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A book has recently been reviewed by Laura Fisher (Feb 19 2016) titled “Junk DNA: a journey through the dark matter of the genome” (Nessa Carey Icon Books 2015 | 352pp ISBN 9781848319158). http://www.rsc.org/chemistryworld/2016/02/junk-dna-genome-nessa-carey-book-review It is important in its focus on, ‘junk DNA’, a term coined in the 1960s that refers to regions of our DNA that don’t code for proteins. It is now known that a large portion of the genome is noncoding. These non-coding areas of our DNA are far from being without function. Whether regulating gene expression and transcription, or providing protein attachment sites, this once-dismissed part of the genome is vital for all life, and this is the focus of Junk DNA. However, in 1869 Friedrich Miescher discovered a new substance (Dahm, 2008) from the cell nuclei that had chemical properties unlike any protein, including a much higher phosphorous content and resistance to proteolysis (protein digestion). He wrote, “It seems probable to me that a whole family of such slightly varying phosphorous-containing substances will appear, as a group of nucleins, equivalent to proteins” (Wolf, 2003). In 1971, Chargaff noted that Miescher’s discovery of nucleic acids was unique among the discoveries of the four major cellular components (i.e., proteins, lipids, polysaccharides, and nucleic acids) in that it could be “dated precisely… [to] one man one place, one date.” We now know that there are two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), each with two fused rings, and the pyrimidines (cytosine [C], thymine [T], and uracil [U]), each with a single ring. Furthermore, it is now widely accepted that RNA contains only A, G, C, and U (no T), whereas DNA contains only A, G, C, and T (no U). Keeping this in mind, the Watson-Crick proposal, as important as it was, was a discovery out of historical proportion, and it set the path of molecular biology for the remainder of the 20th century. A consequence of this seminal event was that the direction of biochemistry and molecular biology became set for several generations into the 21st century, culminating in the Human Genome Project.
As important as this discovery and others related that followed, there were a number of unrelated discoveries that took on huge importance, immediately recognized, but not so soon integrated with the evolving body of knowledge. 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. There was also a relevance of 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. This involves the effect of one ligand on the binding or catalysis of another with no direct interaction between the two ligands. This was 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 binding at a different, nonoverlapping (regulatory) site, the former could inhibit the enzyme without competing with the latter. Pauling (Pauling 1935) had earlier proposed a model for intramolecular control in hemoglobin to explain the positive cooperativity observed in the binding of oxygen molecules. But Monod, Wyman, and Changeux substantially updated the view of allostery in 1965 with their landmark paper. Present day applications of computational methods to biomolecular systems, combined with structural, thermodynamic, and kinetic studies, make possible an approach to that question, so as to provide a deeper understanding of the requirements for allostery. The current view is that a variety of measurements (e.g., NMR, FRET, and single molecule studies) are providing additional data beyond that available previously from structural, thermodynamic, and kinetic results. These should serve to continue to improve our understanding of the molecular mechanism of allostery, particularly when supplemented by simulations and theoretical analyses. A ‘‘dynamic’’ proposal by Cooper and Dryden (1984) is that the distribution around the average structure changes in allostery; which in turn, affects the subsequent (binding) affinity at a distant site. Such a model focuses on the vibrational contribution to the entropy as the origin of cooperativity, as discussed for the CAPN dimer. Why is this important? It is because it brings a different focus into the conception of how living cells engage with their neighbors and external environment. Moreover, this is not all that has to be considered.
What else do we have to consider? Oxidative stress is essentially an imbalance between the production of free radicals and the ability of the body to counteract or detoxify their harmful effects through neutralization by antioxidants. 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. Common mechanisms involving the Fenton reaction, generation of the superoxide radical and the hydroxyl radical appear to be involved for iron, copper, chromium, vanadium and cobalt primarily associated with mitochondria, microsomes and peroxisomes. 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.
In addition to what I have identified, there is substantial work in the last decade to indicate a more complex model of cellular regulatory processes. On the one hand, there is no uncertainty about the importance of “Junk DNA”. Indeed, not only is “Junk DNA” not junk, but it has either a presence that is an evolutionary remnant, or it has a role in cell regulation, much of which has yet to be understood. Moreover, the relationship between the oligonucleotide sequences to their histones are largely unknown. Beyond the DNA sequences, the language of the gene, we now have a large output of research on noncoding RNA. We now have siRNA, miRNA, and others with roles other than transcription. This is a very active field of investigation that requires major revision of our model of cell regulatory processes. The classic model is solely transcriptional. DNA-> RNA-> Amino Acid in a protein. This would now have to be redrawn because DNA-> RNA-> DNA and DNA->RNA-> protein-> DNA.
