNA editing, affecting the miRNA maturation process and activity. ADARs can also edit 3′ UTR of mRNAs, further increasing the interplay between mRNA targets and miRNAs. In this review, we provide a general overview of the ADAR enzymes and their mechanisms of action as well as miRNA processing and function. We then review the more recent findings about the impact of ADAR-mediated activity on the miRNA pathway in terms of biogenesis, target recognition, and gene expression regulation.

Review ADAR Enzyme and miRNA Story: A Nucleotide that Can Make the Difference 

Sara Tomaselli, Barbara Bonamassa, Anna Alisi, Valerio Nobili, Franco Locatelli and Angela Gallo
Int. J. Mol. Sci. 19 Nov 2013; 14, 22796-22816 http://dx.doi.org:/10.3390/ijms141122796

The fermented wheat germ extract (FWGE) nutraceutical (Avemar™), manufactured under “good manufacturing practice” conditions and, fulfilling the self-affirmed “generally recognized as safe” status in the United States, has been approved as a “dietary food for special medical purposes for cancer patients” in Europe. In this paper, we report the adjuvant use of this nutraceutical in the treatment of high-risk skin melanoma patients. Methods: In a randomized, pilot, phase II clinical trial, the efficacy of dacarbazine (DTIC)-based adjuvant chemotherapy on survival parameters of melanoma patients was compared to that of the same treatment supplemented with a 1-year long administration of FWGE. Results: At the end of an additional 7-year-long follow-up period, log-rank analyses (Kaplan-Meier estimates) showed significant differences in both progression-free (PFS) and overall survival (OS) in favor of the FWGE group. Mean PFS: 55.8 months (FWGE group) versus 29.9 months (control group), p  0.0137. Mean OS: 66.2 months (FWGE group) versus 44.7 months (control group), p < 0.0298. Conclusions: The inclusion of Avemar into the adjuvant protocols of high-risk skin melanoma patients is highly recommended.

Adjuvant Fermented Wheat Germ Extract (Avemar™) Nutraceutical Improves Survival of High-Risk Skin Melanoma Patients: A Randomized, Pilot, Phase II Clinical Study with a 7-Year Follow-Up

LV Demidov, LV Manziuk, GY Kharkevitch, NA Pirogova, and EV Artamonova
Cancer Biotherapy & Radiopharmaceuticals 2008; 23(4)

Cancer cells possess unique metabolic signatures compared to normal cells, including shifts in aerobic glycolysis, glutaminolysis, and de novo biosynthesis of macromolecules. Targeting these changes with agents (drugs and dietary components) has been employed as strategies to reduce the complications associated with tumorigenesis. This paper highlights the ability of several food components to suppress tumor-specific metabolic pathways, including increased expression of glucose transporters, oncogenic tyrosine kinase, tumor-specific M2-type pyruvate kinase, and fatty acid synthase, and the detection of such effects using various metabonomic technologies, including liquid chromatography/mass spectrometry (LC/MS) and stable isotope-labeled MS. Stable isotope-mediated tracing technologies offer exciting opportunities for defining specific target(s) for food components. Exposures, especially during the early transition phase from normal to cancer, are critical for the translation of knowledge about food components into effective prevention strategies. Although appropriate dietary exposures needed to alter cellular metabolism remain inconsistent and/or ill-defined, validated metabonomic biomarkers for dietary components hold promise for establishing effective strategies for cancer prevention.

Bioactive Food Components and Cancer-Specific Metabonomic Profiles

Young S. Kim and John A. Milner
Journal of Biomedicine and Biotechnology 2011, Art ID 721213, 9 pages

This reviewer poses the following observation.  The importance of the pyridine nucleotide reduced/oxidized ratio has not been alluded to here, but the importance cannot be understated. It has relevance to the metabolic functions of anabolism and catabolism of the visceral organs.  The importance of this has ties to the pentose monophosphate pathway. The importance of the pyridine nucleotide transhydrogenase reaction remains largely unexplored.  In reference to the NAD-redox state, the observation was made by Nathan O. Kaplan that the organs may be viewed with respect to their primary functions in anabolic or high energy catabolic activities. Thus we find that the endocrine organs are largely tied to anabolic functioning, and to NADP, whereas cardiac and skeletal muscle are highly dependent on NAD. The consequence of this observed phenomenon appears to be related to a difference in the susceptibility to malignant transformation.  In the case of the gastrointestinal tract, the rate of turnover of the epithelium is very high. However, with the exception of the liver, there is no major activity other than cell turnover. In the case of the liver, there is a major commitment to synthesis of lipids, storage of fuel, and synthesis of proteins, which is largely anabolic, but there is also a major activity in detoxification, which is not.  In addition, the liver has a double circulation. As a result, a Zahn infarct is uncommon.  Now we might also consider the heart.  The heart is a muscle syncytium with a high need for oxygen.  Cutting of the oxygen supply makes the myocytes vulnerable to ischemic insult and abberant rhythm abnormalities.  In addition, the cardiomyocyte can take up lactic acid from the circulation for fuel, which is tied to the utilization of lactate from vigorous skeletal muscle activity.  The skeletal muscle is tied to glycolysis in normal function, which has a poor generation of ATP, so that the recycling of excess lactic acid is required by cardiac muscle and hepatocytes.  This has not been a part of the discussion, but this reviewer considers it important to remember in considering the organ-specific tendencies to malignant transformation.

Comment (Aurelian Udristioiu):

Otto Warburg observed that many cancers lose their capacity for mitochondrial respiration, limiting ATP production to anaerobic glycolytic pathways. The phenomenon is particularly prevalent in aggressive malignancies, most of which are also hypoxic [1].
Hypoxia induces a stochastic imbalance between the numbers of reduced mitochondrial species vs. available oxygen, resulting in increased reactive oxygen species (ROS) whose toxicity can lead to apoptotic cell death.
Mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration-.generation in the presence of intact tricarboxylic acid (TCA) enzyme.
One mitochondrial adaptation to increased ROS is over-expression of the uncoupling protein 2 (UCP2) that has been reported in multiple human cancer cell lines [2-3]. Increased UCP2 expression was also associated with reduced ATP production in malignant oxyphilic mouse leukemia and human lymphoma cell lines [4].
Hypoxia reduces the ability of cells to maintain their energy levels, because less ATP is obtained from glycolysis than from oxidative phosphorylation. Cells adapt to hypoxia by activating the expression of mutant genes in glycolysis.
-Severe hypoxia causes a high mutation rate, resulting in point mutations that may be explained by reduced DNA mismatch repairing activity.
The most direct induction of apoptosis caused by hypoxia is determined by the inhibition of the electron carrier chain from the inner membrane of the mitochondria. The lack of oxygen inhibits the transport of protons and thereby causes a decrease in membrane potential. Cell survival under conditions of mild hypoxia is mediated by phosphoinositide-3 kinase (PIK3) using severe hypoxia or anoxia, and then cells initiate a cascade of events that lead to apoptosis [5].
After DNA damage, a very important regulator of apoptosis is the p53 protein. This tumor suppressor gene has mutations in over 60% of human tumors and acts as a suppressor of cell division. The growth-suppressive effects of p53 are considered to be mediated through the transcriptional trans-activation activity of the protein. In addition to the maturational state of the clonal tumor, the prognosis of patients with CLL is dependent of genetic changes within the neoplastic cell population.

1.Warburg O. On the origin of cancer cells. Science 1956; 123 (3191):309-314
PubMed Abstract ; Publisher Full Text

2.Giardina TM, Steer JH, Lo SZ, Joyce DA. Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages. Biochim Biophys Acta 2008, 1777(2):118-129. PubMed Abstract | Publisher Full Text

3. Horimoto M, Resnick MB, Konkin TA, Routhier J, Wands JR, Baffy G. Expression of uncoupling protein-2 in human colon cancer. Clin Cancer Res 2004; 10 (18 Pt1):6203-6207. PubMed Abstract | Publisher Full Text

4. Randle PJ, England PJ, Denton RM. Control of the tricarboxylate cycle and it interactions with glycolysis during acetate utilization in rat heart. Biochem J 1970; 117(4):677-695. PubMed Abstract | PubMed Central Full Text

5. Gillies RJ, Robey I, Gatenby RA. Causes and consequences of increased glucose metabolism of cancers. J Nucl Med 2008; 49(Suppl 2):24S-42S. PubMed Abstract | Publisher Full Text

Shortened version of Comment –

Hypoxia induces a stochastic imbalance between the numbers of reduced mitochondrial species vs. available oxygen, resulting in increased reactive oxygen species (ROS) whose toxicity can lead to apoptotic cell death.
Mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration-.generation in the presence of intact tricarboxylic acid (TCA) enzyme.
One mitochondrial adaptation to increased ROS is over-expression of the uncoupling protein 2 (UCP2) that has been reported in multiple human cancer cell lines. Increased UCP2 expression was also associated with reduced ATP production in malignant oxyphilic mouse leukemia and human lymphoma cell lines.
Severe hypoxia causes a high mutation rate, resulting in point mutations that may be explained by reduced DNA mismatch repairing activity.

Read Full Post »

Innovation in Cancer Biopharmaceutical Intelligence [11.5]

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

The content of this article, with several interesting features is as follows:

11.5.1 Carmen Drahl..A Great Organic Chemist and Science Writer

11.5.2 Anthony Melvin Crasto

11.5.3 Amgen files ‘breakthrough’ leukemia drug in the US

11.5.4 Ginseng fights fatigue in cancer patients, Mayo Clinic-led study finds

11.5.5 The 10-Hydroxy-2-Decenoic Acid (10-2-HDA) content in Royal Jelly, is said to possess strong inhibition of malignant cell growth, namely transferable AKR leukemia, TA3 breast malignancy

11.5.6 A Microcapillary Flow Disc (MFD) Reactor for Organic Synthesis

11.5.7 Pauline Lau. Biochemist, Instrumental Analysis, Molecular and Clinical Diagnostics, and Pharmaceuticals

11.5.8  Kinetic and perfusion modeling of hyperpolarized 13C pyruvate and urea in cancer with arbitrary RF flip angles

11.5.9 ZSTK 474

11.5.10 Marrow-Infiltrating Lymphocytes Safely Shrink Multiple Myelomas


The following content is a series of discussions that identify innovation in therapeutics and individuals who are leaders in pharmaceutical innovation.

11.5.1 Carmen Drahl. A Great Organic Chemist and Science Writer

Her eyes fit a stellar career path. She is a talent in organic and medicinal chemistry, and an informed reporter.

Extract from Dr. Anthony Melvin Castro,  Organic Chemistry

Carmen Drahl

Carmen Drahl


Award-winning science communicator and social media power user based in Washington, DC.

Carmen Drahl is a multimedia science journalist and chemistry communicator based in Washington, DC.



A social media evangelist, Carmen started her first chemistry blog in 2006. Today, she regularly leverages Twitter, Facebook, and Google Plus Hangouts in her reporting.

Carmen has written about how life may have originated on Earth, explained how new medications get their names, and covered the ongoing issues plaguing the forensic science community. Her video on the food science behind 3D printed cocktail garnishes won the 2014 Folio Eddie Award for Best Association Video.

Until December 2014, Carmen worked at Chemical & Engineering News magazine. Her work has also been featured at Scientific American’s blog network, SiriusXM’s Doctor Radio, and elsewhere.

Carmen holds a Ph.D. in chemistry from Princeton University.

Ph.D. with Erik J. Sorensen.  She was on a team that completed the first total synthesis of abyssomicin C, a molecule found in small quantities in nature that showed hints of promise as a potential antibiotic. I constructed molecular probes from abyssomicin for proteomics studies of its biological activity.

M.A. with George L. McLendon worked toward developing a drug conjugate as a potential treatment for cancer. I synthesized a photosensitizer dye-peptide conjugate for targeting the cell death pathway called apoptosis.

Jacobus Fellowship Recipients - Carmen Drahl - Princeton

Jacobus Fellowship Recipients – Carmen Drahl – Princeton

Jacobus Fellowship Recipients – Carmen Drahl – Princeton

At a reception before the Alumni Day luncheon, President Tilghman (third from left) congratulated the winners of the University’s highest awards for students: (from left) Pyne Prize winners Lester Mackey and Alisha Holland; and Jacobus Fellowship recipients Sarah Pourciau, Egemen Kolemen and Carmen Drahl.


interviewing, science writing, social media, Twitter, Storify, YouTube, public speaking, hosting, video production, iPhone videography, non-linear video editing, blogging (WordPress and Blogger), HTML website coding

Carmen Drahl

By the time I discovered science blogs I knew my career goals were changing. I’d already been lucky enough to audit a science writing course at Princeton taught by Mike Lemonick from TIME, and thought that maybe science writing was a good choice for me. After reading chemistry blogs for a while I realized “Hey, I can do this!” and started my own blog, She Blinded Me with Science, in July 2006. It was the typical grad student blog, a mix of posts about papers I liked and life in the lab.

Carmen Drahl pic1

Carmen Drahl

At C&E News I’ve contributed to its C&ENtral Science blog, which premiered in spring 2008. I’ve experimented with a few different kinds of posts- observations and on-the-street interviews when

I run into something chemistry-related in DC, in-depth posts from meetings, and video demos of iPod apps. One of my favorite things to do is toy with new audio/video/etc technology for the blog.

Meant to treat: tumors with loss-of-function in the tumor suppressor protein PTEN (phosphatase and tensin homolog)- 2nd most inactivated tumor suppressor after p53- cancers where this is often the case include prostate and endometrial

Mode of action: inhibitor of phosphoinositide 3-kinase-beta (PI3K-beta). Several lines of evidence suggest that proliferation in certain PTEN-deficient tumor cell lines is driven primarily by PI3K-beta.

Medicinal chemistry tidbits: The GSK team seemed boxed in because in 3 out of 4 animals used in preclinical testing, promising drug candidates had high clearance. It turned out that a carbonyl group that they thought was critical for interacting with the back pocket of the PI3K-beta enzyme wasn’t so critical after all. When they realized they could replace the carbonyl with a variety of functional groups, GSK2636771 eventually emerged. GSK2636771B (shown)



11.5.2 Anthony Melvin Crasto

Principal Scientist, Process research

Glenmark Generics Ltd.

Anthony Melvin Crasto Ph.D

Worlddrugtracker, Principal scientist, Process research, Glenmark-Generics Ltd & Founder of Several Linkedin Gps

Glenmark Generics Ltd., Glenmark Pharmaceuticals

Glenmark Pharmaceuticals, Innovassynth, RPG Life Sciences

Institute of Chemical Technology (UDCT)

December 2005 – Present (9 years 6 months) Mahape, Navimumbai, India,
email  amcrasto@gmail.com

Currently working with GLENMARK GENERICS LTD research centre as Principal Scientist, process research (bulk actives) at Mahape ,Navi Mumbai,and leading a team of scientists in developing APIs for regulated markets, this involves visualization and execution of novel routes, polymorphs, and developing intellectual property to protect the invention. This involves all aspects of synthesis in lab and commercialization on plant , support for DMF filing.

Currently involved in development of several targets for regulated markets. Provide support to US/European marketing team for developing and execution of new projects

Process Development :-

  • Providing guidance and support for process development for challenging of patents in regulated market.
  • Design patent non-infringing scalable synthetic routes/process and scale-up of API’s
  • Bench and Pilot scale synthesis transformations in hands on
  • Optimization of the process, ie,developing industrially feasible process.
  • Preparation of PDR, filing of patent and DMF
  • Lead a group of Scientists and Group Leaders(for docs).

Skill sets:- Technical skills:


  • Development of novel synthetic routes/process for pharmaceuticals and successful implementation of the technology in pilot plant
  • Conducted various reactions at laboratory and production scales.
  • Synthesized various classes of compounds.
  • Experienced to work under cGMP condition

EX Hoechst Marion Roussel(SANOFI AVENTIS), RPG Life Sciences,Innovassynth, SEARLE,AGREVO,IOC

Glenmark Generics Ltd.

Research Activities Covered in Entire Career

1) Extensive range of chemistry and scale of manufacture from laboratory, scale up laboratory, pilot plant, plant scale including third party activity.

Applied intellectual and synthetic skills to the process development of pharmaceutical drugs/their intermediates, and natural products, neutraceuticals, mettalocenes, speciality chemicals, flavours and fragrances in the laboratory and monitor them during plant trials.

Act as a technology transfer man and provide all data required for transfer from lab to commercialization.

Use of Internet and manual literature search methods to decide on non-infringing route

Write DHR for API before implementation of novel route in the plant and assist for all batches for the DMF purposes, very well versed with IPR issues

Ability to develop novel routes for API,s and draft patents,well versed with polymorphism issues.

Several patents filed in US/EU

Total experience 23+ in industry.

Currently working as principal scientist and leading a team of scientists in developing APIs for regulated markets, this involves novel routes, polymorphs, and developing intellectual property to protect the invention. This involves all aspects of synthesis and commercialization and assist in providing support for DMF filing.

11.5.3 Amgen files ‘breakthrough’ leukemia drug in the US

Daily News | Sept 22, 2014

Selina Mckee

Biotechnology giant Amgen has filed its investigational cancer immunotherapy blinatumomab in the US for the treatment of certain forms of acute lymphoblastic leukaemia (ALL).

Specifically, the Biologic License Application seeks approval to market the drug for patients with Philadelphia-negative (Ph-) relapsed/refractory B-precursor forms of the aggressive blood/bone marrow cancer.

Blinatumomab is the first of Amgen’s BiTE antibody constructs, a novel immunotherapy approach under which antibodies are modified to engage two different targets simultaneously. The drug has already been awarded both ‘Orphan’ and ‘Breakthrough’ status by the Food and Drug Administration, indicating that it could offer a significant advance over available therapies on at least one clinically significant endpoint.

The submission includes data from a Phase II which successfully met its primary endpoint, showing a complete response (no leukaemia cells detectable with microscopy) rate of 43% in patients with relapsed/refractory ALL, including those with resistance to previous treatment approaches.

“Currently, there is no broadly accepted standard treatment regimen for adult patients with relapsed or refractory ALL,” noted Anthony Stein, clinical professor, Haematology/Oncology at City of Hope, adding that “blinatumomab has the potential to significantly advance treatment options for patients living with this difficult-to-treat disease”.

In the US, it is estimated that more than 6,000 cases of ALL will be diagnosed in 2014. In adult patients with relapsed or refractory ALL, median overall survival is just three to five months, further highlighting the urgent need for new treatment options.

Read more at: http://www.pharmatimes.com/Article/14-09-22/Amgen_files_breakthrough_leukaemia_drug_in_the_US.aspx#ixzz3aL5d1ZnJ

Follow us: @PharmaTimes on Twitter

11.5.4 Ginseng fights fatigue in cancer patients, Mayo Clinic-led study finds

By Ralph Turchiano on Aug 5, 2014 •

High doses of the herb American ginseng (Panax quinquefolius) over two months reduced cancer-related fatigue in patients more effectively than a placebo, a Mayo Clinic-led study found. Sixty percent of patients studied had breast cancer. The findings are being presented at the American Society of Clinical Oncology’s annual meeting.

Researchers studied 340 patients who had completed cancer treatment or were being treated for cancer at one of 40 community medical centers. Each day, participants received a placebo or 2,000 milligrams of ginseng administered in capsules containing pure, ground American ginseng root.

“Off-the-shelf ginseng is sometimes processed using ethanol, which can give it estrogen-like properties that may be harmful to breast cancer patients,” says researcher Debra Barton, Ph.D., of the Mayo Clinic Cancer Center.

At four weeks, the pure ginseng provided only a slight improvement in fatigue symptoms. However, at eight weeks, ginseng offered cancer patients significant improvement in general exhaustion — feelings of being “pooped,” “worn out,” “fatigued,” “sluggish,” “run-down,” or “tired” — compared to the placebo group.

11.5.5 The 10-Hydroxy-2-Decenoic Acid (10-2-HDA) content in Royal Jelly, is said to possess strong inhibition of malignant cell growth, namely transferable AKR leukemia, TA3 breast malignancy

Royal Jelly - queen larvae

Royal Jelly – queen larvae

Royal Jelly – queen larvae

Royal jelly is a honey bee secretion that is used in the nutrition of larvae, as well as adult queens.[1] It is secreted from the glands in the hypopharynx of worker bees, and fed to all larvae in the colony, regardless of sex or caste.[2]

When worker bees decide to make a new queen, because the old one is either weakening or dead, they choose several small larvae and feed them with copious amounts of royal jelly in specially constructed queen cells. This type of feeding triggers the development of queen morphology, including the fully developed ovaries needed to lay eggs.[3]

Other Common Names:  Apilak, Gelée Royale, Queen Bee Jelly

Royal Jelly has been called the “Crown Jewel” of the beehive that has become extremely popular since the 1950s as a wonderful source of energy and natural way to increase stamina; perhaps that is the reason why the Queen Bee is so strong and enduring.  It is also thought to be a great nutritional source of enzymes, proteins, sugars and amino acids, but there is no scientific proof to verify the supplement’s efficacy for its use as an overall health tonic.

Royal Jelly is a thick, milky material that is secreted from the hypopharyngea- salivary glands in the heads of the young nurse bees between the sixth and twelfth days of life, and when honey and pollen are combined and refined within the nurse bee, Royal Jelly is naturally created.  While all larvæ in a colony are fed Royal Jelly, it is the only food that is fed to the Queen Bee throughout her life; other adult bees do not consume it at all.  All female eggs may produce a Queen Bee, but this occurs only when – during the whole development of the larvæ – she is cared for and fed by this material – in large quantities.