I have provided a series of four mechanisms explanatory for transcription and for regulation of the cell. This is not adequate for a more full comprehension because there is a layer beyond the classic model of metabolic pathways associated with the cytoplasm, mitochondria, endoplasmic reticulum, and lysosome, there are critical paths beyond oxidative phosphorylation and glycolysis, such as the cell death pathways, expressed in a homeostasis between apoptosis and repair. Nevertheless, there is still a missing part of this discussion. The missing piece gets at the time and space interactions of the cell, cellular cytoskeleton and extracellular and intracellular substrate interactions in the immediate environment. This can’t be simply accounted for by genetics or epigenetics. There have been papers that call attention to heterogeneity among cancer cells of expected identical type, which would be consistent with differences in phenotypic expression, aligned with epigenetics. There is now the recent publication of the finding that there is heterogeneity in the immediate interstices between cancer cells, which may seem surprising, but it should not be. This refers to the complexity of the cells arranged as tissues and to their immediate environment, which I shall elaborate on. 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. I did introduce the word gene into this reference, and we are well aware of mutations that occur in cancer progression. In the case of breast cancer, mention is not made of interaction with a hormone, as we refer to in androgen-unresponsive prostate cancer. This is particularly relevant, but incomplete.
The fifth item for discussion is the interaction between enzyme and substrates that may be conditionally unidirectional in defining the activity within the cell. When we speak of the genome, we are dealing with a code defined by an oligonucleotide sequence that has an element of stability, but that can conditionally be altered by a process termed mutagenesis. The altered code can be expected to have a negative, positive, or no effect, depending. In any case, there is a substantial stability inherent in the code that is essential to all living creatures. The activity of the cell is dynamically interacting and at high rates of activity. There are many examples of this. The first example is in a study of energy for reverse pyruvate kinase (PK) reaction. This catalytic activity of the PK reaction was reversed to the thermodynamically unfavorable direction in a muscle preparation by a specific inhibitor. Using the same crude supernatant for the two opposite activities of this enzyme some of the results found in the regulatory assays indicated differences in the active form of pyruvate kinase that were clearly related to the environmental condition – glycolitic or glyconeogenetic – of the assay. The conformational changes indicated by differential regulatory response found in the conditions studied, together with the role of similar factors, for instance, substrates and pH, in the structural states proposed by others, were used together 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 according to its vibrational, translational and rotational components and the activating ions – 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 pyridine nucleotide-linked dehydrogenases. The lactate dehydrogenase (LD) reaction is ordered so that NADH binds to the enzyme before pyruvate can bind. The H-type isoenzyme, but not the M-type, is characterized by substrate inhibition at high pyruvate concentrations. The inhibition of the H4 lactate dehydrogenase, but not the M4, by high concentrations of pyruvate is caused by the formation of an abortive complex consisting of the enzyme, pyruvate, and NADH. An investigation of the structural properties of the ternary complex revealed that the complex possesses an absorption maximum at 335 nm and that a covalent bond was formed between the nicotinamide ring of the NAD+ and the pyruvate moiety. The same study demonstrated that the enol form of pyruvate is responsible for the complex formation. It was suggested that abortive complex formation is a significant metabolic control mechanism, and the different behavior of the H and M forms has been rationalized in terms of different functional roles for the two isoenzymes. However, similar experiments carried out with the mitochondrial malate dehydrogenase suggested a similar inhibition, but in this case only the mitochondrial malate dehydrogenase is sensitive to inhibition by high concentrations of oxaloacetate. Further studies showed the inhibition is promoted by an abortive binary complex formed by the enzymes and the enol form of oxalacetate. Neither the oxidized coenzyme nor the reduced coenzyme appears to be involved in the formation of this complex. These results suggest that the mechanism of substrate inhibition that occurs with the pig heart malate dehydrogenases is different from that observed with the lactate dehydrogenases.
It was established years later that there is an isoenzyme of isocitrate dehydrogenase that is characteristic for cancer cells. IDH1 and IDH2 mutations occur frequently in some types of World Health Organization grades 2–4 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. To date, all IDH1 mutations have been identified at the Arg132 codon. Mutations in IDH2 have been identified at the Arg140 codon, as well as at Arg172, which is aligned with IDH1 Arg132. IDH1 and IDH2 mutations are heterozygous in cancer, and they catalyze the production of α-2-hydroxyglutarate. The study found human IDH1 transitions between an inactive open, an inactive semi-open, and a catalytically active closed conformation. In the inactive open conformation, Asp279 occupies the position where the isocitrate substrate normally forms hydrogen bonds with Ser94. This steric hindrance by Asp279 to isocitrate binding is relieved in the active closed conformation.