As a result of this special nutrition, the Queen develops reproductive organs (while the worker bee develops traits that relate only to work, i.e., stronger mandibles, brood food, wax glands and pollen baskets).  The Queen develops in about fifteen days, while the workers require twenty-one; and finally, the Queen endures for several years, while workers survive only a few months. “10-2 HDA,” thought to be the principle active substance in Royal Jelly, makes the Queen Bee fifty percent larger than the other female worker bees and gives her incredible stamina, ovulation ability and longevity, living four to five years longer than worker bees who only live forty or more days.  Perhaps this is the reason why so many positive qualities have been attributed to Royal Jelly as a truly rare gift of nature, but it should be noted that there is no clinical evidence to support the claims.

There is even great controversy as to the constituents included in the supplement.  Most researchers claim that it includes all the B-vitamins and vitamins A, C, D and E; some disagree.  It does contain proteins, sugars, lipids (essential fatty acids), many essential amino acids, collagen, lecithin, enzymes and minerals, in addition to the very valuable 10-2-HDA (10-Hydroxy-2-Decenoic Acid).  It is said that Royal Jelly may be most effective when combined with honey.

The 10-Hydroxy-2-Decenoic Acid (10-2-HDA) content in Royal Jelly, is said to possess strong inhibition of malignant cell growth, namely transferable AKR leukemia, TA3 breast malignancy, etc., and recent studies indicated immuno-regulation and anti-malignancy activities.  It can promote the growth of T-lymphocyte subsets, Interleukin-2 and the generation of tumor necrosis factor.  Much research is being conducted on this valuable active constituent, which has exhibited positive physiological and pharmacological effects including vasodilative and hypotensive activities, antihypercholesterolemic activity and anti-inflammatory functions.

10-2-HDA (10-Hydroxy-2-Decenoic Acid)

10-2-HDA (10-Hydroxy-2-Decenoic Acid)

11.5.6  A Microcapillary Flow Disc (MFD) Reactor for Organic Synthesis
OCT 28, 2014

A Microcapillary Flow Disc (MFD) Reactor for Organic Synthesis,
C.H. Hornung, M.R. Mackley, I.R. Baxendale and S.V. Ley and, Org. Proc. Res. Dev., 2007, 11, 399-405.


This paper reports proof of concept, development, and trials for a novel plastic microcapillary flow disc (MFD) reactor. The MFD was constructed from a flexible, plastic microcapillary film (MCF), comprising parallel capillary channels with diameters in the range of 80−250 μm. MCFs were wound into spirals and heat treated to form solid discs, which were then capable of carrying out continuous flow reactions at elevated temperatures and pressures and with a controlled residence time. Three reaction schemes were conducted in the system, namely the synthesis of oxazoles, the formation of an allyl-ether, and a Diels−Alder reaction. Reaction scales of up to four kilograms per day could be achieved. The potential benefits of the MFD technology are compared against those of other reactor geometries including both conventional lab-scale and other microscale devices.

11.5.7 Pauline Lau. Biochemist, Instrumental Analysis, Molecular and Clinical Diagnostics, and Pharmaceuticals.

She was born on the China-Russian border, near the end of the rail line.  When they came to US her mother saw bagels and said, look – they have round bread.

At the meetings she always took us to the best Chinese restaurant, and said not to ask what’s in the food.  They always brought out a fish fresh from the tank and showed it to us.  When she went to Roche, where she became a legend. she got a house on the lake. They had to remove the roof to put a round banquet table in her house. At a meeting in Mexico, we saw the amazing too good to be true Monarch butterflies filling the trees.  Her photographic skills are suberb.  She’ll live to 100.

Carl Garber just retired and gave me the address.  I just found your photo calender!

Yes, I have been hiding in Taiwan for the past almost 10 years.  I moved from diagnostic to pharma and selling mostly biosimilar products to pharmaceutical emerging countries which has strong market growth comparing to US/EU.

Pauline Lau Group

Pauline Lau Group

Pauline Lau Group

Pauline Lau Group

I do not go back to US often now.  We have an office in Taipei.  Here is a recent magazine article about our company.  You will see few of my employees and I in front of our 28th floor office window.

I am rushing out for Singapore and will be meeting there for a few days.

11.5.8  Kinetic and perfusion modeling of hyperpolarized 13C pyruvate and urea in cancer with arbitrary RF flip angles

Naeim Bahrami, Christine Leon Swisher, Cornelius Von Morze, Daniel B. Vigneron, Peder E. Z. Larson
Department of Radiology and Biomedical Imaging, University of California – San Francisco, San Francisco, CA, USA
Quant Imaging MedSurg 2014; 4(1):24-32

Abstract: The accurate detection and characterization of cancerous tissue is still a major problem for the clinical management of individual cancer patients and for monitoring their response to therapy. MRI with hyperpolarized agents is a promising technique for cancer characterization because it can non-invasively provide a local assessment of the tissue metabolic profile. In this work, we measured the kinetics of hyperpolarized [1-13C] pyruvate and 13C-urea in prostate and liver tumor models using a compressed sensing dynamic MRSI method. A kinetic model fitting method was developed that incorporated arbitrary RF flip angle excitation and measured a pyruvate to lactate conversion rate, Kpl, of 0.050 and 0.052 (1/s) in prostate and liver tumors, respectively, which was significantly higher than Kpl in healthy tissues [Kpl =0.028 (1/s), P<0.001]. Kpl was highly correlated to the total lactate to total pyruvate signal ratio (correlation coefficient =0.95). We additionally characterized the total pyruvate and urea perfusion, as in cancerous tissue there is both existing vasculature and neovascularization as different kinds of lesions surpass the normal blood supply, including small circulation disturbance in some of the abnormal vessels. A significantly higher perfusion of pyruvate (accounting for conversion to lactate and alanine) relative to urea perfusion was seen in cancerous tissues (liver cancer and prostate cancer) compared to healthy tissues (P<0.001), presumably due to high pyruvate uptake in tumors. Keywords: Hyperpolarized carbon-13; metabolic imaging; cancer; perfusion; kinetic modeling; dynamic MRSI

Hyperpolarization is the nuclear spin polarization of a material far beyond thermal equilibrium conditions. The accurate and correct diagnosis and characterization of cancer is still a major problem for the clinical management of every kind of cancer patients, including individual prostate or liver cancer patients, and also in order to monitor their response to therapy (1-3). Magnetic resonance spectroscopic imaging (MRSI) with hyperpolarized 13C labeled substrates is a new method to study any cancers that may be able to simultaneously and noninvasively assess changes in metabolic intermediates from multiple biochemical pathways of interest. Recent studies have shown a large amount of potential applications of hyperpolarized (HP) 13C MRSI for the in vivo monitoring of cellular metabolism and the characterization of disease. The low natural abundance and sensitivity of 13C compared to protons poses a technical challenge using conventional approaches (4,5). Dynamic nuclear polarization (DNP) of 13C labeled pyruvate and subsequent rapid dissolution generates a contrast agent with a four order-of-magnitude sensitivity enhancement that is injected and gives the ability to monitor the spatial distribution of pyruvate and its conversion to lactate, alanine, and bicarbonate. The conversion of pyruvate to lactate catalyzed by the enzyme lactate dehydrogenase is of particular interest, as the kinetics of this process have been shown to be sensitive to the presence and severity of disease in preclinical models (6,7). HP MRSI can also be used to measure perfusion that in cancer can reflect spatially heterogeneous changes to existing vasculature and neovascularization as tumors surpass the normal blood supply, including microcirculatory disturbance in abnormal vessels. Tumor perfusion data in addition to the metabolic data available from spectroscopic imaging of 13C pyruvate would be of important value in exploring the complex relationship between perfusion and metabolism in cancer at both preclinical and clinical research levels (8-11). The primary purpose of this research was to study the dynamics of simultaneously injected HP [1-13C]-pyruvate and 13C-urea to provide improved characterization of cancerous tissues. To achieve rapid, 2 s temporal resolution, whole mouse MRSI we used a 18-fold accelerated compressed sensing acquisition and reconstruction with smaller flip angles for pyruvate and urea compared to lactate and alanine for efficient usage of the hyperpolarized magnetization by preserving the substrate. This flip angle scheme required using a modified kinetic model that accounts for arbitrary RF flip angles (12-15). Data was acquired in mice with prostate and liver cancer and comparisons were made to normal tissues such as kidney and healthy liver of the metabolite concentrations, including Urea, Pyruvate, and Lactate, the conversion constant (Kpl) between pyruvate to lactate, and the conversion constant (Kpa) between pyruvate to alanine. We also created novel parameterizations of the total pyruvate and urea perfusions in order to assess vascular delivery and tissue uptake. A key new feature of our modeling is the ability to detect metabolic conversion, magnetization exchange between compounds, and perfusion when using arbitrary RF flip angles for different compounds.

We observed a strong correlation between Kpl and the total lactate to total pyruvate ratio, as others have also shown. The ratio is a simpler calculation and easier to implement than the kinetic modeling. However, we have determined through simulation that the total lactate to total pyruvate ratio is highly influenced by the delivery time of pyruvate, so care should be taken when using this ratio if variable vascular delivery rates are expected. Both the kinetic modeling and metabolite ratio are highly influenced by the actual RF flip angles, and precise B1 calibration is important for quantitative measurements. Measurement of urea perfusion can be a marker vascular delivery since urea primarily stays in the vasculature. Liver is a very vascular organ and the opened capillary shape of liver vasculature likely caused high urea perfusion in liver. The kidneys are highly vascularized and are also responsible for concentrating urea for removal in the urine. In tumors, the tissue request for blood is high but in a more uncontrolled way because of the abnormality of blood vasculature and circulation inside most tumors. Thus the urea perfusion in tumors is likely more sporadic and random. Urea cannot perfuse well in some parts of tumor particularly in suspected necrotic regions. On the other hand, some parts of tumor have more metabolic activity and, therefore, these parts need more blood and more vessels, and consequently should have more urea perfusion. Our total pyruvate and urea perfusion parameterizations are different from conventional perfusion modeling, and were designed as a simple representation of the total amount of these compounds that are present in the tissue. In particular, the total pyruvate perfusion also includes any pyruvate or metabolic products that remain in the tissue, in addition to those present in the vasculature. The urea perfusion should primarily represent the vasculature delivery since it primarily stays in the vessels, while the total pyruvate perfusion can also be a marker for vascular delivery but also includes tissue uptake. We hypothesize that when the pyruvate perfusion is higher relative to urea perfusion it represents a higher amount of uptake of the pyruvate that is flowing into the tissue.

Conclusions In this study we fit metabolite T1 values, conversion rates, Kpa, and Kpl, and measured novel pyruvate and urea perfusion parameterizations across cancerous and normal tissues from data acquired with a multiband RF excitation, compressed sensing dynamic MRSI pulse sequence. Our modeling allowed for use of arbitrary RF flip angles between metabolites, which in turn allows for efficient usage of the hyperpolarized magnetization. We observed a high correlation between our Kpl fits and the total lactate to pyruvate signal ratio, suggesting either could be used to characterize pyruvate-lactate metabolism. Through the novel pyruvate and urea perfusion parameterizations we were able to quantify the increased uptake of pyruvate in cancerous tissues, which correlated with increased metabolic conversion to lactate. These provided a more complete characterization of cancerous tissue metabolism and perfusion.

11.5.9  ZSTK474

(Dr. Anthony Melvin Castro)



ZSTK474 is a cell permeable and reversible P13K inhibitor with an IC₅₀ at 6nm. It was identified as part of a screening library, selected for its ability to block tumor cell growth. ZSTK474 has shown strong antitumor activities against human cancer xenographs when administered orally to mice without a significant toxic effect.

Phosphatidylinositol 3-kinase (PI3K) has been implicated in a variety of diseases including cancer. A number of PI3K inhibitors have recently been developed for use in cancer therapy. ZSTK474 is a highly promising antitumor agent targeting PI3K. We previously reported that ZSTK474 showed potent inhibition against four class I PI3K isoforms but not against 140 protein kinases.

However, whether ZSTK474 inhibits DNA-dependent protein kinase (DNA-PK), which is structurally similar to PI3K, remains unknown. To investigate the inhibition of DNA-PK, we developed a new DNA-PK assay method using Kinase-Glo. The inhibition activity of ZSTK474 against DNA-PK was determined, and shown to be far weaker compared with that observed against PI3K. The inhibition selectivity of ZSTK474 for PI3K over DNA-PK was significantly higher than other PI3K inhibitors, namely NVP-BEZ235, PI-103 and LY294002.

PATENT                                                                                                          SUBMITTED GRANTED

Heterocyclic compound and antitumor agent containing the same as active ingredient [US7071189]                                                                                                                                                               2004-06-17   2006-07-04

Treatment of prostate cancer, melanoma or hepatic cancer [US2007244110]                                                                                                                                                                                                   2007-10-18

Heterocyclic compound and antitumor agent containing the same as effective ingredient [US7307077]                                                                                                                                                           2006-11-02   2007-12-11

Immunosuppressive agent and anti-tumor agent comprising heterocyclic compound as active ingredient [us7750001]                                                                                                                                   2008-05-15   2010-07-06

Pyrimidinyl and 1,3,5-triazinyl benzimidazoles and their use in cancer therapy [us2011009405]                                                                                                                                                                       2011-01-13

Substituted pyrimidines and triazines and their use in cancer therapy [us2011053907]                                                                                                                                                                                     2011-03-03

Immunosuppressive agent and anti-tumor agent comprising heterocyclic compound as active ingredient [us2010267700]                                                                                                                             2010-10-21

Amorphous body composed of heterocyclic compound, solid dispersion and pharmaceutical preparation each comprising the same, and process for production of the same [us8227463]                                                                                                                                                                                                                                                                                                                                                                                                                           2010-09-30    2012-07-24

Pyrazolo[1,5-a]pyridines and their use in cancer therapy
[us2010226881]                                                                                                                                                                                                                                                                                                 2010-09-09

Pyrimidinyl and 1,3,5-triazinyl benzimidazole sulfonamides and their use in cancer therapy [us2010249099]                                                                                                                                                   2010-09-30

11.5.10 Marrow-Infiltrating Lymphocytes Safely Shrink Multiple Myelomas

 Medical researchers at the Johns Hopkins Kimmel Cancer Center have published a report that appeared in the journal Science Translational Medicine in which they describe, for the first time, the safe use of a patient’s own immune cells to treat the white blood cell cancer multiple myeloma. There are more than 20,000 new cases of multiple myeloma and more than 10,000 deaths each year in United States. It is the second most common cancer originating in the blood.

The procedure under investigation in this study is called utilizes a specific type of tumor-targeting T cells, known as marrow-infiltrating lymphocytes (MILs). “What we learned in this small trial is that large numbers of activated MILs can selectively target and kill myeloma cells,” says Johns Hopkins immunologist Ivan Borrello, M.D., who led the clinical trial.

According to Borrello, MILs are the foot soldiers of the immune system that attack invading bacteria or viruses. Unfortunately, they are typically inactive and too few in number to have a measurable effect on cancers.

Experiments conducted is Borrello’s laboratory and in the laboratory of competing and collaborating scientists have shown that when myeloma cells are exposed to activated MILs in culture, these cells could not only selectively target the tumor cells, but they could also effectively destroy them.

To move this procedure from the laboratory into the clinic, Borrello and his collaborators enrolled 25 patients with newly diagnosed or relapsed multiple myeloma. Only 22 were able to receive this new treatment, however.

The Hopkins team extracted and purified MILs from the bone marrow of each patient and grew them in the laboratory to increase their numbers. Then they activated the MILs by exposing them to microscopic beads coated with immune activating antibodies. These antibodies bind to specific cell surface proteins on the MILs that induce profound changes in the cells. This induction step wakes the MILs up and readies them to sniff out tumor cells. These laboratory-manipulated MILs were then intravenously injected back into each patient (each of the 22 patients with their own cells). Three days before these injections of expanded MILs, all patients received high doses of chemotherapy and a stem cell transplant, which are standard treatments for multiple myeloma.

One year after receiving the MILs therapy, 13 of the 22 patients had at least a partial response to the therapy (their cancers had shrunk by at least 50 percent) Seven patients experienced at least a 90 percent reduction in tumor cell volume and lived and average of 25.1 months without cancer progression. The remaining 15 patients had an average of 11.8 progression-free months following their MIL therapy. None of the participants experienced serious side effects from the MIL therapy.

According to Borrello, several U.S. cancer centers have conducted similar experimental treatments (adoptive T cell therapy). However, only this Johns Hopkins team has used MILs. Other types of tumor-infiltrating cells can be used for such treatments, but Borrello noted that these cells are usually less plentiful in patients’ tumors and may not grow as well outside the body.

In nonblood-based tumors, such as melanoma, only about half of those patients have T cells in their tumors that can be harvested, and only about one-half of those harvested cells can be grown. “Typically, immune cells from solid tumors, called tumor-infiltrating lymphocytes, can be harvested and grown in only about 25 percent of patients who could potentially be eligible for the therapy. But in our clinical trial, we were able to harvest and grow MILs from all 22 patients,” says Kimberly Noonan, Ph.D., a research associate at the Johns Hopkins Universithttp://www.fiercevaccines.com/special-reports/gvax-pancreasy School of Medicine.

This small trial helped Noonan and her colleagues learn more about which patients may benefit from MILs therapy. As an example, they were able to determine how many of the MILs grown in the lab were specifically targeted to the patient’s tumor and whether they continued to target the tumor after being infused. They also found that patients whose bone marrow before treatment contained a high number of certain immune cells, known as central memory cells, also had better response to MILs therapy. Patients who began treatment with signs of an overactive immune response did not respond as well.

Noonan says the research team has used these data to guide two other ongoing MILs clinical trials. Those studies, she says, are trying to extend anti-tumor response and tumor specificity by combining the MILs transplant with a Johns Hopkins-developed cancer vaccine called GVAX and the myeloma druglenalidomide, which stimulates T cell responses.

These trials also have elucidated new ways to grow the MILs. “In most of these trials, you see that the more cells you get, the better response you get in patients. Learning how to improve cell growth may therefore improve the therapy,” says Noonan.

Kimmel Cancer Center scientists are also developing MILs treatments to address solid tumors such as lung, esophageal and gastric cancers, as well as the pediatric cancers neuroblastoma and Ewing’s sarcoma.

Read Full Post »

Novel Approaches to Cancer Therapy

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

11.1       Novel Approaches to Cancer Therapy

11.1.1 Electrically-driven modulation of surface-grafted RGD peptides for .. cell adhesion

11.1.2 The metabolic state of cancer stem cells—a target for cancer therapy

11.1.3 Regulation of tissue morphogenesis by endothelial cell-derived signals

11.1.4 Novel approach to bis(indolyl)methanes. De novo synthesis of 1-hydroxyimino-methyl derivatives with anti-cancer properties

11.1.5 Synthesis and Biological Evaluation of New 1,3-Thiazolidine-4-one Derivatives of 2-(4-Isobutylphenyl)propionic Acid molecules

11.1.6 Targeting pyruvate kinase M2 contributes to radiosensitivity of NSCLC cells

11.1.7 The tyrosine kinase inhibitor nilotinib has antineoplastic activity in prostate cancer cells but up-regulates the ERK survival signal—Implications for targeted therapies

11.1.8 PAF and EZH2 Induce Wnt.β-Catenin Signaling Hyperactivation

11.1.9 PAF Makes It EZ(H2) for β-Catenin Transactivation

11.1.10 PI3K.AKT.mTOR pathway as a therapeutic target in ovarian cancer

11.1.11 Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway

11.1.12 Curcumin-could-reduce-the-monomer-of-ttr-with-tyr114cys-mutation via autophagy in cell model of familial amyloid polyneuropathy.

11.1.1 Electrically-driven modulation of surface-grafted RGD peptides for .. cell adhesion

Lashkor M1Rawson FJStephenson-Brown APreece JAMendes PM.
Chem Commun (Camb). 2014 Dec 21; 50(98):15589-92

Reported herein is a switchable surface that relies on electrically-induced conformational changes within surface-grafted arginine–glycine–aspartate (RGD) oligopeptides as the means of modulating cell adhesion

Stimuli-responsive surfaces that are capable of modulating their biological properties in response to an external stimuli, including temperature,1,2 light,3 magnetic field4 and electrical potential,59 are of growing interest for a variety of biological and medical applications.10,11 Switchable surfaces that can be controlled on-demand are playing an increasingly important part in the development of highly sensitive biosensors,1215novel drug delivery systems1618 and functional microfluidic, bioanalysis, and bioseparation systems.1922Additionally, dynamic, synthetic surfaces that can control the presentation of regulatory signals to a cell are expected to have a significant impact in the field of tissue engineering and regenerative medicine, and to provide unprecedented opportunities in fundamental studies of cell biology.23,24 The availability of sophisticated and functional switchable surfaces is expected to emulate more complex in vivo like extracellular environments, and provide a powerful means to probe and control the dynamic interactions between the cell and its external environments.

The majority of studies on stimuli-responsive surfaces reported to date either rely2529 on controlling non-specific interactions (i.e., hydrophobic/hydrophilic and electrostatic) of the biomolecules with the active surface, or have focused3032 on demonstrating modulation of specific biomolecular interactions using relatively simple biological systems (e.g. biotin–streptavidin) and conditions (i.e. water or buffer solutions). For example, Zareie et al. 30 fabricated a mixed self-assembled monolayer (SAM) on gold comprising oligo(ethylene glycol) (OEG) thiol molecules and shorter disulfides carrying biotin end-groups that regulated the interaction between biotin and streptavidin in water. The OEG thiols were able to switch in response to a change in temperature below and above their lower critical solution temperature (LCST = 37 °C). At 23 °C the structure of the OEG molecules was fully extended hindering the shorter biotin disulfide components. On the contrary, at 45 °C the OEG backbone collapsed, thus allowing the specific interaction between the biotin molecule on the surface and the protein streptavidin in solution. In our previous work,79 electrically controlled switching has been applied to regulate the conformational changes of modified positively charged oligolysine peptides tethered to a gold surface, such that biotin moieties incorporated into the oligolysines could be reversibly exposed or concealed on demand, as a function of surface potential. Switchable SAMs used to control biomolecular interactions via an electrical stimulus are particularly appealing because of their fast response times, ease of creating multiple individually addressable switchable regions on the same surface, as well as low-drive voltage and electric fields, which are compatible with biological systems.33 Our previous reported electrically switchable surface was able to control directly the biomolecular interactions between biotin and neutravidin in phosphate buffer saline (PBS) solution.