Finally, what does this have to do with personalized medicine? Personalized medicine has been largely view from a lens of genomics. But genomics is only the reading frame, even taking into consideration the mutations that are found in transition. The living activities of cell processes are dynamic and occur at rapid rates. When we refer to homeostasis and to neoplasia, 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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Pauling, L. 1935. The oxygen equilibrium of hemoglobin and its structural interpretation. Proc. Natl. Acad. Sci. 21: 181–191.
Monod, J., Wyman, J., and Changeux, J.P. 1965. On the nature of allosteric transitions: A plausible model. J. Mol. Biol. 12: 88–118.
Cooper, A. and Dryden, D.T.F. 1984. Allostery without conformational change. Eur. Biophys. J. 11: 103–109.
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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.
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.
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:
Cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis
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.
Cancer cells tend to express a partially inhibited splice variant of pyruvate kinase (PK-M2), leading to decreased pyruvate production.
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.
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.
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.
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
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
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
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
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.
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
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
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.
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.
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
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) http://dx.doi.org:/10.1089/cbr.2008.0486
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
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.
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.
The Warburg discoveries from 1922 on, and the influence on metabolic studies for the next 50 years was immense, and then the revelations of the genetic code took precedence. Throughout this period, however, the brilliant work of Briton Chance, a giant of biochemistry at the University of Pennsylvania, opened new avenues of exploration that led to a recent resurgence in this vital need for answers in cancer research. The next two series of presentations will open up this resurgence of fundamental metabolic research in cancer and even neurodegenerative diseases.
2.1.2.1 Cancer Cell Metabolism. Warburg and Beyond
Described decades ago, the Warburg effect of aerobic glycolysis is a key metabolic hallmark of cancer, yet its significance remains unclear. In this Essay, we re-examine the Warburg effect and establish a framework for understanding its contribution to the altered metabolism of cancer cells.
It is hard to begin a discussion of cancer cell metabolism without first mentioning Otto Warburg. A pioneer in the study of respiration, Warburg made a striking discovery in the 1920s. He found that, even in the presence of ample oxygen, cancer cells prefer to metabolize glucose by glycolysis, a seeming paradox as glycolysis, when compared to oxidative phosphorylation, is a less efficient pathway for producing ATP (Warburg, 1956). The Warburg effect has since been demonstrated in different types of tumors and the concomitant increase in glucose uptake has been exploited clinically for the detection of tumors by fluorodeoxyglucose positron emission tomography (FDG-PET). Although aerobic glycolysis has now been generally accepted as a metabolic hallmark of cancer, its causal relationship with cancer progression is still unclear. In this Essay, we discuss the possible drivers, advantages, and potential liabilities of the altered metabolism of cancer cells (Figure 1). 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.
Figure 1. The Altered Metabolism of Cancer Cells
Drivers (A and B). The metabolic derangements in cancer cells may arise either from the selection of cells that have adapted to the tumor microenvironment or from aberrant signaling due to oncogene activation. The tumor microenvironment is spatially and temporally heterogeneous, containing regions of low oxygen and low pH (purple). Moreover, many canonical cancer-associated signaling pathways induce metabolic reprogramming. Target genes activated by hypoxia inducible factor (HIF) decrease the dependence of the cell on oxygen, whereas Ras, Myc, and Akt can also upregulate glucose consumption and glycolysis. Loss of p53 may also recapitulate the features of the Warburg effect, that is, the uncoupling of glycolysis from oxygen levels. Advantages (C–E). The altered metabolism of cancer cells is likely to imbue them with several proliferative and survival advantages, such as enabling cancer cells to execute the biosynthesis of macromolecules (C), to avoid apoptosis (D), and to engage in local metabolite-based paracrine and autocrine signaling (E). Potential Liabilities (F and G). This altered metabolism, however, may also confer several vulnerabilities on cancer cells. For example, an upregulated metabolism may result in the build up of toxic metabolites, including lactate and noncanonical nucleotides, which must be disposed of (F). Moreover, cancer cells may also exhibit a high energetic demand, for which they must either increase flux through normal ATP-generating processes, or else rely on an increased diversity of fuel sources (G).
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).
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 (Wiesener et al., 2001; Zhong et al., 1999). Therefore, the Warburg effect—that is, an uncoupling of glycolysis from oxygen levels—cannot be explained solely by upregulation of HIF.
Recent work 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; Samanathan 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. Loss of the tumor suppressor protein p53 prevents expression of the gene encoding SCO2 (the synthesis of cytochrome c oxidase protein), which interferes with the function of the mitochondrial respiratory chain (Matoba et al., 2006). A second p53 effector, TIGAR (TP53-induced glycolysis and apoptosis regulator), inhibits glycolysis by decreasing levels of fructose-2,6-bisphosphate, a potent stimulator of glycolysis and inhibitor of gluconeogenesis (Bensaad et al., 2006). Other work also suggests that p53-mediated regulation of glucose metabolism may be dependent on the transcription factor NF-κB (Kawauchi et al., 2008).
It has been shown that inhibition of lactate dehydrogenase A (LDH-A) prevents the Warburg effect and forces cancer cells to revert to oxidative phosphorylation in order to reoxidize NADH and produce ATP (Fantin et al., 2006; Shim et al., 1997). While the cells are respiratory competent, they exhibit attenuated tumor growth, suggesting that aerobic glycolysis might be essential for cancer progression. In a primary fibroblast cell culture model of stepwise malignant transformation through overexpression of telomerase, large and small T antigen, and the H-Ras oncogene, increasing tumorigenicity correlates with sensitivity to glycolytic inhibition. This finding suggests that the Warburg effect might be inherent to the molecular events of transformation (Ramanathan et al., 2005). However, the introduction of similar defined factors into human mesenchymal stem cells (MSCs) revealed that transformation can be associated with increased dependence on oxidative phosphorylation (Funes et al., 2007). Interestingly, when introduced in vivo these transformed MSCs do upregulate glycolytic genes, an effect that is reversed when the cells are explanted and cultured under normoxic conditions. These contrasting models suggest that the Warburg effect may be context dependent, in some cases driven by genetic changes and in others by the demands of the microenvironment. Regardless of whether the tumor microenvironment or oncogene activation plays a more important role in driving the development of a distinct cancer metabolism, it is likely that the resulting alterations confer adaptive, proliferative, and survival advantages on the cancer cell.
Altered Metabolism Provides Substrates for Biosynthetic Pathways
Although studies in cancer metabolism have largely been energy-centric, rapidly dividing cells have diverse requirements. Proliferating cells require not only ATP but also nucleotides, fatty acids, membrane lipids, and proteins, and a reprogrammed metabolism may serve to support synthesis of macromolecules. Recent studies have shown that several steps in lipid synthesis are required for and may even actively promote tumorigenesis. Inhibition of ATP citrate lyase, the distal enzyme that converts mitochondrial-derived citrate into cytosolic acetyl coenzyme A, the precursor for many lipid species, prevents cancer cell proliferation and tumor growth (Hatzivassiliou et al., 2005). Fatty acid synthase, expressed at low levels in normal tissues, is upregulated in cancer and may also be required for tumorigenesis (reviewed in Menendez and Lupu, 2007). Furthermore, cancer cells may also enhance their biosynthetic capabilities by expressing a tumor-specific form of pyruvate kinase (PK), M2-PK. Pyruvate kinase catalyzes the third irreversible reaction of glycolysis, the conversion of phosphoenolpyruvate (PEP) to pyruvate. Surprisingly, the M2-PK of cancer cells is thought to be less active in the conversion of PEP to pyruvate and thus less efficient at ATP production (reviewed in Mazurek et al., 2005). A major advantage to the cancer cell, however, is that the glycolytic intermediates upstream of PEP might be shunted into synthetic processes.
Biosynthesis, in addition to causing an inherent increase in ATP demand in order to execute synthetic reactions, should also cause a decrease in ATP supply as various glycolytic and Krebs cycle intermediates are diverted. Lipid synthesis, for example, requires the cooperation of glycolysis, the Krebs cycle, and the pentose phosphate shunt. As pyruvate must enter the mitochondria in this case, it avoids conversion to lactate and therefore cannot contribute to glycolysis-derived ATP. Moreover, whereas increased biosynthesis may explain the glucose hunger of cancer cells, it cannot explain the increase in lactic acid production originally described by Warburg, suggesting that lactate must also result from the metabolism of non-glucose substrates. Recently, it has been demonstrated that glutamine may be metabolized by the citric acid cycle in cancer cells and converted into lactate, producing NADPH for lipid biosynthesis and oxaloacetate for replenishment of Krebs cycle intermediates (DeBerardinis et al., 2007).