However, switchable surfaces have been scarcely used, thus far, to control biomolecular interactions on more complex systems such as those involving modulation of cell responsiveness.3437 Jonkheijm and co-workers35 have reported a cucurbit[8]uril-based SAM system to electrochemically control the release of cells. Charged end groups on SAM surfaces have been exploited to electrically control the early stages of bacterial cell adhesion37 and form patterned surfaces with two independent dynamic functions for inducing cell migration.36 In spite of these efforts, given cellular complexity and diversity, such studies are very limited in number, as are the opportunities to further understand and control the complex interplay of events and interactions occurring within living cells.

Herein, we report on a stimuli-responsive surface that relies on electrically-induced conformational changes within surface-grafted arginine–glycine–aspartate (RGD) oligopeptides as the means of modulating cell adhesion. RGD, which is present in most of the adhesive ECM proteins (e.g. fibronectin, vitronectin, laminin and collagen), is specific for integrin-mediated cell adhesion.38 The RGD modified electrode is used here to dynamically regulate the adhesion of immune macrophage cells. The stimuli-responsive surface is fabricated on a gold surface and comprises a mixed SAM consisting of two components (Fig. 1): (i) an oligopeptide containing a terminal cysteine for attachment to the gold surface, three lysine residues as the main switching unit, and a glycine–arginine–glycine–aspartate–serine (GRGDS) as the recognition motif for cell adhesion –C3K-GRGDS, and (ii) an ethylene glycol-terminated thiol (C11TEG) to space out the oligopeptides. Since the charged backbone of the oligopeptide can be potentially harnessed79 to induce its folding on the surface upon an application of an electrical potential, we reasoned that such conformational changes can be employed to selectively expose under open circuit (OC) conditions (bio-active state) or conceal under negative potential (bio-inactive state) the RGD to the cell and dynamically regulate cell adhesion.



RDG oligopeptide SAM utilised for controlling specific cellular interactions


Fig. 1 Schematic of the dynamic RDG oligopeptide SAM utilised for controlling specific cellular interactions. The electrically switchable SAM exposes the RGD peptide and supports cell adhesion under open circuit (OC) conditions (no applied potential), while …

Mixed SAMs of C3K-GRGDS : C11TEG were formed from a solution ratio of 1 : 40 and characterised by X-ray photoelectron spectroscopy (XPS) (Fig. S2, ESI). XPS analysis confirmed the formation of the C3K-GRGDS:C11TEG mixed monolayer and displayed signals from S, N, C and O. The chemical state of the sulphur atom was probed using the XPS spectra of the S 2p emission (Fig. S2, ESI). The S 2p spectrum (Fig. S2a, ESI) consists of two doublet peaks, with one doublet peak at 162.0 eV (S 2p3/2) and 163.2 eV (S 2p1/2), indicating that the sulphur is chemisorbed on the gold surface.39 A second small doublet peak can be observed at 163.8 eV and 165.0 eV, which can be attributed to the S–H bond, indicating a small presence of unbound sulphur. No sulphur peaks above 166 eV were observed, indicating that no oxidised sulphur is present at the surface. The N 1s spectrum (Fig. S2b, ESI) can be de-convoluted into two peaks, which support the presence of the peptide on the surface. The first peak centred at 400.5 eV is attributed to amino (NH2) and amide (CONH) moieties. The second peak centred at 402.8 eV is ascribed to protonated amino groups.40 Note that no nitrogen peak was observed for pure C11TEG SAMs. The C 1s spectrum (Fig. S2c, ESI) can be de-convoluted into three peaks, which are attributed to five different binding environments. The peak at 285.0 eV is attributed to C–C bonds,41 while the peak at 286.7 eV corresponds to C 1s of the three binding environments of C–S, C–N and C–O.41 The third and smaller peak (288.6 eV) is assigned to the C 1s photoelectron of the carbonyl moiety, C O.41 The O 1s spectrum (Fig. S2d, ESI) is de-convoluted into two different peaks, corresponding to two different binding environments, arising from the C–O (533.3 eV) and C O (532.0 eV) bonds.41 From integrating the area of the S 2 p and N 1s peaks and taking into consideration that the C3K-GRGDS oligopeptide consists of 15 N atoms and 1 S atom and C11TEG has no N and 1 S atom only, it was possible to infer that the ratio of C3K-GRGDS:C11TEG on the surface is 1 : 10 ± 2. The presence of C11TEG was utilised not only to ensure sufficient spatial freedom for molecular reorientation of the surface bound oligopeptide, but also to stop non-specific binding to the surface.

The C3K-GRGDS:C11TEG mixed SAMs were shown to support adhesion of immune macrophage cells as determined by cell counting42,43 (Fig. 2). When RAW 264.7 mouse macrophages were cultured on theC3K-GRGDS:C11TEG mixed SAM in supplemented Dulbecco’s Modified Eagle Medium (DMEM), the number of cells adhered to the surface increased with incubation time, reaching 1792 ± 157 cells per mm2after 24 hours. This is in contrast with the weak cell adhesion observed in two control surfaces, pureC11TEG SAMs and clean gold, in which the number of cells that adhere was 60% and 50% lower, respectively, after 24 hours (Fig. 2).



Microscopic images and density of adhered cells on C3K-GRGDS:C11TEG mixed SAM, pure C11TEG SAM and bare gold surfaces


Fig. 2 Microscopic images and density of adhered cells on C3K-GRGDS:C11TEG mixed SAM, pure C11TEG SAM and bare gold surfaces that were normalized against the density of cells adherent onto the C3K-GRGDS:C11TEG mixed SAM. The surfaces were cultured in RAW 264.7 mouse macrophage cells under OC conditions for 24 hours.

In order to demonstrate that the C3K-GRGDS:C11TEG mixed SAMs can support or resist cell adhesion on demand, the macrophage cells were cultured on the C3K-GRGDS:C11TEG mixed SAM in DMEM medium under OC conditions and applied negative potential (–0.4 V) for a period of 1 h. Note that DMEM contains a mixture of inorganic salts, amino acids, glucose and vitamins. On application of the applied potential of –0.4 V the number of adherent cells was 70% less compared to the C3K-GRGDS:C11TEGmixed SAMs under OC conditions, Fig. 3. Similar switching efficiencies have been observed in another oligopeptide system using different DMEM solutions.44 These findings suggest that the negative potential induces the conformational changes in the C3K moiety of C3K-GRGDS in the SAM which in turn leads to the RGD moiety being concealed and hence reducing the binding of the cells.



Density of adhered cells on C3K-GRGDS:C11TEG, C11TEG, C6EG-GRGDS:C11TEG mixed SAMs


Fig. 3 Density of adhered cells on C3K-GRGDS:C11TEG, C11TEG, C6EG-GRGDS:C11TEG mixed SAMs that were normalized against the density of cells adherent onto the C3K-GRGDS:C11TEG mixed SAM. The surfaces were cultured in RAW 264.7 for 1 h under OC conditions or while applying –0.4 V.

Previous studies have shown that small conformational and orientational changes in proteins and peptides modulate the availability and potency of the active sites for cell surface receptors.4547 Thus, in a similar manner, small changes in the conformation/orientation of the RGD peptide on the surface induced by application of an electrical potential are able to affect the binding activity of the peptide. Recently, we have conducted detailed theoretical8 and experimental9 studies aimed at understanding the switching mechanism of oligopeptide-based switchable surfaces, that similarly as in the case of the C3K-GRGDS:C11TEG mixed SAMs, use lysine residues to act as the switching unit. These previous studies unraveled that the surface-appended oligolysines undergo conformational changes between fully extended, partially extended and collapsed conformer structures in response to an applied positive potential, open circuit conditions and negative electrical potential, respectively. Thus, these previous findings allow us to propose that when a negative potential is applied to the GRGDS:C11TEG mixed SAM surface, the oligopeptide chain adopts a collapsed conformation on the surface and the RGD binding motif is partially embedded on the C11TEGmatrix, thus showing no bioactivity (“OFF” state).

In order to verify that the changes in adhesion upon application of a negative surface potential occur due to changes in the conformational orientation of the RGD instead of cell repulsion or cell damage due to the presence of an electrical potential, control mixed SAMs were also prepared using C11TEG and a peptide where the 3 lysine residues as the switching unit were replaced by 6 non-switchable ethylene glycol units –C6EG-GRGDS (Fig. S1, ESI). Fig. 3 demonstrates that cells adhered in similar numbers to the C11TEGand C6EG-GRGDS:C11TEG mixed SAMs under OC conditions and an applied negative potential. These results provide strong evidence that control over cell adhesion using the C3K-GRGDS:C11TEG mixed SAM is due to a conformational behaviour of the lysine-containing oligopeptide that can either expose or conceal the RGD moiety.

Cell viability was checked following application of –0.4 V for 1 h by performing a trypan blue assay. Cells that were dead were stained blue due to a break down in membrane integrity. Incubation of the cells under a negative potential had negligible effect on cell viability, which was greater than 98%. Cyclic voltammetric studies (outlined in detail in the Fig. S3, ESI) were also performed to demonstrate that no significant faradaic process occur over the potential range studied, and thus ions are not participating in redox reactions and consequently redox chemistry is not being significantly affected by application of the potential used. In agreement with other studies,35,36,48 we conclude that the electrical modulation of the surface neither affected cell viability nor induced any redox process in the medium that could have had an effect on cells.

We then addressed the question of whether the C3K-GRGDS:C11TEG surfaces could be switched between different cell adhesive states (cell-resistant and cell-adhesive states). To begin with, we investigated the switching from a cell-adhesive state to a cell-resistant state, and the possibility to detach the cells from the substrate upon the application of a negative potential. Cells were incubated in the C3K-GRGDS:C11TEGmixed SAMs for 1 h under OC conditions, thereby exposing the RGD moiety and allowing for cell attachment. This step was followed by the application of a potential of –0.4 V for 1 h in order to detach the cells from the surface, by concealing the RGD moieties. Cell counts showed no significant differences between the pre and post application of the –0.4 V, suggesting that the electrostatic force generated by the applied negative electrical potential might not be sufficient to disrupt the RGD–integrin interaction. These results were to a certain extent expected since adherent cells are able to withstand strong detachment forces due to the adhesion being mediated by multiple RGD–integrin bonds in parallel.49

In contrast, a reversal of the switching sequence demonstrated that our surfaces can be dynamically switched from a non-adhesive to cell-adhesive state. Cells were incubated in the C3K-GRGDS:C11TEG mixed SAMs for 1 h while holding the potential at –0.4 V for 1 h making the RGD peptide inaccessible for recognition by the corresponding integrin. As above, the number of adherent cells when a negative potential of –0.4 V was applied was 70% of the number that adhered to the C3K-GRGDS:C11TEG mixed SAMs under OC conditions, Fig. 4. The potential was then shifted to open circuit conditions for 1 h on those exposed to a potential of –0.4 V, which resulted in a significant increase in the number of cells as a result of the exposure of the RGD moiety to the cells (Fig. 4). These values were similar to those obtained for the samples that were only incubated for 1 hour under OC conditions (Fig. 4), indicating that the surfaces were highly effective at switching from a non-adhesive to cell-adhesive state.



Microscopic images and density of adhered cells on C3K-GRGDS:C11TEG mixed SAMs


Fig. 4  Microscopic images and density of adhered cells on C3K-GRGDS:C11TEG mixed SAMs that were incubated with cells for 1 h while applying –0.4 V and subsequently in OC conditions for 1 h. The density was normalized against the density of cells adherent onto C3K-GRGDS:C11TEG mixed SAMs that were incubated with cells in OC conditions for 1 h.

In summary, an electrically switchable surface has been devised and fabricated that is capable of efficiently exposing and concealing the RGD cell adhesion motif and dynamically regulate the adhesion of immune macrophage cells. This study will no doubt be useful in developing more realistic dynamic extracellular matrix models and is certainly applicable in a wide variety of biological and medical applications. For instance, macrophage cell adhesion to surfaces plays a key role in mediating immune response to foreign materials.50 Thus, development of such dynamic in vitro model systems that can control macrophage cell adhesion on demand are likely to provide new opportunities to understand adhesion signaling in macrophages51 and develop effective approaches for prolonging the life-span of implantable medical devices and other biomaterials.52

11.1.2 The metabolic state of cancer stem cells—a target for cancer therapy

Vlashi E1Pajonk F2.
Free Radic Biol Med. 2015 Feb; 79:264-8


  • Bulk tumor cell populations rely on aerobic glycolysis.
  • Cancer stem cells are in a specific metabolic state.
  • Cancer stem cells in breast cancer, glioblastoma, and leukemia rely on oxidative phosphorylation of glucose.

In the 1920s Otto Warburg first described high glucose uptake, aerobic glycolysis, and high lactate production in tumors. Since then high glucose uptake has been utilized in the development of PET imaging for cancer. However, despite a deepened understanding of the molecular underpinnings of glucose metabolism in cancer, this fundamental difference between normal and malignant tissue has yet to be employed in targeted cancer therapy in the clinic. In this review, we highlight attempts in the recent literature to target cancer cell metabolism and elaborate on the challenges and controversies of these strategies in general and in the context of tumor cell heterogeneity in cancer.



11.1.3 Regulation of tissue morphogenesis by endothelial cell-derived signals

Saravana K. RamasamyAnjali P. KusumbeRalf H. Adams
Trends Cell Biol  Mar 2015; 25(3):148–157


  • Endothelial cells lining blood vessels induce organ formation and other morphogenetic processes in the embryo.
  • Blood vessels are also an important source of paracrine (angiocrine) signals acting on other cell types in organ regeneration.
  • Vascular niches and endothelial cell-derived signals generate microenvironments for stem and progenitor cells.

Endothelial cells (ECs) form an extensive network of blood vessels that has numerous essential functions in the vertebrate body. In addition to their well-established role as a versatile transport network, blood vessels can induce organ formation or direct growth and differentiation processes by providing signals in a paracrine (angiocrine) fashion. Tissue repair also requires the local restoration of vasculature. ECs are emerging as important signaling centers that coordinate regeneration and help to prevent deregulated, disease-promoting processes. Vascular cells are also part of stem cell niches and have key roles in hematopoiesis, bone formation, and neurogenesis. Here, we review these newly identified roles of ECs in the regulation of organ morphogenesis, maintenance, and regeneration.


Figure 1. Role of endothelial cells (ECs) during organogenesis


Figure 2. Endothelial cells (ECs) in lung regeneration


Figure 3. Liver endothelium in regeneration and fibrosis.

Vascular cells have key roles in morphogenesis and regeneration

Vascular cells have key roles in morphogenesis and regeneration


Figure 4. Functional roles of the bone vasculature


Figure 5. Vascular niche for neurogenesis.

Concluding remarks

The examples provided in this review highlight the important roles of ECs in tissue development, patterning, homeostasis, and regeneration. The endothelium often takes a central position in these processes and there are many reasons why ECs are ideally positioned as the source of important instructive, angiocrine signals. The vascular transport network extends into every organ system and needs to be embedded in those tissues in a certain spacing or pattern, which places ECs in central and, therefore, strategic positions for the regulation of morphogenesis and organ homeostasis.

Given that ECs and other cell types frequently form functional units, such as kidney glomeruli, liver lobules, or lung alveoli, the assembly, differentiation, and function of the different cellular components needs to be tightly coordinated. In addition, because circulating blood cells extensively rely on the vascular conduit system and frequently interact with the endothelium, it is perhaps not surprising that ECs contribute to niche microenvironments. During tissue repair, proliferative cell expansion processes are sometimes temporally separated from cell differentiation and tissue patterning events. The latter has to involve the restoration of a fully functional vascular network so that ECs appear ideally suited as the source of molecular signals that can trigger or suppress processes in the surrounding tissue.


11.1.4 Novel approach to bis(indolyl)methanes. De novo synthesis of 1-hydroxyimino-methyl derivatives with anti-cancer properties

Grasso C, et al.
Eur J Medicinal Chem 01/2015; 93:9-15.

A versatile and broad range approach to previously unknown bis(indolyl)methane oximes based on two consecutive hetero Diels-Alder cycloaddition reactions of electrophilic conjugated nitrosoalkenes with indoles is disclosed. The cytotoxic properties and selectivity of some adducts against several human cancer cell lines pointing to a promising role in the development of anti-tumoural drugs, in particular for leukemia and lymphoma.

Novel approach to bis(indolyl)methanes: De novo synthesis of 1-hydroxyiminomethyl derivatives with anti-cancer properties. Available from:

_Novel_approach_to_bis-28indolyl-29methanes_De_novo_synthesis_of_1-hydroxyiminomethyl_ derivatives_with_anti-cancer_properties [accessed Apr 11, 2015].

The one-pot synthetic strategy to bis(indolyl)methanes is outlined in Scheme 3. The starting a,a 0-dihalogenooximes 3 were efficiently prepared from the respective ketones by known procedures [58,61]. These compounds, in the presence of base, were converted, in situ, into the corresponding transient and reactive nitrosoalkenes 4, which were intercepted bya first molecule of the appropriate indole 5 originating the intermediate indole oximes 6. The initially formed tetrahydroxazines undergo ring-opening to the corresponding oximes, under the driving force of the energy gain on rearomatisation. Subsequent dehydro-halogenation of 6 produces nitrosoalkenes 7 which reacted with a second molecule of indole, producing the target bis(indolyl)methanes 8. The results obtained are summarised in Table 1.

The reaction yields may be considered generally good, taking into account that the synthetic process involves a sequence of reactions. On the other hand, no other products could be obtained, which indicates that the reactions were regioselective. The results have shown also that both alkyl and aryl oximes can be used in the synthesis of bis(indolyl)methanes. Starting from aryl oximes 3aef the expected (E) oximes 9 were obtained as single or major products (Entries 1e11) whereas alkyl oxime 3g reacted with indole to give the (Z)-oxime 10g as the major product (Entries 12e13). The stereochemistry assignment of oximes 9 and 10 was confirmed by analysis of the NOESY spectra of 9d, 9g, 10d and 10g. In the spectra of 10d and 10g, connectivity was observed between the hydroxyl proton and the phenyl protons and the methyl protons, respectively, whereas in the case of 9d and 9g no connectivity was observed. Moreover, oximes 9 and 10 are also characterized by 1H NMR spectra with different features. The chemical shift of the methylenic proton appears at higher value for (E)-oximes 9 (9b: δ  6.81 ppm; 9d: δ  = 6.82 ppm; 9g: δ = 6.39 ppm) than for the corresponding (Z) oximes 10 (10b: δ = 5.74 ppm; 10d: δ = 5.77 ppm; 10g: δ = 5.41 ppm).

The synthesis of two isomeric oximes from the reaction of arylnitrosoethylenes with pyrrole and dipyrromethanes has been previously observed [62]. The process was rationalized considering the conjugate addition of the heterocycle to the nitrosoalkene, at the s-cis or s-trans conformation, followed by rearomatization of the pyrrole unit leading to (E)- and (Z)-oxime, respectively. Thus, the synthesis of the BIM oximes via 1,4-conjugate addition of indole to the nitrosoelkene cannot be ruled out.

The use of water as solvent in Diels- Alder reactions has been shown to be advantageous, not only in environmental terms but also inducing critical improvements in reaction times, yields and selectivity [51,63]. We observed that carrying out the synthesis of bis(indolyl)methanes in water using dichloromethane as co-solvent is a valuable alternative to the use of dichloromethane as the only solvent. Generally the yields were better or comparable to those obtained in dichloromethane and reaction time significantly shorter (the reaction time was reduced from 36 h to 3 h). Clearly the efficiency of the reaction, using H2O/CH2Cl2 system, amongst the nitrosoalkenes bearing halogenated aryl substituents increases in the order F > Cl > Br > H the order of electron withdrawing ability and consequently the order of the expected effectiveness for an inverse electron demand Diels-Alder reaction (entries 2, 5, 7 and 9). However, the isolated yields from the reaction carried out in CH2Cl2 do not reflect the expected reactivity, which can be explained considering differences in the efficiency of the purification process.

The cytotoxicity of compounds 9a, 9e and 9d was evaluated in different tumorl cell lines, namely HepG2 (hepatocellular carcinoma), MDA-MB-468 (human breast carcinoma), RAW 264.7 (murine leukemic monocyte macrophages), THP1 (human acute monocytic leukaemia), U937 (human leukaemic monocytic lymphoma) and EL4 cells (murine T-lymphoma). The compounds’ selectivity towards tumoural cells was assessed determining their cytotoxicity with respect to two non-tumoural derived cell lines S17 (murine bone marrow) and N9 cells (murine microglial). Results of the half maximal concentrations (IC50) are shown in Table 2 together with the toxicity of etoposide, a known antitumoural drug. Compound 9e was considerably less cytotoxic on tumor cell lines than the other two compounds, with IC50 values ranging from 35.7 (HepG2) to 124 mM (THP1) and was not selective. Compounds 9a and 9d, however, were considerably cytotoxic to all cells tested, with IC50 values ranging from 1.62 (THP1) to 23.9 mM (RAW) and from 10.7 (MDA) to 34.1 mM (U937), respectively. Compound 9a was particularly active against non-adherent cell lines with IC50 values ranging from 1.62 in THP1 to 1.65 mM in EL4.

Some conclusions regarding structure activity relationships can be redrawn based on the biological evaluation of these bis(indolyl)methanes. There is a dramatic difference in anticancer activitybetweenN-unsubstituted bis(indolyl)methanes 9a and the Nmethyl substituted derivative 9e, the latter characterized by high IC50 values. On the other hand, the significantly lower IC50 values observed for 9a for non-adherent cell lines in comparisonwith the ones obtained for 9d demonstrates that the presence of the bromo substituent leads to higher cytotoxic activity.

The observed high cytotoxicity of compound 9a against THP1, EL4 and U937 cell lines led us to extend the study to BIMs 9c, 9g and 10g (Table 3). Compound 9c, bearing a 4-fluorophenyl substituent, showed moderate anti-cancer activity which reinforces the observation that the 4-bromophenyl group is crucial to ensure low IC50 values. On the other hand, alkyl oximes 9g and 10g were even less cytotoxic against THP1, EL4 and U937 cell lines. None of these compounds were selective towards the tumor cell lines (selectivity index calculated for non-tumour cell line S17). In addition to having displayed higher toxicity towards the nontumor cell lines than all the studied compounds, compound 9a demonstrated the highest selectivity indexes: 9.86-14.2. Further studies using 9a as scaffold in the development of anti-tumoural drugs for leukaemia and lymphoma is worth pursuing since it presents lower IC50 and higher selectivity than etoposide.


The reliable preparation of a variety of unknown BIMs bearing different oxime substituents at the methylene bridge was presented. This strategy, supported on the robust and proved methodology of Diels-Alder cyclo addition reactions of electrophilic nitrosoalkenes with electron rich indoles, may pave the way for the synthesis of a vast library of new compounds.

Table 1 Preparation of bis(indolyl)methane oxime

Scheme 1. Selected biological active bis(indolyl)methanes.

Scheme 2. Common methods for BIMs’ preparation [27e44].

Scheme 3. Synthetic strategy towards BIM oximes.

Synthesis of a new bis(indolyl)methane that inhibits growth and induces apoptosis in human prostate cancer cells

Marrelli M., et al.
Natural product research 08/2013; 27(21).

The synthesis and the antiproliferative activity against the human breast MCF-7, SkBr3 and the prostate LNCaP cancer cell lines of a series of bis(indolyl)methane derivatives are reported. The synthesis of new compounds was first accomplished by the reaction of different indoles with trimethoxyacetophenone in the presence of catalytic amounts of hydrochloric acid. A second procedure involving the use of oxalic acid dihydrate [(CO2H)2·2H2O] and N-cetyl-N,N,N-trimethylammonium bromide in water was carried out and led to better yields. Compound 5b significantly reduced LNCaP prostate cancer cell viability in a dose-dependent manner, with an IC50 of 0.64 ± 0.09 μM. To determine whether the growth inhibition was associated with the induction of apoptosis, treated cells were stained using DAPI. LNCaP cells treated with 1 μM of 5b showed the morphological changes characteristic of apoptosis after 24 h of incubation.

11.1.5 Synthesis and Biological Evaluation of New 1,3-Thiazolidine-4-one Derivatives of 2-(4-Isobutylphenyl)propionic Acid molecules

Vasincu IM1Apotrosoaei M2Panzariu AT3Buron F4Routier S5Profire L6
Molecules. 2014 Sep 18; 19(9):15005-25

New thiazolidine-4-one derivatives of 2-(4-isobutylphenyl)propionic acid (ibuprofen) have been synthesized as potential anti-inflammatory drugs. The structure of the new compounds was proved using spectral methods (FR-IR, 1H-NMR, 13C-NMR, MS). The in vitro antioxidant potential of the synthesized compounds was evaluated according to the total antioxidant activity, the DPPH and ABTS radical scavenging assays. Reactive oxygen species (ROS) and free radicals are considered to be involved in many pathological events like diabetes mellitus, neurodegenerative diseases, cancer, infections and more recently, in inflammation. It is known that overproduction of free radicals may initiate and amplify the inflammatory process via upregulation of genes involved in the production of proinflammatory cytokines and adhesion molecules. The chemical modulation of acyl hydrazones of ibuprofen 3a–l through cyclization to the corresponding thiazolidine-4-ones 4a–n led to increased antioxidant potential, as all thiazolidine-4-ones were more active than their parent acyl hydrazones and also ibuprofen. The most active compounds are the thiazolidine-4-ones 4e, m, which showed the highest DPPH radical scavenging ability, their activity being comparable with vitamin E.

In order to improve the anti-inflammatory effect and safety profile of representative NSAIDs, one research strategy is derivatization of the carboxylic acid group with various heterocyclic systems (oxazole, izoxazole, pyrazole, oxadiazole, thiazole, thiadiazole, triazole, etc.) [9,10]. In the past two decades there has been considerable interest in the role of reactive oxygen species (ROS) in inflammation [11]. ROS mediate the oxidative degradation of cellular components and alteration of protease/antiprotease balance with damage to the corresponding tissue. In the early stages of the inflammatory process, ROS exert their actions through activation of nuclear factors, such as NFkB or AP-1, that induce the synthesis of cytokines. In later stages, endothelial cells are activated due to the synergy between free radicals and cytokines, promoting the synthesis of inflammatory mediators and adhesion of molecules. In the last step free radicals react with different cellular components (trypsin, collagen, LDL, DNA, lipids) inducing the death of cells [12,13].

The thiazolidine-4-one moiety is a heterocycle that has received more attention in the last years due its important biological properties [14]. Many effects have been found, including anti-inflammatory and analgesic [15], antitubercular [16], antimicrobial and antifungal [17], antiviral, especially as anti-HIV agents [18], anticancer, antioxidants [19], anticonvulsants [20] and antidiabetic activity [21]. In the present study, some new derivatives of ibuprofen that contain thiazolidine-4-one scaffolds were synthesized in order to obtain compounds with double effect—antioxidant and anti-inflammatory properties. The structures of the compounds were assigned based on their spectral data (FT-IR, 1H-NMR, 13C-NMR, MS) and the compounds were screened for their in vitro antioxidant potential.

The 1,3-thiazolidine-4-one derivatives 4am were synthesized in several steps using the method summarized in Scheme 1 and Table 1. First 2-(4-isobutylphenyl)propionic acid (ibuprofen, 1) was reacted with thionyl chloride, followed by treatment with dry ethanol to get 2-(4-isobutylphenyl)propionic acid ethyl ester, which was turned in 2-(4-isobutylphenyl)propionic acid hydrazide (2) by reaction with 66% hydrazine hydrate [22]. The condensation of compound 2 with various aromatic aldehydes allowed the preparation of the corresponding hydrazone derivatives 3al in satisfactory yields. Finally, the hydrazone derivatives of ibuprofen upon reaction with mercaptoacetic acid led to the thiazolidine-4-one derivatives 4al in moderate to good yields. By reduction of compound 4g in presence of tin chloride and few drops of acetic acid in ethanol, the thiazolidine-4-one 4m was obtained in 90% yield. Acetylation of 4m with acetyl chloride gave thiazolidine-4-one 4n in moderate yield.

In the acyl hydrazone series most of the the tested compounds showed a radical scavenging ability comparable with ibuprofen (Table 4). The most active compounds were 3e and 3f which are about three times and two times more active than their parent compound, respectively. The scavenging ability of the acyl hydrazones was improved by cyclization to the corresponding thiazolidine-4-one derivatives, these compounds all being more active than ibuprofen, except for compound 4j which contains a CF3 group in the metaposition of phenyl ring (Table 5). The most active compounds were 4e and 4m which contain NO2 and NH2 groups in ortho and paraposition of the phenyl ring, respectively. For these compounds the radical scavenging ability (%) was 94.42 ± 0.43 and 94.88 ± 0.57, which means that the compounds are about 23 times more active than ibuprofen (4.15 ± 0.22). The activity of these compounds is comparable with that of vitamin E used as positive control. Important radical scavenging ability was also shown by compound 4b(81.31 ± 0.55), which contains a Cl group in the para position of the phenyl ring, the compound being 20 times more active than ibuprofen.

The acyl hydrazone derivatives showed an antioxidant activity comparable with ibuprofen. The most active compound in this series was 3h, with radical scavenging activity of 13.31 ± 0.81, which means that this compound is three times more active than ibuprofen (4.42 ± 0.18). In the thiazolidine-4-one series the most active compounds were 4b4e and 4k, which contain Cl(4), NO2(2) and CN(4), respectively, as substituents on the phenyl ring. These compounds, which showed a scavenging ability of around 50%, are 12 times more active than ibuprofen. In comparison with the corresponding acyl hydrazones 3b3e and 3k the thiazolidine-4-ones were 10 times (4b), seven times (4e) and 13 times (3k) more active. The improved antiradical activity of acyl hydrazones by cyclization to form thiazolidine-4-ones was also observed for compounds 3d3f and 3g. The most favorable influence was observed for acyl hydrazone 4g, which contains a NO2 in the para position of the phenyl ring. The corresponding thiazolidine-4-one (4g, 37.14 ± 1.10) is 22 times more active than 3g (1.67 ± 0.35). These data strongly support the favorable influence of the thiazolidine-4-one ring on the antioxidant potential of these compounds. The tested compounds were less active than vitamin E.

In this study new heterocyclic compounds that combine the thiazolidine-4-one structure with the arylpropionic acid one have been synthesized. The structure of the new compounds was proved using spectral methods (IR, 1H-NMR, 13C-NMR, MS). The compounds were evaluated for their antioxidant effects using in vitro assays: total antioxidant activity, DPPH and ABTS radical scavenging ability. All thiazolidin-4-one derivatives 4an showed improved antioxidant effects in comparison with the corresponding acyl hydrazones 3al and ibuprofen, the parent compound. The encouraging preliminary results illustrate the antioxidant potential of the synthesized compounds and motivate our next research focused on their anti-inflammatory effects in chronic and acute inflammation models.

11.1.6 Targeting pyruvate kinase M2 contributes to radiosensitivity of NSCLC cells

Meng MB1Wang HH2Guo WH3Wu ZQ2Zeng XL2Zaorsky NG4, et al.
Cancer Lett. 2015 Jan 28; 356(2 Pt B):985-93

Aerobic glycolysis, a metabolic hallmark of cancer, is associated with radioresistance in non-small cell lung cancer (NSCLC). Pyruvate kinase M2 isoform (PKM2), a key regulator of glycolysis, is expressed exclusively in cancers. However, the impact of PKM2 silencing on the radiosensitivity of NSCLC has not been explored. Here, we show a plasmid of shRNA-PKM2 for expressing a short hairpin RNA targeting PKM2 (pshRNA-PKM2) and demonstrate that treatment with pshRNA-PKM2 effectively inhibits PKM2 expression in NSCLC cell lines and xenografts. Silencing of PKM2 expression enhanced ionizing radiation (IR)-induced apoptosis and autophagy in vitro and in vivo, accompanied by inhibiting AKT and PDK1 phosphorylation, but enhanced ERK and GSK3β phosphorylation. These results demonstrated that knockdown of PKM2 expression enhances the radiosensitivity of NSCLC cell lines and xenografts as well as may aid in the design of new therapies for the treatment of NSCLC.

Knockdown of PKM2 expression increases the sensitivity of NSCLC cells to radiotherapy in vitro

To examine PKM2 expressions levels in the normal lung epithelial cell and the NSCLC cell lines, we evaluated the expression levels of PKM2 in normal lung bronchial epithelial cell BEAS-2B and five NSCLC cell lines including A549, H460, H1299, H292, and H520 by Western blotting assays, and our results demonstrated that PKM2 expression was elevated in almost five NSCLC cell lines examined compared to autologous normal lung bronchial epithelial cell, although the expression levels fluctuated slightly depending on the different cell lines (Fig.1A). To test the role of PKM2 in the sensitivity of NSCLC to radiotherapy, we generated plasmids of pshRNA-PKM2 and control pshRNA-Con by inserting the DNA fragment for a pshRNA specifically targeting the PKM2 or control into the pGenesil2 vector. After demonstrating the authenticity, A549 and H460 cells were transfected with the plasmid for 48h and the levels of PKM2 expression were tested by Western blot assays. Obviously, transfection with control plasmid did not significantly modulate PKM2 expression; while transfection with pshRNA-PKM2 reduced the levels of PKM2 expression (Fig.1B and Appendix: Supplementary Fig.S1A). Quantitative analysis revealed that transfection with pshRNA-PKM2 significantly reduced PKM2 expressions as compared with that in the mock-treated and control pshRNA-Con plasmid-transfected cells, respectively (p<0.05, Fig.1C). Mock-treated and pshRNA-PKM2-trasnfected A549 and H460 cells were subjected to IR (4Gy), and 12 and 24h after IR, these cells, together with un-irradiated mock-treated, pshRNA-Con-transfected, and pshRNA-PKM2-trasnfected cells, were tested for cell viability by trypan blue staining. Knockdown of PKM2 reduced the percentage of A549 viable cells by 12.6–20% and IR treatment decreased the frequency of viable cells by 17.1–28.2%. However, the percentages of viable cells in the PKM2-silencing and irradiated cells were reduced by 27.7–48.7%, which were significantly lower than that in other groups (Fig.1D, p<0.05). Furthermore, it was consistent with the above results of A549 cells that knockdown of PKM2 significantly reduced the percentage of H460 viable cells (Appendix: Supplementary Fig.S1B). In addition, to further validate PKM2 silencing on their radiosensitivity,unirradiated control, mock-treated, and pshRNA-PKM2 transfected A549 cells were subjected to IR (0, 2, 4, 6, and 8Gy), and two weeks after IR, these cells were tested for the capacity for colony formation. The results showed that the numbers of colonies formed by pshRNA-PKM2 cells were significantly decreased compared with that of mock-treated and control cells; however, there was no significant change in mock-treated cells compared with control cells. These results suggested that pshRNA-PKM2 cells were more sensitive to IR than mock-treated and control cells (Fig.1E and F). Given that IR usually causes DNA double-strand breaks [28], we characterized the frequency of γ-H2AX nuclear foci positive cells by immunofluorescent assays. While IR treatment dramatically increased the frequency of γ-H2AX+ cells, the same dose of IR further significantly increased the percentages of γ-H2AX+ cells when combined with PKM2 silencing at 12 and 24h after IR, and there was a significant difference in γ-H2AX+ cells between these two groups at 12 and 24 h after IR (Fig. 1G and H, p < 0.05).

Fig. 1. The PKM2 expression levels in the normal lung epithelial cell and the NSCLC cell lines and knockdown of PKM2 expression enhance the radiosensitivity of A549 cells in vitro. The expression levels of PKM2 in normal lung bronchial epithelial cell BEAS-2B and five NSCLC cell lines including A549, H460, H1299, H292, and H520 were determined by Western blotting assay (A). A549 cells were transfected with pshRNA-PKM2 or pshRNA-Con plasmid for 48h, and the levels of PKM2 expression were determined by Western blot assays using a PKM2-specific antibody and β-actin as an internal control (B and C). Data are representative images or expressed as mean±SD of the relative levels of PKM2 to control β-actin in individual groups of cells from three separate experiments. # p

Knockdown of PKM2 enhances IR-induced apoptosis in NSCLC cells

Next, we tested the impact of PKM2-silencing on IR-induced cell death types. One day after IR, the apoptotic cells in the irradiatedmock-treated,pshRNA-PKM2-trasnfected cells, and one group of cells that had been pre-treated with 30μM Z-VAD for 1h prior to IR, together with mock-treated, unirradiated pshRNA-Contransfected, and pshRNA-PKM2-trasnfected groups of cells were characterized by TUNEL assays and/or FACS analysis (Fig.2A and C). In comparison with that in mock-treated and control plasmid transfected cells, the frequency of apoptotic cells in the PKM2 silencing or IR-treated cells increased moderately, while the percentages of apoptotic cells in the cells receiving combined treatment with IR and PKM2-silencing were significantly greater. However, the frequency of apoptotic cells in the Z-VAD-pretreated cells was partially reduced. Apparently, knockdown of PKM2 and IR induced apoptosis in NSCLC cells in vitro (Fig. 2B and D, and Appendix: Supplementary Fig.S1C).

Fig. 2. Knockdown of PKM2 expression enhances IR-induced apoptosis in A549 cells. A549 cells were transfected with, or without, pshRNA-Con or pshRNA-PKM2 for 48h and treated with, or without, Z-VAD for 1h. Subsequently, the cells were subjected to IR, and 24h later, the frequency of apoptotic cells was determined by TUNEL assays and FACS. (A and C) TUNEL and FACS analyses of apoptotic cells. (B and D) Quantitative analysis of the percentage of apoptotic cells. Data are representative images or expressed as mean%±SD of individual groups of cells from three independent experiments. * p

Knockdown of PKM2 enhances IR-induced autophagy in NSCLC cells

The cell autophagy is characterized by the formation of numerous autophagic vacuoles, autophagosome, in the cytoplasm and elevated levels of the microtubule-associated protein 1 light chain 3 (LC3)-II [29]. To test the impact of PKM2 silencing on IR-induced autophagy, the presence of autophagosome in mock-treated, pshRNACon-transfected, pshRNA-PKM2-transfected, IR-treated alone, IR + pshRNA-PKM2-transfected, and 1 mM 3-MA-pretreated IR + pshRNA-PKM2-transfected cells was characterized by electronic microphotography (EM). Intriguingly and importantly, numerous autophagosomes were detected in the IR + pshRNAPKM2-transfected cells, and only a few were detected in the sensitivity of the NSCLC cells to radiotherapy in vitro. It was noted that pshRNA-Con had almost no effect on A549 cells, therefore, some subsequently experiments did not set this group.

Fig. 3. Knockdown of PKM2 and IR induce A549 cell autophagy. A549 cells were transfected with, or without, pshRNA-Con or pshRNA-PKM2 for 48h and treated with, or without, 3-MA for 1h. Subsequently, the cells were subjected to IR, and 2h later, the presence of autophagic vacuoles and autolysosomes in A549 cells was determined by EM and the relative levels of LC3-I, LC3-II, AKT, ERK1/2, and control β-actin expression and AKT, ERK1/2, GSK3β, PDK1 phosphorylation were determined by Western blot assays using specific antibodies. Data are representative images and expressed as mean values of the relative levels of target protein to control in individual groups of cells from three separate experiments. The relative levels of target protein to control in mock-treated cells were designated as 1. (A) EM analysis of autophagic vacuoles and autophagosomes. Black arrows point to autophagic vacuoles and autophagosomes in the cytoplasma of A549 cells. (B) Western blot analysis of LC3-I and LC3-II expression. The values indicate the ratios of the relative levels of LC3-II to LC3-I in individual groups. (C) Western blotting analysis of individual signal events. The values indicate the relative levels of target protein to control β-actin in individual groups of cell

Fig. 4. The impact of 3-MA or/and V-ZAD on cell viability, colony formation, apoptosis and autophagy in A549 cells. A549 cells were transfected with, or without, pshRNACon or pshRNA-PKM2 for 48h and pre-treated with, or without, 3-MA or V-ZAD for 1h, respectively. Subsequently, the cells were subjected to IR. Twenty-four hours later and two weeks, the viability, apoptosis, and colony formation were determined. Two hours after treatment, autophagy and the relative levels of LC3-I and LC3-II expression in different groups of cells were determined. Data are representative images and expressed as mean%±SD of individual groups of cells from three separate experiments. (A) The percentages of viable cells. (B) The capacity of cell colony formation. (C) Quantitative analysis of apoptotic cells. (D) Western blot analysis of LC3-I and LC3-II expression. The values indicate the ratios of LC3-II to LC3-I in individual groups of cells. * p

Fig. 5. Treatment with pshRNA-PKM2 enhances the IR-inhibited growth of implanted tumors in mice. The nude mice were inoculated with A549 cells and when the tumor grew at 50mm3 in one dimension, the mice were randomized and treated with vehicle (PS), plasmid of pshRNA-Con or pshRNA-PKM2 alone or IR (4Gy×7f) alone or in combination with pshRNA-PKM2 and IR, respectively. The body weights and tumor growths of individual mice were monitored longitudinally. At the end of the in vivo experiment, the tumor tissues were dissected out and the frequency of apoptotic cells, the presence of autophagosomes and the expression of PKM2 were determined by TUNEL, EM and immunohistochemistry, respectively. Data are representative images or expressed as mean±SD of individual groups of mice (n=6 per group). (A) The body weights of mice. (B and C) The tumor growth curve of implanted tumors and the log-transformed tumor growth curve of implanted tumors in mice. (D) Quantitative analysis of the frequency of apoptotic cells.(E) EM analysis of autophagy. (F)The expression of PKM2.(G) Quantitative analysis of PKM2 expression.The cells with brown cytoplasma were considered as positive anti-PKM2 staining and the percentage of PKM2-positive cells was obtained by dividing the numbers of the PKM2-positive cells by the total number of cancer cells in the same field.

11.1.7 The tyrosine kinase inhibitor nilotinib has antineoplastic activity in prostate cancer cells but up-regulates the ERK survival signal—Implications for targeted therapies

Schneider M1Korzeniewski N2Merkle K2Schüler J, et al.
Urol Oncol. 2015 Feb; 33(2):72.e1-7

Background: Novel therapeutic options beyond hormone ablation and chemotherapy are urgently needed for patients with advanced prostate cancer. Tyrosine kinase inhibitors (TKIs) are an attractive option as advanced prostate cancers show a highly altered phosphotyrosine proteome. However, despite favorable initial clinical results, the combination of the TKI dasatinib with docetaxel did not result in improved patient survival for reasons that are not known in detail. Methods: The National Cancer Institute-Approved Oncology Drug Set II was used in a phenotypic drug screen to identify novel compounds with antineoplastic activity in prostate cancer cells. Validation experiments were carried out in vitro and in vivo. Results: We identified the TKI nilotinib as a novel compound with antineoplastic activity in hormone-refractory prostate cancer cells. However, further analyses revealed that treatment with nilotinib was associated with a significant up-regulation of the phospho-extracellular-signal-regulated kinases (ERK) survival signal. ERK blockade alone led to a significant antitumoral effect and enhanced the cytotoxicity of nilotinib when used in combination. Conclusions: Our findings underscore that TKIs, such as nilotinib, have antitumoral activity in prostate cancer cells but that survival signals, such as ERK up-regulation, may mitigate their effectiveness. ERK blockade alone or in combination with TKIs may represent a promising therapeutic strategy in advanced prostate cancer.

Identification of nilotinib as a novel antineoplastic compound in prostate cancer cells

Using the NCI-Approved Oncology Drug Panel II for a phenotypic drug screen of normal prostate epithelial cells and prostate cancer cell lines (Fig. 1) [7], we identified the TKI nilotinib as a positive hit in hormone-refractory DU-145 prostate cancer cells.

Fig. 1. Discovery of nilotinib as a novel antineoplastic agent in prostate cancer cells using a phenotypic drug screen. Overview of the drug screen procedure (see text for details).

Results were confirmed using annexin V staining, which showed a significant induction of apoptosis beginning at 24 hours (Fig. 2A). The IC50 of nilotinib against DU-145 cells was determined at 10 μM using an MTT cell viability assay (Fig. 2B). Immunoblot experiments confirmed an induction of apoptosis using PARP cleavage in DU-145 cells and in hormonerefractory PC-3 prostate cancer cells at this drug concentration (Fig. 2C). An onset of apoptosis at 24 hours was likewise confirmed using PARP cleavage at a nilotinib concentration of 10 μM(Fig. 2D). PWR-1E prostate epithelial cells and hormone-sensitive prostate LNCaP prostate cancer cells were not found to undergo enhanced apoptosis when treated with nilotinib (not shown).

Fig. 2. Antitumoral effects of nilotinib in prostate cancer cells: (A) flow cytometric analysis of DU-145 prostate cancer cells for annexin V to detect apoptotic cells after treatment with 10 μM of nilotinib for the indicated intervals; (B) cell viability (MTT) assay to determine the IC50 of nilotinib in DU-145 cells (24-h treatment); (C and D) immunoblot analysis of DU-145 and PC-3 prostate cancer cells for PARP cleavage (arrow) at nilotinib concentrations and time intervals as indicated. GAPDH is shown for protein loading; and (E) colony growth assay of DU-145 cells after drug treatment and washout as shown. Cells grown in 60-mm dishes were stained with crystal violet to visualize viable cells at the time points indicated. (Color version of figure is available online.

To further confirm the effect of nilotinib on prostate cancer cell growth, we performed a colony growth assay in which DU-145 cells were treated with nilotinib for 72 hours followed by a washout of the drug and continued culture for additional 9 days (Fig. 2E). We found that nilotinib induced significant cytotoxicity after 72 hours and that a minor regrowth of cancer cells did not occur until 6 to 9 days after the washout, which is comparable to other TKIs [8]. Next, we sought to identify the targets of nilotinib in DU-145 prostate cancer cells. Overall, 5 well-established targets, including ABL1, KIT, PDGFRA, DDR1, and NQO2, were analyzed for their role in the drug response. We found that protein expression of 3 of these targets (ABL1, KIT, and PDGFRA) was not detectable in DU-145 cells and that small interfering RNA–mediated knockdown of the remaining 2 targets, DDR1 and NQO2, did not result in apoptosis (not shown). Collectively, these results show a significant antitumoral activity of nilotinib in prostate cancer cells. However, this effect was associated with a relatively high IC50 and was independent of known nilotinib targets.

Nilotinib up-regulates the ERK survival signal in prostate cancer cells

To further investigate why relatively high concentrations of nilotinib were required to induce cytotoxicity, we analyzed 40,6-diamidino-2-phenylindole–stained DU-145 cells treated with 10 μM of nilotinib for 24 hours using fluorescence microscopy (Fig. 3A).

Fig. 3. Nilotinib up-regulates the ERK survival signal in prostate cancer cells. (A) Fluorescence microscopic analysis of DAPI-stained DU-145 cells. (B and C) Immunoblot analyses of DU-145 cells (B) or DU-145 cells in comparison with LNCaP and PC-3 cells (C) treated with nilotinib for the expression of phospho-ERK1/2 T202/Y204 and total ERK. Immunoblot for GAPDH is shown as a loading control. (D) Immunohistochemical staining of xenografted DU-145 cells after 21 days of treatment with 75 mg/kg/d of nilotinib for phospho-ERK1/2 T202/Y204 expression. It can be noted that tumors explanted from vehicle-treated mice showed mostly positivity at the tumor periphery, whereas tumors explanted from nilotinib-treated mice showed a more evenly distributed phospho-ERK immunostaining (left panels). Quantification of phospho-ERK–positive DU-145 xenografts explanted after 21 days of treatment. Mean and standard errors of positive cells per high-power field (HPF; [1]40) from at least 3 tumors are given (right panel). (E) Immunoblot analysis of DU-145 cells treated with U0126 alone or in combination with nilotinib shows abrogation of phospho-ERK1/2 T202/Y204 expression by U0126. (F) Quantification of viable cells compared with that of controls using the MTT assay after treatment with U0126 (10 μM) or nilotinib (10 μM) or both and after either pretreatment (24 h) or simultaneous treatment (72 h). DAPI ¼ 40,6-diamidino-2-phenylindole. (Color version of figure is available online.)

We found that, despite the presence of apoptotic cells, there was also a population of actively dividing tumor cells in the presence of nilotinib as well as a population of viable but multinucleated cells (Fig. 3A). We interpreted these results as evidence that a subset of tumor cells has the ability to resist TKI treatment. To reconcile these results, we analyzed the activation of ERK1/2, which is known to function as a prosurvival signal in TKI-treated tumor cells [9,10]. We detected a robust overexpression of phospho-ERK1/2 T202/Y204 in nilotinib-treated DU-145 cells (Fig. 3B). An up-regulation of phospho-ERK1/2 T202/Y204 was also detectable in nilotinib-treated LNCaP cells, albeit at a lower level, and was not found in PC-3 cells (Fig. 3C). To further corroborate the evidence of phospho-ERK upregulation in vivo, we analyzed explanted DU-145 xenografts from a representative experiment in which nilotinib was used at a 75-mg/kg/d concentration. This initial dosage was based on published animal experiments [11] but yielded no or incomplete tumor control in our experiment (data not shown).

In vivo antitumoral activity of nilotinib and ERK blockade

Our results raised 2 important questions First, can a higher dose of nilotinib induce improved tumor control, and second, is a combination of nilotinib with the MEK inhibitor U0126 to block ERK activity superior to nilotinib alone?

Fig. 4. In vivo antitumoral activity of nilotinib and ERK blockade in prostate cancer cells: (A) tumor growth curves of DU-145 xenografts in NMRI-nude mice and (B) analysis of tumor volumes on day 21. Asterisks indicate statistical significance (**P r 0.01 and ***P r 0.001). (Color version of figure is available online.)

11.1.8 PAF and EZH2 Induce Wnt.β-Catenin Signaling Hyperactivation

Jung HY1Jun SLee MKim HCWang XJi HMcCrea PDPark JI
Mol Cell. 2013 Oct 24; 52(2):193-205

Fine-control of Wnt signaling is essential for various cellular and developmental decision making processes. However, deregulation of Wnt signaling leads to pathological consequences including cancer. Here, we identify a novel function of PAF, a component of translesion DNA synthesis, in modulating Wnt signaling. PAF is specifically overexpressed in colon cancer cells and intestinal stem cells, and required for colon cancer cell proliferation. In Xenopus laevis, ventrovegetal expression of PAF hyperactivates Wnt signaling, developing secondary axis with β-catenin target gene upregulation. Upon Wnt signaling activation, PAF is dissociated from PCNA, and directly binds to β-catenin. Then, PAF recruits EZH2 to β-catenin transcriptional complex, and specifically enhances Wnt target gene transactivation, independently of EZH2’s methyltransferase activity. In mouse, conditional expression of PAF induces intestinal neoplasia via Wnt signaling hyperactivation. Our studies reveal an unexpected role of PAF in regulating Wnt signaling, and propose a novel regulatory mechanism of Wnt signaling during tumorigenesis. Fine-control of Wnt signaling is essential for various cellular and developmental decision making processes. However, deregulation of Wnt signaling leads to pathological consequences including cancer. Here, we identify a novel function of PAF, a component of translesion DNA synthesis, in modulating Wnt signaling. PAF is specifically overexpressed in colon cancer cells and intestinal stem cells, and required for colon cancer cell proliferation. In Xenopus laevis, ventrovegetal expression of PAF hyperactivates Wnt signaling, developing secondary axis with β-catenin target gene upregulation. Upon Wnt signaling activation, PAF is dissociated from PCNA, and directly binds to β-catenin. Then, PAF recruits EZH2 to β-catenin transcriptional complex, and specifically enhances Wnt target gene transactivation, independently of EZH2’s methyltransferase activity. In mouse, conditional expression of PAF induces intestinal neoplasia via Wnt signaling hyperactivation. Our studies reveal an unexpected role of PAF in regulating Wnt signaling, and propose a novel regulatory mechanism of Wnt signaling during tumorigenesis.

Keywords: Wnt, β-catenin, PAF, KIAA0101, EZH2

Strict regulation of stem cell proliferation and differentiation is required for mammalian tissue homeostasis, and its repair in the setting of tissue damage. These processes are precisely orchestrated by various developmental signaling pathways, with dysregulation contributing to disease and genetic disorders, including cancer (Beachy et al., 2004). Cancer is initiated by the inactivation of tumor suppressor genes and activation of oncogenes. For instance, colon cancer cells harbor genetic mutations in Wnt/β-catenin pathway constituents such as adenomatous polyposis coli (APC), Axin, and β-catenin (Polakis, 2007). In mouse models, inactivation of APC or activation of β-catenin results in the development of intestinal hyperplasia and adenocarcinoma (Moser et al., 1990), indicating that hyperactivation of Wnt signaling promotes intestinal tumorigenesis.

In canonical Wnt signaling, Wnt ligand induces stabilization of β-catenin protein via inhibition of the protein destruction complex (glycogen synthase kinase 3, APC, casein kinase I, and Axin). Then, activated β-catenin is translocated into the nucleus and binds to its nuclear interacting partners, TCF/LEF. Finally, β-catenin-TCF/LEF transactivates the expression of its target genes (Clevers and Nusse, 2012).

Although various Wnt/β-catenin modulators have been identified (Wnt homepage; wnt.stanford.edu), the pathological relevance of these modulators to tumorigenesis remains elusive. Also, many reports have suggested that mutation-driven Wnt signaling activation can be enhanced further (Goentoro and Kirschner, 2009He et al., 2005Suzuki et al., 2004Vermeulen et al., 2010), which implies the presence of an additional layer of Wnt-signaling regulation in cancer beyond genetic mutations in APC or β-catenin. Here, we unraveled a novel function of the DNA repair gene, PAF (PCNA-associated factor) /KIAA0101). PAF was shown to be involved in translesion DNA synthesis (TLS), an error-prone DNA repair process that permits DNA replication machinery to replicate DNA lesions with specialized translesion DNA polymerase (Emanuele et al., 2011Povlsen et al., 2012Sale et al., 2012). Our comprehensive approaches uncover that cancer-specifically expressed PAF hyperactivates Wnt/β-catenin signaling and induces intestinal tumorigenesis.

Mitogenic role of PAF via Wnt signaling

To identify colon cancer-specific Wnt signaling regulators, we analyzed multiple sets of human colon cancer tissue samples using the publicly available database (www.oncomine.org), and selected genes that are highly expressed in colon cancer cells (fold change > 2; P < 0.0001; top 10% ranked). Among several genes, we investigated the biological role of PAF, based on its significant overexpression in human colon adenocarcinoma with correlated expression of Axin2, a well-established specific target gene of β-catenin (Jho et al., 2002Lustig et al., 2002)(Figure 1A). To validate our in silico analysis, we performed immunostaining of colon cancer tissue microarray, and confirmed that PAF was highly expressed in colon cancer cells, whereas its expression was barely detectable in normal intestine (Figure 1B). Consistently, PAF was strongly expressed and mainly localized in the nucleus of colon cancer cell lines (Figure 1C). Additionally, we found that PAF was not expressed in non-transformed cells such as NIH3T3, mouse embryonic fibroblasts, and mammary epithelial cells (data not shown). Next, to assess the relevance of PAF upregulation in colon cancer cell proliferation, we depleted endogenous PAF using short hairpin RNAs (shRNAs) in these cell lines. Intriguingly, PAF knockdown (sh-PAF) inhibited colon cancer cell proliferation (Figures 1D and 1E). Given that PAF was shown to interact with PCNA via PIP box (Yu et al., 2001), we also examined whether PAF-PCNA interaction is required for mitogenic effects of PAF. In reconstitution experiments, sh-PAF-induced cell growth inhibition was rescued by ectopic expression of both shRNA non-targetable wild-type PAF (nt-PAF) and PIP mutant PAF (mutPIP-PAF) (Figure 1F), indicating that the PAF-PCNA interaction is not necessary for PAF-mediated colon cancer cell proliferation. Interestingly, PAF knockdown downregulated cell proliferation–related genes (Cyclin D1 and c-Myc) (Figure 1G). Given that Cyclin D1 and c-Myc are β-catenin direct target genes (He et al., 1998Tetsu and McCormick, 1999), PAF likely participates in regulating Wnt/β-catenin signaling. Interestingly, PAF depletion-induced downregulation of Cyclin D1 andc-Myc was only observed in SW620 colon cancer cells, but not in Panc-1 and MDA-MB-231 cells (Figure 1G), indicating the specific effects of PAF on Cyclin D1 and c-Myc expression in colon cancer cells. We also assessed the effects of PAF knockdown on Axin2. Indeed, PAF knockdown suppressed Axin2transcription in colon cancer cells (Figure 1H). Moreover, as nt-PAF did, β-catenin ectopic expression reverted sh-PAF–induced cell growth arrest (Figure 1I), implying that PAF might be functionally associated with Wnt/β-catenin. We also examined whether other mitogenic signaling pathways mediate PAF’s mitogenic role. Of note, except HT29, other colon cancer cell lines (SW620, HCT116, HCC2998, and HCT15) harbor oncogenic mutations in K-Ras gene. Nonetheless, PAF depletion induced the suppression of cell growth on all five colon cancer cells (Figure 1D), indicating that PAF’s mitogenic function is independent of Ras/MAPK signaling activation. Additionally, overexpression of wild-type Akt or constitutively active form of Akt (myristoylated form of Akt [Myr-Akt]) did not rescue sh-PAF-induced inhibition of cell proliferation (Figure 1I). Moreover, β-catenin activation did not revert cell proliferation suppression resulted from MAPK or PI3K inhibition (Figure 1J), indicating that β-catenin-mediated mitogenic function is independent of MAPK and PI3K signaling pathways. These results suggest that PAF contributes to colon cancer cell proliferation, possibly via Wnt/β-catenin signaling.


Figure 1 Mitogenic role of PAF in colon cancer cells

PAF positively modulates Wnt signaling

Given that many cancers develop as a result of deregulation of developmental signalings (Beachy et al., 2004), analyzing PAF expression during development may provide insights into the mechanisms of PAF-mediated signaling regulation. Whole mount immunostaining of mouse embryos, showed that PAF was specifically enriched in the apical ectodermal ridge (AER) of the limb bud, midbrain, hindbrain, and somites (Figure 2A and data not shown). During limb development, AER induction is specifically coordinated by active Wnt signaling (Figure 2B)(Kengaku et al., 1998). Using, Axin2-LacZ, a β-catenin reporter (Lustig et al., 2002), mouse embryos, we confirmed the specific activation of Wnt signaling in AER (Figure 2C). Intriguingly, Wnt signaling activity as exhibited in the AER, overlapped with the pattern of PAF expression (Figures 2A and 2C). Given that (1) Wnt signaling is deregulated in most colon cancer, (2) PAF is highly overexpressed in colon cancer cells, (3) PAF is required for colon cancer cell proliferation (Figure 1D), and (4) PAF is enriched in AER where Wnt signaling is active (Figure 2A), we hypothesized that PAF modulates the Wnt signaling pathway. To test this, we first examined the impact of PAF on β-catenin transcriptional activity using TOPFLASH reporter assays. In HeLa cells, PAF knockdown decreased β-catenin reporter activation by 6-bromoindirubin-3′-oxime, a GSK3 inhibitor (Figure 2D). Similarly, Wnt3A-induced transcriptional activation of Axin2 was also inhibited by PAF depletion (Figure 2E). These data suggest that PAF might be required for Wnt target gene expression.


Figure 2 Activation of Wnt signaling by PAF

To gain better insight of PAF’s role in Wnt signaling regulation, we utilized Xenopus laevis embryos for axis duplication assays (Funayama et al., 1995), as previously performed (Park et al., 2009). Because of Wnt signaling’s pivotal role in vertebrate anterior-posterior axis development, the effects of Xenopus PAF (xPAF) on Wnt signaling can be monitored and quantified on the basis of secondary axis formation following injection of in vitro transcribed mRNAs. xβ-catenin mRNA, titrated to a subphenotypic level when expressed in isolation, was co-injected with xPAF mRNA into ventrovegetal blastomeres. Unlike the controls (β-catenin and β-galactosidase mRNA), the experimental group (β-catenin and xPAF mRNA) displayed axis-duplications (Figures 2F-H). Of note, the ventrovegetal injection of xPAF mRNA alone failed to induce secondary axes (data not shown), showing that PAF hyperactivates Wnt/β-catenin signaling only in the presence of active β-catenin. Consistent with the results of axis duplication assays, qRT-PCR assays showed that xPAF expression upregulated expression of Siamois and Xnr3, β-catenin targets in frogs (Figure 2I). Furthermore, we examined the specificity of PAF on Wnt/β-catenin signaling activity, using various luciferase assays. Ectopic expression of PAF hyperactivates Wnt3A or LiCl, a GSK3 inhibitor, -induced activation of β-catenin target gene reporter activity (MegaTOPFLASH, Siamoisc-Myc, and Cyclin D1). Of note, BMP/Smad pathway also plays an essential role in the developing limb AER (Ahn et al., 2001). However, PAF knockdown or overexpression did not affect BMP/Smad or FoxO signalings, respectively, (Figure 2J) indicating the specificity of PAF in regulating Wnt signaling. These results suggest that PAF positively modulates Wnt/β-catenin signaling in vitro and in vivo.

PAF-EZH2-β-catenin transcriptional complex formation

Next, we investigated the molecular mechanism underlying PAF hyperactivation of Wnt signaling. Given that stabilization of β-catenin protein is a key process in transducing Wnt signaling, we asked whether PAF affects β-catenin protein level. However, we found that the level of β-catenin protein was not altered by PAF knockdown or overexpression (Figures 2E and ​and3A),3A), leading us to test whether PAF controls the β-catenin/TCF transcriptional complex activity. Owing to the nuclear specific localization of PAF in colon cancer cells (Figure 1C), we tested whether PAF interacts with β-catenin transcriptional complex. Using a glutathione S-transferase (GST) pull-down assay, we found that PAF bound to β-catenin and TCF proteins (Figure 3B). Also, endogenous PAF interacted with β-catenin and TCF3 in SW620 cells that display constitutive hyperactivation of Wnt signaling by APC mutation (Figure 3C). Moreover, binding domain mapping assays showed that the Armadillo repeat domain of β-catenin was essential for its interaction with PAF (Figure 3D). Although PAF is a cell cycle-regulated anaphase-promoting complex substrate (Emanuele et al., 2011), PAF-β-catenin interaction was not affected (Figure S1). These data suggest that PAF directly binds to β-catenin transcriptional complex and this interaction is independent of cell cycle. Next, due to interaction of PAF with β-catenin and TCF, we tested whether PAF is also associated with Wnt/β-catenin target genes. First, we analyzed the subnuclear localization of PAF by chromatin fractionation. We found that PAF was only detected in the chromatin fraction of HCT116 cells (Figure 3E). Additionally, chromatin immunoprecipitation (ChIP) assays showed that both ectopically expressed and endogenous PAF occupied the TCF-binding element (TBE)-containing proximal promoter of the β-catenin targets (c-Myc and Cyclin D1) in HCT116 cells (Figures 3F and 3G). These data show that PAF is specifically associated with the promoters of Wnt/β-catenin target genes.


Figure 3 PAF-EZH2-β-catenin transcriptional complex at target gene promoters

In intestine, Wnt/β-catenin signaling constitutively activates intestinal stem cells (ISCs) to give rise to progenitor cells, which replenishes intestinal epithelium (Figure 3H). Given the involvement of PAF on Wnt/β-catenin signaling regulation (Figure 2), we analyzed the spatial expression of PAF in intestinal epithelium. Immunostaining showed that PAF was specifically expressed in B lymphoma Mo-MLV insertion region 1 homolog (Bmi1) positive intestinal stem cells (ISCs)(Figures 3I and 3J). Bmi1 and its associated components in Polycomb-repressive complex 1 (PRC1) and 2 (PRC2) are shown to epigenetically regulate gene expression (Sparmann and van Lohuizen, 2006). Due to (1) specific association of PAF with TBEs of β-catenin target promoters (Figures 3F and 3G) and (2) co-localization with Bmi1 positive ISCs (Figure 3J), we asked whether PAF is associated with components of PRC1 and PRC2, using co-immunoprecipitation (co-IP) assays. Intriguingly, PAF interacted with both Bmi1 and enhancer of zeste homolog 2 (EZH2) in SW620 cells (Figure 3K), which led us to test whether either Bmi1 or EZH2 is functionally associated with PAF-mediated Wnt signaling hyperactivation. To do this, we assessed the effects of Bmi1 and EZH2 on β-catenin transcriptional activity, using β-catenin reporter assays. We observed that ectopic expression of EZH2 upregulated β-catenin transcriptional activity, but Bmi1 overexpression did not (data not shown), implying that EZH2 might be associated with Wnt signaling activation. Binding domain mapping analysis showed that EZH2 bound to PAF via the middle region of EZH2 including the CXC cysteine-rich domain (Figure 3L). In conjunction with the Bmi1-containing PRC1, EZH2-containing PRC2 catalyzes histone H3 lysine 27 trimethylation (H3K27me3) via histone methyltransferase domain. Despite the crucial role of EZH2 in H3K27me3-meidated gene regulation, we found that other core components of PRC2, EED, and Suz12 were not associated with PAF (Figure 3K). Moreover, although EZH2 overexpression in cancer induces PRC4 formation in association with the NAD+-dependent histone deacetylase Sirt1 (Kuzmichev et al., 2005), the PAF-EZH2 complex did not contain Sirt1 (Figure 3K). These data indicate that PAF-EZH2 complex is distinct from the conventional PRCs in cancer cells. Also, we questioned whether PCNA is required for PAF’s interaction with either PAF or β-catenin. Interestingly, β-catenin-PAF and EZH2-PAF complexes existed only in PCNA-free fractions (Figure 3M, compare lanes 1 and 2), which is consistent with PCNA-independent mitogenic role of PAF in colon cancer cell proliferation (Figure 1I). Due to exclusive interaction of PAF with either PCNA or β-catenin, we asked whether Wnt signaling activation affects either PAF-β-catenin or PAF-PCNA interaction. Co-IP assays showed that, in HeLa cells, PAF-β-catenin interaction was only detected upon LiCl treatment, while PAF-EZH2 interaction remained constant. Moreover, PAF-PCNA association was decreased by LiCl or Wnt3A treatment (Figures 3N and 3O, compare lanes 3 and 4). These data suggest that Wnt signaling activation is required for PAF-β-catenin interaction. Due to absence of endogenous Wnt signaling activity in HeLa cells, we also assessed the effects of active Wnt/β-catenin signaling on PAF-PCNA binding in colon cancer cell lines that exhibit hyperactivation of Wnt signaling by genetic mutations in APC or β-catenin alleles. Surprisingly, PAF-PCNA interaction was barely detectable in colon cancer cell lines, whereas 293T and HeLa cells displayed strong PAF-PCNA association (Figure 3P), implying that active β-catenin may sequester PAF from PCNA. In binding domain mapping analysis, we also found that N-terminal and PIP regions are required for β-catenin interaction (Figure S2), suggesting that β-catenin competes with PCNA for PAF interaction. These results suggest that, upon Wnt signaling activation, PAF is conditionally associated with β-catenin transcriptional complex.

PAF activates β-catenin target genes by recruiting EZH2 to promoters

Previous studies showed that EZH2 interacts with β-catenin (Li et al., 2009Shi et al., 2007). Also, we found that PAF is physically associated with EZH2, independently of PRC2 complex (Figure 3). These evidences prompted us to ask whether EZH2 mediates PAF-induced Wnt signaling hyperactivation. Given PAF-EZH2-β-catenin complex formation, we tested whether EZH2 is also associated with the promoters of β-catenin target genes. Intriguingly, PAF, EZH2, and β-catenin steadily co-occupied the promoters of c-Myc,Cyclin D1, and Axin2 in HCT116 cells carrying β-catenin mutation, whereas PCNA, EED, and Suz12 did not (Figure 4A), which recapitulates PRC2 complex-independent association of EZH2 with PAF (see Figures 3K and 3N). Next, we asked whether PAF, EZH2, and β-catenin are recruited to β-catenin target gene promoter upon Wnt signaling activation, as PAF-β-catenin interaction was dependent of Wnt signaling activation (Figure 3N). In HeLa cells, we found that PAF, EZH2, and β-catenin conditionally bound to TBEs in the c-Myc and Axin2 promoters, only upon LiCl treatment (Figure 4B), indicating that Wnt signaling activation is a prerequisite for PAF-β-catenin-EZH2’s promoter association. To further confirm the specificity of PAF-EZH2-β-catenin’s recruitment to β-catenin target promoters, we performed ChIP promoter scanning of 10 kb of the c-Myc promoter, and found that PAF, EZH2, and β-catenin specifically co-occupied the proximal promoter containing TBEs of the c-Myc gene (at -1037 and -459 bp) (He et al., 1998) in HCT116 cells (Figure 4C). Also, the analysis of EZH2 ChIP-sequencing data from mouse embryonic stem cells showed that EZH2 was specifically enriched in the proximal promoters of β-catenin targets (Lef1Lgr5c-Myc, and Axin2) (Figure 4D).


Figure 4 PAF promotes EZH2-β-catenin interaction

Next, we asked whether EZH2 promoter recruitment is necessary for activation of β-catenin target gene transcription. Previously, depletion of EZH2 was shown to inhibit c-Myc expression in DLD-1 colon cancer cells (Fussbroich et al., 2011). Consistently, EZH2 knockdown downregulated β-catenin target genes, Axin2and Cyclin D1 in HCT116 cells (Figure 4E), and decreased LiCl-induced β-catenin reporter activation (Figure 4F), suggesting that EZH2 is required for PAF-mediated Wnt target gene hyperactivation. These results are also supported by previous finding that EZH2 enhances β-catenin transcriptional activity by connecting β-catenin with the Med1/RNA polymerase II (Pol II) complex (Shi et al., 2007). Indeed, Med1/TRAAP220 and Pol II conditionally binds to c-Myc and Axin2 promoters in LiCl-treated HeLa cells (Figure 4G). Given that PRC2-indepednent interaction between EZH2 and PAF (Figures 3K and 3N), we asked whether EZH2’s histone methyltransferase activity is dispensable in β-catenin regulation. We utilized an EZH2 point mutant (F681I) that disrupts the contact between the EZH2 hydrophobic pocket and histone lysine residue H3K27 (Joshi et al., 2008). Ectopic expression of either EZH2 or EZH2-F681I enhanced β-catenin reporter activity (Figure 4H). Also, PAF knockdown did not change the H3K27 methylation status (H3K27me3) of proximal promoters of c-MycAxin2Cyclin D1, and DCC in HCT116 cells (Figure 4I). These results indicate a methyltransferase-independent role of EZH2 in transactivating β-catenin targets.

Due to PAF’s (1) small size (111 amino acids, one α-helix), (2) lack of a specific catalytic domain, and (3) binding to both β-catenin and EZH2, PAF may facilitate the interaction between EZH2 and β-catenin through recruiting EZH2 to the promoter. We tested this using ChIP assays for EZH2 in the setting of PAF depletion. Indeed, PAF-depleted HCT116 cells displayed decreased EZH2-association at the c-Myc promoter (Figure 4J), suggesting that PAF assists or is needed to recruit EZH2 to β-catenin transcriptional complex. Also, β-catenin knockdown decreased recruitment of PAF and EZH2 to promoters (Figure 4K), showing that PAF and EZH2 occupy target promoters via β-catenin. We then asked whether PAF promotes β-catenin-EZH2 binding. In vitro binding assays showed that the addition of GST-PAF protein increased EZH2-β-catenin association (Figure 4L). Moreover, ectopic expression of PAF promoted the EZH2-β-catenin interaction in HeLa cells treated with LiCl (Figure 4M). Additionally, we tested whether Wnt signaling-induced post-translational modification of either β-catenin or PAF is required for EZH2 interaction. However, in GST pull-down assays, we found that bacterially expressed either GST-β-catenin or –PAF bound to EZH2 (Figure S3). Due to the lack of post-translational modification in GST protein expression system, these data indicate that post-translation modification of either β-catenin or PAF is not necessary for EZH2 interaction. Together, these results suggest that PAF acts as a molecular adaptor to facilitate EZH2-β-catenin complex, and subsequently enhances the transcriptional activity of the β-catenin transcriptional complex at Wnt target promoters (Figure 4N).

Intestinal tumorigenesis following PAF conditional expression

Having determined that PAF is overexpressed in colon cancer cells and hyperactivates Wnt/β-catenin signaling, we aimed to determine whether mimicking PAF overexpression drives intestinal tumorigenesis, using genetically engineered mouse models. To conditionally express PAF, we generated doxycycline (doxy)-inducible PAF transgenic mice (TetO-PAF-IRES-emGFP [iPAF]). For intestine-specific expression of PAF, we used iPAF:Villin-Cre:Rosa26-LSL-rtTA mouse strains. Villin-Cre is specifically expressed in intestinal epithelial cells (IECs), including ISCs and progenitor cells. Cre removes a floxed stop cassette (loxP-STOP-loxP [LSL]) from the Rosa26 allele and induces rtTA expression. Upon doxy treatment, rtTA drives the transcriptional activation of the tetracycline-responsive element promoter, resulting in conditional transactivation of PAF selectively in IECs. We also utilized the Rosa26-rtTA strain for ubiquitous expression of PAF (Figure 5A and Figure S4). First, we examined the effects of PAF induction on IEC proliferation using a crypt organoid culture system (Figure S5A). Intriguingly, PAF conditional expression (2 weeks) induced expansion of the crypt organoids (Figures 5B and 5C), which recapitulates the mitogenic function of PAF (Figure 1). In mouse, IEC-specific PAF expression (iPAF:Villin-Cre:Rosa26-LSL-rtTA; 2 months) developed adenoma in both small intestine and colon (Figure 5D). Also, microscopic analysis using hematoxylin and eosin (H&E) staining showed aberrant IEC growth and crypt foci formation (Figures 5E and 5F), with disorganized epithelial cell arrangements (Figure S5B). Consistently, PAF-induced IEC hyperproliferation was manifested by increased Ki67 expression, a mitotic marker (Figure 5G). Importantly, these lesions exhibited the upregulation of CD44, a β-catenin target gene, whereas CD44 was expressed strictly in the crypts of normal intestine (Figure 5H). Next, we examined whether PAF directly hyperactivates Wnt/β-catenin in vivo using BAT-gal, a β-catenin reporter transgenic mouse carrying multiple TBEs followed by a LacZ reporter. To quantify the early effects of PAF on β-catenin activity, we treated mice with doxy for 1 week, and found that short-term induction of PAF increased β-catenin transcriptional activity as represented by enhanced X-gal staining (Figure 5I). Moreover, conditional PAF expression upregulated the β-catenin target genes, Axin2Lgr5CD44Cyclin D1, and c-Myc in crypt organoids (Figure 5J). Additionally, mice ubiquitously expressing PAF exhibited intestinal hypertrophy (Figure S5C), which is similar to that induced by R-Spondin1, a secreted Wnt agonist (Kim et al., 2005). These data strongly suggest that PAF expression is sufficient to initiate intestinal tumorigenesis via Wnt signaling hyperactivation.


Figure 5 Induction of intestinal neoplasia by PAF expression

Herein we reveal the unexpected role of PAF in modulating Wnt/β-catenin signaling. PAF enhances the transcription of Wnt targets by recruiting EZH2 to the β-catenin transcriptional complex. This is similar to the mechanism by which Lgl/BCL9 binds to β-catenin and thereby recruits the PHD-finger protein Pygopus, to bridge the β-catenin/TCF complex to Med12 and Med13 (Carrera et al., 2008). Importantly, due to specific overexpression of PAF in cancer cells, our studies identified an additional layer of the regulatory mechanism of β-catenin target gene transactivation.

In cancer cells, the upregulation of EZH2 contributes to tumorigenesis through the epigenetic repression of various genes including tumor suppressor genes, Wnt antagonists, and DNA repair genes (Chang et al., 2011Cheng et al., 2011Kondo et al., 2008). Our results propose a noncanonical function of EZH2 in activating β-catenin target genes in conjunction with PAF. Consistently, recent study also suggests methyltransferase activity-independent function of EZH2 in gene activation (Xu et al., 2012). Moreover, this non-canonical role of EZH2 is supported by several lines of evidence: (a) EZH2 transactivates β-catenin target genes (Li et al., 2009Shi et al., 2007) (Figures 4E and 4F); (b) EZH2 overexpression in murine mammary epithelium induces ductal hyperplasia (Li et al., 2009), which phenocopies that in a ∆Nβ-catenin (constitutively active form of β-catenin) mouse model (Imbert et al., 2001); (c) EZH2 occupies β-catenin target promoters (Figures 4A-D); and (d) EZH2’s methyltransferase activity is dispensable for β-catenin target activation (Figures 4H and 4I). Moreover, similar to PAF expression in the AER (Figure 2A), EZH2 is also specifically expressed there to maintain of Hox cluster gene transcription (Wyngaarden et al., 2011). Thus, it is plausible that EZH2 and PAF cooperatively control Hox gene activation in the developing limb. Interestingly, despite the presence of a physical and functional connection between Bmi1 and EZH2 in H3K27me3-mediated gene repression, EZH2 is expressed only in crypt IECs including ISCs (Figure S6), whereas Bmi1 is expressed in ISCs at position 4 (Figure 3J), implying a Bmi1-independent role for EZH2 in gene regulation. These results demonstrate the novel function of EZH2 in β-catenin target gene activation, independent of the histone methyltransferase activity of EZH2.

Previously, we found that TERT, a catalytic subunit of telomerase, positively modulates Wnt signaling (Park et al., 2009), elucidating a non-telomeric function of telomerase in development and cancer. Here our results propose that one component of DNA damage bypass process also functions in regulating Wnt signaling, dependent of context. In cancer, PAF overexpression may play a dual role in inducing (a) cell hyperproliferation (via Wnt signaling hyperactivation) and (b) the accumulation of mutations arising from DNA lesion bypass (by PAF-mediated TLS) (Povlsen et al., 2012). Importantly, PAF is only expressed in cancer cells, but not in normal epithelial cells. Thus, upon DNA damage, instead of cell growth arrest to permit high-fidelity DNA repair, the PAF overexpression in cancer cells is likely to induce DNA lesion bypass by facilitating TLS. However, in the setting of Wnt signaling deregulation, nuclear β-catenin sequesters PAF from PCNA and utilize PAF as a co-factor of transcriptional complex, which induces Wnt signaling hyperactivation and possibly lead to increased mutagenesis.

We observed that PAF marked the stemness of ISCs and mouse embryonic stem cells (Figure S7), implicating its roles in stem cell regulation under physiological conditions. In a previous study, a PAFgermline knockout mouse model displayed defects in hematopoietic stem cell self-renewal (Amrani et al., 2011), suggesting a crucial role of PAF in stem cell maintenance and activation. In the intestine, β-catenin activation in Lgr5-positive or Bmi1-positive cells is sufficient to develop intestinal adenoma (Barker et al., 2009Sangiorgi and Capecchi, 2008), suggesting an essential role of tissue stem cells in tumor initiation. Considering PAF expression in Bmi1-positive ISCs, PAF upregulation in ISCs likely hyperactivates the Wnt/β-catenin signaling and contributes to intestinal tumor initiation.

Despite the critical role of Wnt signaling in early vertebrate, development PAF germline knockout mice are viable (Amrani et al., 2011). It is noteworthy that, whereas deletion of any core component in the Wnt signaling pathway causes embryonic lethality, mice with germline knockout of Wnt signaling modulators, including Nkd1/2Pygo1/2, and BCL9/9-2, exhibit no lethal phenotypes (Deka et al., 2010Schwab et al., 2007Zhang et al., 2007). This may result from the robustness of Wnt signaling during embryogenesis because of functional compensation not only via the presence of multiple Wnt signaling regulators per se but also via other types of signaling crosstalk. Therefore, as described previously in pRb studies (Sage et al., 2003), acute deletion of PAF in a conditional knockout mouse model may disrupt the developmental balance or tissue homeostasis, and then reveal the full spectrum of the physiological and pathological roles of PAF in tumorigenesis. Taken together, our findings reveal unexpected function of PAF and EZH2 in modulating Wnt signaling, and highlight the impacts of PAF-induced Wnt signaling deregulation on tumorigenesis.

11.1.9 PAF Makes It EZ(H2) for β-Catenin Transactivation

Xinjun Zhang1 and Xi He1
Mol Cell. 2013 Oct 24; 52(2)

In this issue of Molecular Cell, Park and colleagues (Jung et al., 2013) show that PAF (PCNA-associatedfactor) binds to and hyperactivates transcriptional function of β-catenin in colon cancer cells by recruiting EZH2 to the coactivator complex. PAF-β-catenin and PAF-PCNA interactions are competitive, raising the question of whether β-catenin might regulate PCNA-dependent DNA replication and repair.

Wnt signaling through stabilization of transcription co-activator β-catenin plays critical roles in animal development and tissue homeostasis, and its deregulation is involved in myriad human diseases including cancer (Clevers and Nusse, 2012). Notably, most colorectal cancers (CRCs) have elevated β-catenin signaling caused by mutations of Wnt pathway components such as the tumor suppressor APC (Adenomatosis polyposis coli) and β-catenin itself (Clevers and Nusse, 2012). Much effort has focused on studying β-catenin-dependent transactivation in CRCs, including the current study by Park and colleagues that identifies PAF as an unexpected β-catenin co-activator (Jung et al., 2013).

PAF, for PCNA (proliferating cell nuclear antigen)-associated factor (also known as KIAA0101 or p15PAF), is an interacting partner of PCNA (Yu et al., 2001). PCNA has a key role in DNA replication and repair by assembling various DNA polymerase and repair complexes at the replication fork (Mailand et al., 2013). Dynamic regulation of PAF abundance and/or interaction with PCNA appears to be important for engaging DNA damage repair and bypass pathways (Emanuele et al., 2011Povlsen et al., 2012). PAF is overexpressed in many types of cancers and required for cell proliferation (e.g., Yu et al., 2001).

In the current study (Jung et al., 2013), Jung et al. show that PAF is overexpressed in CRCs in a manner that parallels expression of Axin2, an established Wnt/β-catenin target gene. PAF knockdown inhibits CRC proliferation, and this effect is independent of PAF-PCNA interaction and can be rescued by a PAF mutant that does not binds to PCNA or by β-catenin overexpression. PAF knockdown downregulates the expression of Wnt/β-catenin target genes Cyclin D1c-Myc, and Axin2 in a CRC line, leading the authors to hypothesize that PAF participates in Wnt/β-catenin signaling. Indeed PAF knockdown reduces, and its overexpression augments, Wnt/β-catenin responsive TOPFLASH reporter and target gene expression induced by Wnt3a or by pharmacological agents that stabilize β-catenin. In Xenopus embryos, PAF synergizes with β-catenin to induce Wnt target gene expression and axis duplication (a hallmark of Wnt/β-catenin activation). In mouse embryos, PAF is highly expressed in regions known for Wnt/β-catenin signaling such as the apical ectodermal ridge of the limb bud. Therefore PAF appears to be a positive regulator of Wnt/β-catenin signaling in CRCs and vertebrate embryos.

PAF does not affect β-catenin protein levels and is localized in the nucleus. Protein binding assays show that PAF interacts, directly or indirectly, with β-catenin (via the Armadillo-repeat domain) and its DNA-bound partner TCF (T Cell factor). Indeed PAF is associated with promoters of Wnt/β-catenin target genes in chromatin in CRC cells. Interestingly in the mouse intestine, the PAF protein is enriched in Bmi (B lymphoma Mo-MLV insertion region 1 homolog)-positive stem cells (at the “+4” position) (Sangiorgi and Capecchi, 2008). Bmi1 is a component of Polycomb Repressive Complex 1 (PRC1), which, together with the PRC2 complex that modifies Histone H3, has critical functions in transcriptional epigenetic silencing. Previous studies have suggested that a core PRC2 component, EZH2 (enhancer of zeste homolog 2), is a partner and paradoxically a co-activator of β-catenin, acting in a manner that is independent of EZH2’s methyltransferase activity (Li et al., 2009Shi et al., 2007). Jung et al. found that PAF indeed interacts with both Bmi1 and EZH2, but not other PRC2 components, and EZH2 overexpression augments β-catenin transcriptional activity. PAF, EZH2, and β-catenin are found to co-occupy promoters of several Wnt/β-catenin target genes in CRC and mouse ES cells, and PAF depletion decreases EZH2 association with the c-Myc promoter, and β-catenin depletion decreases the association of both PAF and EZH2 with the promoter. Thus the β-catenin-PAF-EZH2 complex appears to constitute a chain of co-activators (Figure 1), and indeed PAF, which binds to both β-catenin and EZH2, enhances β-catenin-EZH2 co-immunoprecipitation. Together with an earlier study (Shi et al., 2007), these results suggest a model that PAF brings EZH2 and the associated RNA polymerase II Mediator complex to β-catenin target genes for transactivation in CRCs (Figure 1). Consistent with this model, transgenic overexpression of PAF in the mouse intestine induces β-catenin-dependent target and reporter gene expression, intestinal overgrowth, and adenoma formation in vivo and crypt organoid expansion in vitro, resembling Wnt/β-catenin signaling activation in the gastrointestinal tract.



β-catenin transactivation


Figure 1 β-catenin transactivation mediated by PAF and EZH2 in the G1 phase and a speculative role of β-catenin in modulating PAF-PCNA-dependent DNA replication and repair/bypass pathways in the S phase.

PAF and EZH2 represent newer additions to β-catenin’s plethora of co-activators (Mosimann et al., 2009), which offer multiple routes to engage the basal transcription apparatus. These co-activators may have partially redundant and/or context-dependent functions for numerous Wnt/β-catenin-dependent gene programs. Mouse mutants that lack an individual β-catenin co-activator are often viable (MacDonald et al., 2009Mosimann et al., 2009). Paf−/− mice are viable but exhibit defects in hematopoietic stem cell properties (Amrani et al., 2011). PAF is also expressed in self-renewing mouse ES cells but the expression is downregulated upon ES cell differentiation (Jung et al., 2013). Whether PAF has a general role in self-renewal of embryonic and adult stem cells through its role in β-catenin signaling or DNA replication and repair pathways remains to be investigated.

PAF-β-catenin interaction is observed under Wnt stimulation, likely as a consequence of β-catenin accumulation (Jung et al., 2013). In some cell types PAF is ubiquitinated and degraded by the anaphase promoting complex and thus exhibits the lowest level in the G1 phase of the cell cycle (Emanuele et al., 2011). In these cells PAF may have a limited role as a co-activator for β-catenin-dependent transcription, which primarily occurs in G1. But in CRC and other cancers where PAF is overexpressed, PAF may have a prominent role as a β-catenin co-activator.

PAF-PCNA interaction is well documented (e.g., Yu et al., 2001). Surprisingly however, in CRCs with high levels of β-catenin, PAF-PCNA interaction is barely detectable (Jung et al., 2013). Conversely, in cells where the basal level of Wnt/β-catenin signaling is low, PAF-PCNA interaction is detected but is diminished by Wnt3a or pharmacological agents that stabilize β-catenin (Jung et al., 2013). PAF seems to interact with β-catenin and PCNA via an overlapping domain (although this remains to be better defined), offering a possible explanation why PAF-β-catenin and PAF-PCNA complexes appear to be mutually exclusive (Jung et al., 2013). This may simply reflect the fact that PAF-β-catenin (for RNA transcription) and PAF-PCNA (for DNA replication/repair) complexes act in G1 and S, respectively (Figure 1). However, when β-catenin levels are high in Wnt-stimulated cells or in CRCs, one may speculate that β-catenin accumulation could inhibit PAF-PCNA complex formation in the S phase, thereby enabling Wnt/β-catenin signaling to modulate PAF-PCNA-dependent DNA replication and repair/bypass pathways (Figure 1). This would constitute an unsuspected role for Wnt/β-catenin signaling in genomic stability beyond its established transcriptional function and could have implications to tumorigenesis.

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11.1.10 PI3K.AKT.mTOR pathway as a therapeutic target in ovarian cancer

Li H1Zeng JShen K.
Arch Gynecol Obstet. 2014 Dec; 290(6):1067-78

Background: Ovarian cancer is one of the major causes of death in women worldwide. Despite improvements in conventional treatment approaches, such as surgery and chemotherapy, a majority of patients with advanced ovarian cancer experience relapse and eventually succumb to the disease; the outcome of patients remains poor. Hence, new therapeutic strategies are urgently required. The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) is activated in approximately 70 % of ovarian cancers, resulting in hyperactive signaling cascades that relate to cellular growth, proliferation, survival, metabolism, and angiogenesis. Consistent with this, a number of clinical studies are focusing on PI3K pathway as an attractive target in the treatment of ovarian cancer. In this review, we present an overview of PI3K pathway as well as its pathological aberrations reported in ovarian cancer. We also discuss inhibitors of PI3K pathway that are currently under clinical investigations and the challenges these inhibitors face in future clinical utility.Methods: PubMed was searched for articles of relevance to ovarian cancer and the PI3K pathway. In addition, the ClinicalTrials.gov was also scanned for data on novel therapeutic inhibitors targeting the PI3K pathway. Results: Genetic aberrations at different levels of PI3K pathway are frequently observed in ovarian cancer, resulting in hyperactivation of this pathway. The alterations of this pathway make the PI3K pathway an attractive therapeutic target in ovarian cancer. Currently, several inhibitors of PI3K pathway, such as PI3K/AKT inhibitors, rapamycin analogs for mTOR inhibition, and dual PI3K/mTOR inhibitors are in clinical testing in patients with ovarian cancer. Conclusions: PI3K pathway inhibitors have shown great promise in the treatment of ovarian cancer. However, further researches on selection patients that respond to PI3K inhibitors and exploration of effective combinatorial therapies are required to improve the management of ovarian cancer.

Fig.1. Inputs from receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCR) to class I PI3K.

Fig. 2. Schematic representation of the PI3K/AKT/mTOR signaling pathway.

Fig.3. PI3K/AKT/mTOR inhibitors.

AKT inhibitors

AKT inhibitors can be grouped into three classes including lipid based phosphatidylinositol (PI) analogs, ATP-competitive inhibitors, and allosteric inhibitors. Perifosine, which is the most clinically studied AKT inhibitor, is a lipid-based PIanalog that targets the pleckstrin homology domain of AKT, preventing its translocation to the cell membrane. Amongthe three classes of AKT inhibitors, allosteric AKT inhibitors display highly specific selectivity for AKT isoforms. Considering the genetic background of ovarian cancer, allosteric AKT inhibitors such as MK2206 that can target both AKT1 and AKT2 might be the best agents for treating ovarian cancer.In clinical trials, AKT inhibitors have shown similar toxicities to those caused by PI3K inhibitors, such as hyperglycemia, rashes, stomatitis, and gastrointestinal side effects [25].

mTOR inhibitors

Rapamycin and its analogs Rapamycin (sirolimus), a potent inhibitor of mTORC1, was first isolated in 1975 from the bacterium Streptomyces hygroscopicus. Rapamycin inhibits mTORC1 by first binding to the intracellular protein FK506 binding protein 12 (FKBP12). The resultant rapamycin–FKBP12 complex then binds to the FKBP12–rapamycin-binding domain (FRB) of mTORC1 and inhibits the serine/threonine kinase activity of mTORC1 via an allosteric mechanism. In contrast to mTORC1, the rapamycin–FKBP12 complex cannot interact with the FRB domain of mTORC2, and thus,mTORC2 is generally resistant to rapamycin treatment [12]. As rapamycin displays very poor water solubility, which limits its clinical use, several soluble ester analogs of rapamycin (rapalogs) have been developed [12]. Currently, these analogs include temsirolimus, everolimus, and ridaforolimus. Temsirolimus and everolimus are formulated for intravenous and oral administration, respectively. Ridaforolimus was initially developed as an intravenous formulation, but an oral formulation was subsequently produced [12,28]. Clinically, rapalogs are generally well tolerated, with the most common side effects including stomatitis, rashes, fatigue, hyperglycemia, hyperlipidemia, hypercholesterolemia, and myelosuppression [3,12,25].

ATP-competitive inhibitors

Different from rapalogs, ATP-competitive inhibitors do not require co-factors such as FKBP12 to bind to mTOR. By competingwith ATP for theATP-binding sites of mTOR, this class of mTOR inhibitors can inhibit the kinase activity of both mTORC1 and mTORC2. Although there is a concern that the simultaneous inhibition of mTORC1 and mTORC2 might result in greater toxicities in normal tissues, ATP-competitive mTOR inhibitors have been shown to display stronger anti-proliferative activity than rapalogs across a broad range of cancers includingovarian cancer [12,15].


Metformin,the most commonly prescribed oral anti-diabetic agent, has been shown to reduce the incidence of malignancies in patients with diabetes. The activation of 5′ adenosine monophosphateactivated protein kinase (AMPK) by metformin plays an important role in mediating the drug’s effects. AMPK activation results in the phosphorylation and activation of TSC2, which exerts inhibitory effects on mTORC1. Metformin-induced AMPK activation also reduces AKT activity by inhibiting insulin receptor substrate 1 (IRS-1). Ultimately, AMPK activation results in the inhibition of the PI3K/AKT/mTOR signaling pathway, making metformin an effective treatment for cancer [28].

mTORC1 inhibitors              mTORC1                      Dual PI3K/mTOR inhibitors

PI3K inhibitors                     Class I PI3K                   mTORC2

AKT inhibitors                        AKT                              mTORC ½  inhibitors

PI3K inhibitors

Pan-class I PI3K inhibitors Pan-class IPI3K inhibitors can inhibit the kinase activity ofall 4 isoforms of classI PI3K.The main advantage of pan-class IPI3K inhibitors is that most cancer cells express multiple PI3K isoforms with redundant oncogenic signaling functions. Early clinical trials have suggested that the most common toxicitiesof pan-class IPI3K inhibitors are hyperglycemia, skin toxicities, stomatitis, and gastrointestinal side effects. Of these, hyperglycemia is likely to be a mechanism-based toxicity given the well described role of PI3K in insulin receptor signaling [3,25].

Isoform-selective PI3K inhibitors

This class of agents target the specific PI3K p110 isoforms involved in particular types of cancer. The p110α isoform (which is encoded by the PIK3CA gene) is a frequent genetic driver (PIK3CA mutations) of ovarian cancer, whereas p110β activity is known to be essential in cancer cells lacking PTEN. As for the p110δ isoform, it plays a fundamental role in the survival of normal B cells and is implicated in malignancies affecting this lineage. Thus, the main theoretical advantage of these inhibitors is that they have the potential to completely block the relevant target whilst causing limited toxicities compared with pan-PI3K inhibitors. Consistent withthese findings, preclinical studies have detected significant activities of PI3Kα inhibitor in tumors exhibiting PIK3CA mutations, PI3Kβ inhibitors in tumors with PTEN loss, and PI3Kδ inhibitors in hematologic malignancies. In addition, PI3Kδ inhibitors have already shown very promising activity in patients with chronic lymphocytic leukemia [26].

Dual PI3K/mTOR inhibitors

Structural similarities between the ATP-binding domain of p110 and the catalytic domain of mTOR have led to the development of a class of agents that inhibit both class I PI3K and mTORC1/2. Theoretically, dual mTOR/PI3K inhibitors should lead to more complete suppression of the PI3K/AKT/mTOR pathway than targeting either component independently.In agreement with this, in preclinical studies of ovarian cancer dual PI3K/mTOR inhibitors were found to exhibit greater in vitro and in vivo anti-tumor activity than mTOR inhibitors alone [27]. The safety profile of these inhibitors is similar to that of pan-PI3K inhibitors, with common adverse events including nausea, diarrhea, fatigue, and vomiting [3,25]. 


11.1.11 Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway

Mira JP1Benard VGroffen JSanders LCKnaus UG.
Proc Natl Acad Sci U S A. 2000 Jan 4; 97(1):185-9.

Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3-Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth.

Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3–Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth.

Rac proteins are members of the Rho GTPase family and act as molecular switches in regulating a variety of biological response pathways, including cell motility, gene transcription, cell transformation, and cell-cycle progression (1). The Rac family includes Rac1, the myeloid-lineage-specific Rac2, and the recently cloned Rac3 proteins (2). Rac3 differs from Rac1 and Rac2 in two domains, the insert region and the C terminus, which influence transformation (34), interaction with guanine nucleotide exchange factors (GEFs) (56), and subcellular localization (78). Small GTPases, including Rac, cycle between an inactive GDP-bound state and an active GTP-bound state. Two classes of regulatory factors, GTPase-activating proteins (GAPs) and GEFs, determine by their opposing effects the ratio of GDP versus GTP, which is bound to the GTPase (1). GAP proteins increase the intrinsic rate of GTP hydrolysis, rendering the GTPase inactive, whereas GEFs enhance the exchange of bound GDP for GTP, thereby activating the protein. Active Rac regulates distinct downstream signaling pathways by interacting with specific effector proteins, including a family of serine-threonine protein kinases termed Paks (p21-activated kinases) (911).

Apart from its well documented role in cytoskeletal rearrangements in growth factor-stimulated cells (12), Rac1 is required for Ras-induced malignant transformation and is involved in transcription and growth control (11314). Recently, the importance of the Rac effector Pak in cell transformation has been highlighted by inhibiting RasV12- and Rac1V12-induced transformation of Rat-1 fibroblasts with a catalytically inactive form of Pak (1516). The involvement of Rac1 in driving cell-cycle progression through the G1 phase and stimulating DNA synthesis has been shown by introducing dominant-active and -negative Rac1 mutants into fibroblasts (1718). However, the signaling pathways used by Rac to control mitogenesis and proliferation still remain poorly understood. Overexpression of constitutively active Rac-effector-domain mutants in fibroblasts indicated that although Rac1 mediated cyclin D1 transcription by Pak in NIH 3T3 cells (19), Pak was not involved in the DNA synthesis of Swiss 3T3 cells (20). Accumulating evidence, however, suggests higher complexity where Pak-binding proteins, such as the GEF Pix, contribute to the Rac–Pak interaction in vivo and influence subsequent cellular functions (2123).

All biological functions listed above have been attributed to Rac1 in experimental cell systems using overexpression or microinjection of mutant forms. Endogenously active Rho GTPases, including Rac, have not yet been observed. In this paper, we describe a consistently active Rac3 GTPase leading to hyperactivity of its effector protein kinase, Pak, in human breast cancer-derived epithelial cell lines. Analysis of growth properties and DNA synthesis revealed that both proteins are required to convey the highly proliferative phenotype displayed by these cells.

Highly Proliferating Cancer Cells Contain Hyperactive Rac3.

Comparison of growth rates among several breast cancer cell lines showed that three lines (MDA-MB 435, T47D, and MCF 7) grew faster under normal and low-serum conditions (Fig. ​(Fig.1).1). Interestingly, in contrast to MDA-MD 231 and Hs578T cells, these three highly proliferative cell lines do not possess mutated Ras (2829). To assess whether Rho GTPases drive this cellular phenotype, we determined whether these cell lines contained active GTP-bound Rac or Cdc42. We used a recently described assay, the PBD-pulldown assay (24), which is based on the specific binding of the GTP-bound forms of Rac and Cdc42 to the PBD of Pak (10). Neither active Rac1 (Fig. ​(Fig.22A) nor active Cdc42 (data not shown) could be detected in any of the cell lysates obtained from serum-starved cells. However, both proteins were detected if the PBD-pulldown assay was performed with in vitro guanosine 5′-[γ-thio]triphosphate (GTP[γS])-loaded cell lysates, confirming that Rac1 and Cdc42 were present in their inactive GDP-bound forms in these cells (Fig. ​(Fig.22A for Rac1). Next we wanted to determine whether active Rac3 was present in breast cancer cell lines. Because Rac3 effectors have not yet been characterized, we demonstrated by overlay binding and kinase assays that Rac3 bound to and activated Pak as efficiently as Rac1 (data not shown). We verified that the PBD-pulldown assay specifically detected the active GTP-bound form of Rac3 (GTP[γS]-loaded Rac3wt or Rac3V12, Fig. ​Fig.22B) and not the inactive form. To probe for Rac3 protein in breast cell lysates, a Rac3-specific antibody was used. GST-PBD-pulldown experiments from cell lysates revealed the presence of hyperactive Rac3 in highly proliferative cell lines (MDA-MB 435, T47D, and MCF 7), but not in normal breast cell lines or in less proliferative breast cancer cells (Fig. ​(Fig.22C). Additionally, as indicated by the virtual absence of Rac3 in the supernatant of the PBD pulldown, all the Rac3 protein present in these cell lines was active (Fig. ​(Fig.22C). To demonstrate that consistent Rac3 activation is not limited to cell lines, we performed an initial screening of human metastatic breast cancer tissues and found active Rac3 in one of three samples, underlining the potential clinical relevance of the cellular findings (Fig. ​(Fig.22D).

Differential growth rates of human breast cell lines.  pq0104939001

Differential growth rates of human breast cell lines. pq0104939001

Differential growth rates of human breast cell lines.

Figure 1 Differential growth rates of human breast cell lines. Human breast cell lines, including HMEC 184 (○), MDA-MB 231 (▵), Hs578T (□), MDA-MB 435 (●), T47D (▴), and MCF 7 (♦), were grown in 10% serum (A) or 0.5% serum (B) conditions. The cells were split in duplicate over 6-well plates at 5 × 105 cells per well and counted daily with a hemocytometer for 4 days. Data shown in A and B are representative of three independent experiments.


Figure 2 Active Rac3 is present in highly proliferative cell lines and in human breast cancer tissue. (A and C) Cell lysates from serum-starved breast cancer cell lines without (A and C) or after (+) GTP[γS] loading (A) were incubated with 10 μg of GST-PBD. Active Rac proteins (PBD pulldown) were detected by immunoblot with anti-Rac1 (A) or anti-Rac3 antibodies (C). Blotting of PBD supernatants revealed the GDP-bound form of Rac3 in lysates. Equal amounts of Rac3 protein were detected by immunoblot (IB) in all cell lines. (B) A PBD-pulldown assay of extracts from HeLa cells expressing Myc-Rac3wt or -Rac3 mutants, followed by an anti-Myc immunoblot, detected only active Rac3 (GTP[γS] loading or Rac3V12). (D) PBD pulldown of lysates obtained from three different human metastatic breast cancer tissues, followed by anti-Rac1 and anti-Rac3 immunoblots, revealed active Rac3 in tissue 1. (E) PBD pulldown of lysates derived from MDA-MB 435 and MDA-MB 231 cells expressing LacZ control or Myc-Rac3wt without or after in vitro GTP[γS] loading. Consistent activation of Myc-Rac3wt occurred only in MDA-MB 435 cells. (F) Subcellular localization of Rac1 and Rac3. Cytosol (c) and membranes (m) were obtained after nitrogen cavitation and fractionation of breast cancer cell lines and immunoblotted with anti-Rac1 and anti-Rac3 antibodies. All blots are representative of at least three experiments.

Subcellular Localization and GTPase-Regulatory Factors Influence Rac3 Activity.

Constitutive activation of Ras proteins in cancer cells is often caused by activating point mutations at the switch I or II regions (29). cDNA cloning and complete sequence analysis of full-length Rac3 did not reveal any mutations in the breast cell lines studied and did not explain the observed Rac3 activation. GTPase-regulatory proteins such as GEFs and GAPs, which are usually regulated by upstream stimuli, control cycling between the active and inactive forms of Rac. To confirm the presence of an altered regulatory mechanism involved in Rac3 activation, we used the PBD-pulldown assay to analyze the activation state of Myc-tagged Rac3wt transfected into either MDA-MB 231, a cell line harboring only GDP-Rac3, or MDA-MB 435, a cell line that contains endogenous, active GTP-Rac3. Fig. ​Fig.22E shows that activated Myc-Rac3 was detected only in the MDA-MB 435 cell line, confirming that the regulation of the GDP/GTP state of Rac3 was altered in these cells. We then investigated several upstream stimuli that have been shown to affect GTPase-regulatory proteins (283032). We excluded the possibility of an autocrine growth-stimulatory loop by culturing MDA-MB 231 cells with the conditioned medium from MDA-MB 435, which did not affect the Rac3 activation state (data not shown). Treatment of cell cultures with phosphatidylinositol 3-kinase or tyrosine kinase inhibitors, including wortmannin, LY294002, and genistein, did not decrease Rac3 activation (data not shown). At this point, we speculated that an oncogenic, Rac3-specific GEF is present in certain breast cancer cells. GEFs possess a pleckstrin homology domain that is essential for membrane localization and for their oncogenic properties (533). Analysis of the subcellular localization of the Rac family members revealed that Rac3 is located in the membranes of breast epithelial cell lines, independently of its activation state (Fig. ​(Fig.22F). In contrast, endogenous Rac1 in its inactive GDP-bound state was essentially cytosolic (Fig. ​(Fig.22F). Thus, the distinct localization of Rac3 and Rac1 may contribute to their different activation states in certain breast cancer cell lines. It is conceivable that the highly proliferative cell lines (Fig. ​(Fig.1)1) express a constitutively active, membrane-bound Rho GEF that activates adjacent Rac3 protein. This hypothesis was further supported by using an hydroxymethylglutaryl-CoA reductase inhibitor, lovastatin, that interferes with isoprenoid synthesis and thereby with posttranslational processing of GTPases. Unprocessed Rac3 from lovastatin-treated MDA-MB 435 cells was predominantly cytosolic and inactive (GDP-Rac3) (data not shown). The requirement of membrane localization for consistent Rac3 activity was further supported by using a Rac3S189 mutant. Replacing cysteine-189 of the CAAX box with serine abolishes isoprenoid incorporation, rendering the GTPase cytosolic. This Rac3 mutant remained in its inactive GDP-bound state when transfected into MDA-MB 435 cells (data not shown).

Several Rho GTPase-regulating GEFs have been identified (5), including the Rac1-specific GEF Tiam-1, which has been linked to tumors such as invasive T-lymphomas (34). Although Tiam-1 is expressed in virtually all tissues, no evidence of oncogenic truncations or alternative splicing of Tiam-1 transcripts has been found (35). A variation of Tiam-1 transcript levels in certain cancer cell lines might lead to overexpression and possibly activation of Tiam-1 protein. However, the activation state of Rac3 protein in the cell lines used in this study does not seem to correlate with Tiam-1 expression levels as reported by Habets et al. (35). Hyperactivity of Rac3 in cancer cells could also result from an absent or dysfunctional Rac3-specific GAP protein. By accelerating the intrinsic GTP hydrolysis rate, GAPs render the GTPase inactive and act as tumor suppressors. Deletion or mutations in the RasGAP gene NF1 and the RhoGAP homologs bcr and DLC-1 have been reported in cancer cells (3637).

Active Rac3 Drives Epithelial Cell Proliferation.

To study whether active Rac3 could account for the high proliferation rate of certain breast cancer cells, we expressed a constitutively active Rac3 mutant (Rac3V12) in normal mammary epithelial cells (HMEC 184) that contain only GDP-Rac3 (Fig. ​(Fig.22C). Rac3V12 expression significantly increased the incorporation of BrdUrd into nascent DNA (Fig. ​(Fig.3),3), emphasizing that transfection of active Rac3 drives epithelial cell proliferation.

Rac3V12 induces DNA synthesis in human mammary epithelial cells pq0104939003

Rac3V12 induces DNA synthesis in human mammary epithelial cells pq0104939003


Rac3V12 induces DNA synthesis in human mammary epithelial cells

Figure 3 Rac3V12 induces DNA synthesis in human mammary epithelial cells. HMEC 184 cells, infected with recombinant LacZ or Rac3V12 Semliki Forest virus, were allowed to express protein for 14 h in serum-free medium containing 10 μM BrdUrd. Cells were fixed and stained with anti-Myc antibody for Myc-Rac3V12 expression level (Upper) or with FITC-conjugated anti-BrdUrd antibody for BrdUrd incorporation (Lower). The presence of bright fluorescent nuclei indicates BrdUrd-positive cells. The percentage was calculated after counting 400 cells in each of three independent experiments.

Hyperactive Pak and c-Jun Kinases in Cancer Cells.

The signaling cascade utilized by Rac proteins to control cell proliferation still remains to be identified (19), but might involve Paks. We analyzed Pak activity in cell lysates derived from serum-starved breast cancer cell lines by using in-gel kinase assays and by usingin vitro kinase assays after immunoprecipitation with Pak-specific antibodies. Pak activity was increased 4- to 6-fold in the three cell lines containing active Rac3 (Fig. ​(Fig.44A). This increased kinase activity was mainly associated with the Pak2 isoform, which can phosphorylate and positively regulate Raf-1 activity, another key component in cell proliferation (3840).


Figure 4 Rac3 activates Pak and JNK by two different pathways. (A) Breast cancer cell lysates from serum-starved cells were analyzed for Pak and JNK activities. Pak activities in cell lysates were analyzed by in-gel kinase assays. JNK activity was determined by 

Intracellular Rac-regulated signaling pathways impinge on distinct mitogen-activated protein kinase cascades. Constitutively active Rac has been shown to positively regulate the activity of the stress-activated kinases JNK and p38 (1). Moreover, ERK activity can be indirectly stimulated by Rac or mediated by crosstalk between the distinct mitogen-activated protein kinase cascades (141). Determination of distinct mitogen-activated protein and stress-activated protein kinase activities in the breast cell lines studied here showed that consistent Rac3 and Pak kinase activities were associated with enhanced JNK activity (Fig. ​(Fig.44A). In contrast, no correlation existed between p38 or ERK kinase activities and active Rac3 or Pak (data not shown).

Rac3 Triggers Pak and JNK Activities by Separate Pathways.

To determine whether the highly proliferative phenotype of breast cancer cells depends directly on a consistently active Rac3-Pak-JNK cascade, we used virus-mediated protein expression in MDA-MB 435 cells to examine the ability of Rac3 and Paks to control JNK activation and cellular proliferation. The importance of Pak as an effector protein in Rac-mediated activation of JNK is still controversial and seems to be cell-type-dependent (42). Expression of the PBD domain, which controls the activity of both Rac and Pak (21), completely inhibited Pak and JNK stimulation (Fig. ​(Fig.44B). The mutation of leucine to phenylalanine at position 107 of the PBD domain suppresses the autoinhibitory function of the PBD (21). Thus, PBD F107 will act only to sequester active Rac3 and blocks its ability to bind and activate endogenous effectors. Expression of either dominant-negative Rac3N17 or PBD F107 almost completely blocked Pak and JNK activities, demonstrating that Rac3 is upstream of these proteins (Fig. ​(Fig.44B). Moreover, Pak kinase activity can be inhibited independently of Rac3 by overexpressing the kinase autoinhibitory domain, PID, which does not interact with Rac (2143). Transfection of PID into MDA-MB 435 cells dramatically inhibited Pak activity as expected, but did not decrease JNK activation (Fig. ​(Fig.44B). Our results indicate that in MDA-MB 435 cells, consistent stimulation of JNK by Rac3 is independent of PAK activity and that Rac3 initiates two different pathways involving Pak and JNK, respectively.

Rac3 and Pak Are Both Required for Breast Cancer Cell Proliferation.

We subsequently determined which of these two Rac3 pathways promoted the increased cell proliferation in breast cancer cell lines with hyperactive Rac3. We studied the consequence of expressing inhibitory Rac mutants or Pak fragments on DNA synthesis. LacZ-expressing MDA-MB 435 cells still proliferated in low-serum conditions and 35% incorporated BrdUrd (Fig. ​(Fig.5).5). This percentage increased to 50% when Rac3wt, which will be partially activated in these cells (Fig. ​(Fig.22E), is expressed (Fig. ​(Fig.55 Bottom Right). In contrast, expression of inhibitory proteins, including Rac3N17 or the PBD that suppressed Pak and JNK activation (Fig. ​(Fig.44B), almost completely blocked S-phase entry, as indicated by the absence of BrdUrd incorporation (Fig. ​(Fig.5).5). Expression of the PID that inhibited Pak kinase activity without affecting JNK stimulation (Fig. ​(Fig.44B) also arrested proliferation in MDA-MB 435 cells (Fig. ​(Fig.5).5). These experiments emphasize the crucial role of active Rac3 for DNA synthesis in breast cancer cell lines and demonstrate that Pak kinase activity is necessary for Rac3-induced proliferation.

Rac3 mediates proliferation in MDA-MB 435 cells  pq0104939005

Rac3 mediates proliferation in MDA-MB 435 cells pq0104939005

Rac3 mediates proliferation in MDA-MB 435 cells


Figure 5 Rac3 mediates proliferation in MDA-MB 435 cells by a Pak-dependent pathway. MDA-MB 435 cells growing in 0.5% FBS were infected with Semliki Forest virus encoding for LacZ, Rac3N17, Pak1-PBD, Pak1-PBD F107, Pak1-PID, or Rac3wt. After 12 to14 h of protein expression in serum-free medium, 20 μM BrdUrd was added for 20 min before the cells were fixed and stained with anti-Myc antibody and phalloidin for expression (Top) or with FITC-conjugated anti-BrdUrd antibody for BrdUrd incorporation (Lower five micrographs). The presence of bright fluorescent nuclei indicates BrdUrd-positive cells. The percentage was calculated after counting 400 cells in each of four independent experiments.

Our results establish the persistent activation of a small Rho GTPase, Rac3, and the effector kinase Pak in human breast cancer cells. In contrast to Rac1, endogenous Rac3 is localized at the plasma membrane in both guanine nucleotide states. It seems likely that a Rac3 regulatory protein is altered or deleted in highly proliferating cancer cells, and that its specificity toward Rac3 results from the adjacent location of both proteins at the membrane and/or from discrete Rac3 domains, which convey a specific interaction. The cytoskeletal phenotypes of serum-starved breast cancer cells, such as ruffles or lamellipodia typical of Rac1 protein activation, did not seem to correlate with the GDP versus GTP state of endogenous Rac3. This may suggest that Rac family members are specialized in certain cellular functions, as already reported for Rac2 in leukocyte phagocytosis (44) and now demonstrated by us for Rac3 in cancer cell proliferation. Our studies establish further that endogenous, active Rac3 is essential for breast cancer cell proliferation via a Pak-dependent pathway. Paks have been shown to directly phosphorylate Raf kinase, which binds to retinoblastoma protein and regulates its function (45), and to interact with cyclin-dependent kinases to up-regulate cyclin D1 expression (46). Initial screening of various human cancer-derived cell lines revealed the presence of hyperactive Rac3 and Pak kinase in other types of highly proliferating tumors (data not shown). Further investigations, primarily in animal models and clinical settings, will be necessary to assess whether loss of Rac3 and Pak regulation correlates with certain breast tumor stages and is accompanied by specific alterations in cell-cycle regulators. Approaches to inhibit Rac3 or Pak activity would then open a new avenue for cancer therapeutics.

11.1.12 Curcumin-could-reduce-the-monomer-of-ttr-with-tyr114cys-mutation via autophagy in cell model of familial amyloid polyneuropathy.

Li H1Zhang Y1Cao L1Xiong R1Zhang B1Wu L1Zhao Z1Chen SD2
Drug Des Devel Ther. 2014 Oct 31; 8:2121-8

Transthyretin (TTR) familial amyloid polyneuropathy (FAP) is an autosomal dominant inherited neurodegenerative disorder caused by various mutations in the transthyretin gene. We aimed to identify the mechanisms underlying TTR FAP with Tyr114Cys (Y114C) mutation. Our study showed that TTR Y114C mutation led to an increase in monomeric TTR and impaired autophagy. Treatment with curcumin resulted in a significant decrease of monomeric TTR by recovering autophagy. Our research suggests that impairment of autophagy might be involved in the pathogenesis of TTR FAP with Y114C mutation, and curcumin might be a potential therapeutic approach for TTR FAP.

Transthyretin (TTR) familial amyloid polyneuropathy (FAP) is an autosomal dominant inherited disease, characterized clinically by progressive sensory, motor, and autonomic impairment, which typically lead to death around a decade after diagnosis.1 Since the first identification of TTR with Val30Met mutation (TTR V30M), the most common gene mutation in FAP patients, more than 100 TTR mutations have been found to cause FAP.2 However, the detailed pathogenesis underlying TTR FAP remains undefined. Previous studies of the TTR V30M mutant have shown that misfolding and self-aggregation of TTR are implicated in the pathogenesis of TTR FAP involving abnormal endoplasmic reticulum (ER) stress.3

Corresponding to the various TTR gene mutations and a wide range of geographical distributions, FAP presents diverse characteristics in genotype-phenotype in different regions. We have recently published the first report of a TTR Tyr114Cys (TTR Y114C) mutation in a Chinese family with TTR FAP.4 Compared with TTR V30M, the TTR Y114C mutation showed different clinical manifestations, and was also observed in a Japanese family.5,6 This suggests that the pathogenesis of the TTR Y114C and TTR V30M mutations might be different. Studies focused on monomer generation and tetramer depolymerization have been performed.1,2 However, the mechanisms underlying the clearing of the abnormally increased monomer are unknown.

Autophagy is the major lysosomal pathway via which cells degrade intracytoplasmic protein. It is widely accepted that autophagy plays a key role in the process of amyloid deposition in certain neurodegenerative diseases, including alpha-synuclein, beta peptides, tau oligomers, and misfolded prion protein.7 Therefore, autophagy may be involved in degradation of the TTR monomer in TTR FAP.

Curcumin and its analogs have demonstrated a protective effect in many diseases involving antimicrobial, antitubercular,8 and anticancer mechanisms,9 and they can also modulate innate immunity.10 Of note, curcumin has been shown to promote autophagy.11 Therefore, we hypothesized that autophagy might be involved in the pathogenetic mechanism of the TTR Y114C mutation in TTR FAP and curcumin might have potential therapeutic role in this disease. In this study, we aimed to identify the role of autophagy in the pathogenetic mechanism of TTR FAP and to assess the therapeutic effect of curcumin in the disease.

TTR Y114C mutation led to increased monomeric TTR and impaired autophagy in vitro

To investigate the alteration of monomeric TTR with different mutations, we generated HEK293T cell lines with wild-type TTR, TTR Y114C, and stable overexpression of TTR V30M. Wild-type TTR represented the normal control and TTR V30M represented the positive control. Western blotting analysis of the TTR level in the cells when cultured for 24 hours showed that the monomer of TTR Y114C and TTR V30M was increased by approximately 2.3 times and 2.78 times, respectively, compared with wild-type TTR (Figure 1A and B). Mutation of TTR Y114C was related to the increase in monomeric TTR, as well as the mutation of TTR V30M.

Changes in autophagy and endoplasmic reticulum stress related to wild-type TTR, TTR V30M, and TTR Y114C dddt-8-2121Fig1

Changes in autophagy and endoplasmic reticulum stress related to wild-type TTR, TTR V30M, and TTR Y114C dddt-8-2121Fig1

Changes in autophagy and endoplasmic reticulum stress related to wild-type TTR, TTR V30M, and TTR Y114C


Figure 1 Changes in autophagy and endoplasmic reticulum stress related to wild-type TTR, TTR V30M, and TTR Y114C.

Next we investigated the activation of several markers associated with ER stress, including ER-resident chaperone BiP and p-eIF2α. Our results showed the levels of BiP and p-eIF2α is higher in TTR V30M than those in wild-type TTR. In contrast, BiP and p-eIF2α levels in TTR Y114C were similar to those in wild-type TTR (Figure 1A and C), indicating ER stress might not be the main pathogenetic mechanism for the TTR Y114C mutation. We then investigated whether autophagy plays a role in the mechanism of TTR Y114C mutation. LC3-II is well known to be a robust marker of autophagosomes, and immunofluorescent staining of LC3-II can be used to assay for autophagosome formation. A high ratio of LC3-II to LC3-I would indicate induction of autophagy. Our results revealed that the ratio of LC3-II/I was markedly decreased for TTR Y114C, but less suppressed for TTR V30M (Figure 1A and D). Likewise, a significant decrease in LC3-II immunoreactivity was detected in TTR Y114C (Figure 1E). The results of Western blotting and immunofluorescence indicated that autophagy in TTR Y114C was significantly downregulated. Therefore, impaired autophagy might be responsible for the pathogenesis of TTR Y114C mutation.

Curcumin decreased monomeric TTR by promoting autophagy

The effects of curcumin were investigated in TTR Y114C and wild-type TTR stable overexpressed HEK293T cells. Curcumin did not show toxic effects in the stable overexpressed cell lines at curcumin concentrations below 10 µM (Figure 2A and B). We chose 5 µM as the experimental concentration, because it is the minimal effective concentration of curcumin in these cell lines. Further, we wanted to determine whether curcumin could decrease monomeric TTR by promoting autophagy at the minimal effective concentration. Therefore, we used curcumin (2.5 µM and 5 µM) as a protective agent to assess whether it could decrease monomeric TTR with mutation by promoting autophagy. Quantification of LC3-II and LC3-I indicated markedly higher activation of LC3 in TTR Y114C treated with curcumin 5 µM for 24 hours (Figure 2D). In contrast, treatment with curcumin at different concentrations could not activate LC3 in wild-type TTR (Figure 2C, E). We next examined the ratio of monomers to tetramers in TTR Y114C, which was significantly decreased after 24 hours of treatment with 5 µM curcumin compared with no treatment with curcumin (Figure 2D and F). However, for wild-type TTR, the ratio of monomers to tetramers was unchanged after treatment with curcumin (Figure 2C and E). These results indicate that treatment with curcumin 5 µM for 24 hours was able to decrease the monomer in the TTR Y114C mutation by promoting autophagy.

Curcumin decreased monomeric TTR by promoting autophagy dddt-8-2121Fig2

Curcumin decreased monomeric TTR by promoting autophagy dddt-8-2121Fig2

Curcumin decreased monomeric TTR by promoting autophagy


Figure 2 Curcumin decreased monomeric TTR by promoting autophagy.

Protective effect of curcumin on TTR Y114C could be partially blocked by 3-MA

To further validate whether the decrease in monomer by curcumin in our experiments was mediated by autophagy, 3-MA, an inhibitor of autophagosome formation, was implied to negatively regulate autophagy. 3-MA (1 mM) was added to the cell culture medium 2 hours before curcumin and incubated for 24 hours. Analysis of LC3, tetrameric TTR, and monomeric TTR from TTR Y114C revealed that 3-MA partly reversed the LC3 II activation induced by curcumin and increased the monomer of TTR Y114C (Figure 3). These results confirm that curcumin induced the decrease in the TTR Y114C monomer by promoting the autophagy pathway.


Figure 3 Protective effect of curcumin on TTR Y114C could be partially blocked by 3-MA.


TTR FAP is a severe autosomal dominant inherited disease, for which the treatment options are limited. Liver transplantation performed early in the course of the disease is the only therapeutic strategy known to stabilize this neuropathy.1,13 More recently, tafamidis meglumine, a potent inhibitor of misfolding and deposition of mutated TTR, has completed an 18-month, placebo-controlled Phase II/III clinical trial for the treatment of FAP.14 However, in June 2012, the US Food and Drug Administration Peripheral and Central Nervous System Drugs Advisory Committee rejected this drug, stating a lack of convincing data supporting its efficacy.15 Hence, it is important to identify the pathogenetic mechanism of FAP to find an alternative effective treatment strategy.

Accumulating studies focused on the TTR mutation gene and protein have provided insights into the pathogenesis of TTR FAP, including decreased stability of TTR tetramers, conformational change in the crystal structure of variant TTR, altered kinetics of denaturation, and disturbing endoplasmic ER quality control system.1,1618 Previous studies have demonstrated that increased levels of ER stress are correlated with extracellular TTR deposition. Two ER stress markers, BiP and p-eIF2α, have been observed to be present and upregulated in the salivary gland tissue of FAP patients.3 However, the precise molecular mechanisms underlying TTR FAP and its phenotypic heterogeneity are not yet fully understood.

Our current study investigated whether the two mutations, TTR Y114C and TTR V30M, share the same pathogenesis and evaluated the effect of pathogenic mutations on the clearance of the monomer. Our results show that the ratio of LC3-II/I was markedly decreased, while BiP and p-eIF2α levels remained constant in TTR Y114C when compared with wild-type TTR and TTR 30M. The results of our research indicate the impaired autophagy contributed to the TTR Y114C mutation, but not ER stress. This observation indicates that abnormal accumulation of TTR caused by a different mutation might be cleared by different pathways, and more studies are necessary to confirm whether this difference applies to other TTR mutations.

Curcumin is known to have neuroprotective properties through a variety of mechanisms.811 Our research indicates that curcumin decreased the monomeric TTR by promoting autophagy, and without toxic effects. Moreover, this protective effect of curcumin on TTR Y114C could be partially blocked by 3-MA. Pullakhandam et al showed that curcumin binds to wild-type TTR and prevents urea-induced perturbations in the tertiary structure of TTR in vitro.19 Recently, Ferreira et al reported that dietary curcumin modulated TTR amyloidogenicity.20 Therefore, curcumin might be an effective therapy for FAP involving multiple molecular pathways.

Overall, our findings show that abnormal accumulation of TTR caused by different mutations might be cleared in different ways, and curcumin might be an effective therapy for FAP by promoting autophagy. Further studies are necessary to determine whether this phenomenon exists in other TTR mutations.

Stephen Williams, PhD

For PI3K and related inhibitors of PI3K/AKT/mTOR i would refer you to two people who should be in the discussion of this signaling pathway and PI3K/AKT inhibitors used for chemotherapy. The first is Dr. Mien-Chie Hung and the second is Dr. Gordon Mills. They both had been at MD Anderson and developed some of the first inhibitors as well as the earliest discoveries of overactivity of PI3K/AKT in ovarian cancer.
Next the field had never progressed any inhibitors past Stage II as there has been some serious toxicities seen in preclinical phases (most long term tox studies are done after patients are enrolled in phase I).

I would refer to three papers

Discovery of GSK2126458, a Highly Potent Inhibitor of PI3K and the Mammalian Target of Rapamycin http://pubs.acs.org/doi/abs/10.1021/ml900028r

A new mutational AKTivation in the PI3K pathwayhttp://www.researchgate.net/publication/6146395_A_new_mutational_AKTivation_in_the_PI3K_pathway

These will show how inhibitors of certain isoforms of PI3K (namely delta) had to be developed to circumvent some of the severe toxicity seen with the earliest inhibitors (wortmanin and LY294002.

Take your PIK: phosphatidylinositol 3-kinase inhibitors race through the clinic and toward cancer therapy http://mct.aacrjournals.org/content/8/1/1.full

Targeting the phosphoinositide 3-kinase (PI3K) pathway in cancerhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3142564/

Development of PI3K Inhibitors in Breast Cancer http://www.onclive.com/publications/contemporary-oncology/2014/November-2014/Development-of-PI3K-Inhibitors-in-Breast-Cancer by Aggerwal nice review

Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeuticshttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3843585/ will explain about some of the toxicities and describes the one PI3K that has made it to phase II

Most of them have failed and I believe now are being thought as an adjuvant not front line therapy

Aurelian Udristioiu


Aurelian Udristioiu

Lab Director at Emergency County Hospital Targu Jiu

In experimental models, disrupting the MDM2–p53
interaction restored p53 function and sensitized tumors to
chemotherapy or radiotherapy. (Kojima et al., 2005). This
strategy could be particularly beneficial in treating
cancers that do not harbor TP53 mutations. For example
in hematologic malignancies, such as multiple myeloma,
chronic lymphocytic leukemia (CLL), acute lymphoblastic
leukemia (ALL), acute myeloid leukemia (AML), and
Hodgkin’s disease, the induction of p53 – using a small
MDM2-inhibitor molecule, nutlin-3 – can induce the
apoptosis of malignant cells. Nutlins are a group of cisimidazoline
analogs, first identified by Vassilev et al.
(2004), which have a high binding potency and selectivity
for MDM2. Crystallization data have shown that nutlin-3
mimics the three residues of the helical region of the
trans-activation domain of p53 (Phe19, Trp23 and
Leu26), which are conserved across species and critical
for binding to MDM2 (Wade et al., 2010). Nutlin-3
displaces p53 by competing for MDM2 binding. It has
also been found that nutlin-3 potently induces apoptosis
in cell lines derived from hematologic malignancies,
including AML, myeloma, ALL, and B-cell CLL (Secchiero
et al., 2010).

Stephen J Williams, PhD

Now as far as PKM2 you would want to look at a company called Synta Pharmaceuticals and their inhibitor Elesclomal. elesclomol binds copper ions causing a change in conformation that enables its uptake through membranes and into cells. Elesclomol binds copper in an oxidative, positively charged state called Cu(II). Once inside mitochondria, the elesclomol-Cu(II) complex interacts with the energy production mechanism of the cell, or the electron transport chain. This interaction reduces the copper from Cu(II) to Cu(I), resulting in a cascade of reduction-oxidation, or redox, reactions, that causes a rapid increase of oxidative stress, disruption of mitochondrial energy production, and ultimately, triggering of the mitochondrial apoptosis pathway.

The important part is that it seemed, to prefer tumors which had lower LDH activity, meaning that these tumor cells actually did have a more active electron transport chain than tumors with high LDH (Warburg) and therefore in clinical trials the tumors with lower LDH activity responded more favorably.

http://www.drugs.com/clinical_trials/synta-pharmaceuticals-announces-updated-elesclomol-symmetry-data-presented-melanoma-xiii-8223.html for press release and study results

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