Metabolic Pathways Regulate Apoptosis
In addition to involvement in proliferation, altered metabolism may promote another cancer-essential function: the avoidance of apoptosis. Loss of the p53 target TIGAR sensitizes cancer cells to apoptosis, most likely by causing an increase in reactive oxygen species (Bensaad et al., 2006). On the other hand, overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) prevents caspase-independent cell death, presumably by stimulating glycolysis, increasing cellular ATP levels, and promoting autophagy (Colell et al., 2007). Whether or not GAPDH plays a physiological role in the regulation of cell death remains to be determined. Intriguingly, Bonnet et al. (2007) have reported that treating cancer cells with dichloroacetate (DCA), a small molecule inhibitor of pyruvate dehydrogenase kinase, has striking effects on their survival and on xenograft tumor growth.
DCA, a currently approved treatment for congenital lactic acidosis, activates oxidative phosphorylation and promotes apoptosis by two mechanisms. First, increased flux through the electron transport chain causes depolarization of the mitochondrial membrane potential (which the authors found to be hyperpolarized specifically in cancer cells) and release of the apoptotic effector cytochrome c. Second, an increase in reactive oxygen species generated by oxidative phosphorylation upregulates the voltage-gated K+ channel, leading to potassium ion efflux and caspase activation. Their work suggests that cancer cells may shift their metabolism to glycolysis in order to prevent cell death and that forcing cancer cells to respire aerobically can counteract this adaptation.
Cancer Cells May Signal Locally in the Tumor Microenvironment
Cancer cells may rewire metabolic pathways to exploit the tumor microenvironment and to support cancer-specific signaling. Without access to the central circulation, it is possible that metabolites can be concentrated locally and reach suprasystemic levels, allowing cancer cells to engage in metabolite-mediated autocrine and paracrine signaling that does not occur in normal tissues. So called androgen-independent prostate cancers may only be independent from exogenous, adrenal-synthesized androgens. Androgen-independent prostate cancer cells still express the androgen receptor and may be capable of autonomously synthesizing their own androgens (Stanbrough et al., 2006).
Metabolism as an Upstream Modulator of Signaling Pathways
Not only is metabolism downstream of oncogenic pathways, but an altered upstream metabolism may affect the activity of signaling pathways that normally sense the state of the cell. Individuals with inherited mutations in succinate dehydrogenase and fumarate hydratase develop highly angiogenic tumors, not unlike those exhibiting loss of the VHL tumor suppressor protein that acts upstream of HIF (reviewed in Kaelin and Ratcliffe, 2008). The mechanism of tumorigenesis in these cancer syndromes is still contentious. However, it has been proposed that loss of succinate dehydrogenase and fumarate hydratase causes an accumulation of succinate or fumarate, respectively, leading to inhibition of the prolyl hydroxylases that mark HIF for VHL-mediated degradation (Isaacs et al., 2005; Pollard et al., 2005; Selak et al., 2005). In this rare case, succinate dehydrogenase and fumarate hydratase are acting as bona fide tumor suppressors.
There are many complex questions to be answered: Is it possible that cancer cells exhibit “metabolite addiction”? Are there unique cancer-specific metabolic pathways, or combinations of pathways, utilized by the cancer cell but not by normal cells? Are different stages of metabolic adaptations required for the cancer cell to progress from the primary tumor stage to invasion to metastasis? How malleable is cancer metabolism?
2.1.2.2 Cancer metabolism. The Warburg effect today
One of the first studies on the energy metabolism of a tumor was carried out, in 1922, in the laboratory of Otto Warburg. He established that cancer cells exhibited a specific metabolic pattern, characterized by a shift from respiration to fermentation, which has been later named the Warburg effect. Considerable work has been done since then, deepening our understanding of the process, with consequences for diagnosis and therapy. This review presents facts and perspectives on the Warburg effect for the 21st century.
Research highlights
► Warburg first established a tumor metabolic pattern in the 1920s. ► Tumors’ increased glucose uptake has been studied since then. ► Cancer bioenergetics’ study provides insights in all its hallmarks. ► New cancer diagnostic and therapeutic techniques focus on cancer metabolism.
Introduction
Contestation to Warburg’s ideas
Glucose’s uptake and intracellular fates
Lactate production and induced acidosis
Hypoxia
Impairment of mitochondrial function
Tumour microenvironment
Proliferating versus cancer cells
More on cancer bioenergetics – integration of metabolism
Perspectives
2.1.2.3 New aspects of the Warburg effect in cancer cell biology
Altered cellular metabolism is a defining feature of cancer [1]. The best studied metabolic phenotype of cancer is aerobic glycolysis–also known as the Warburg effect–characterized by increased metabolism of glucose to lactate in the presence of sufficient oxygen. Interest in the Warburg effect has escalated in recent years due to the proven utility of FDG-PET for imaging tumors in cancer patients and growing evidence that mutations in oncogenes and tumor suppressor genes directly impact metabolism. The goals of this review are to provide an organized snapshot of the current understanding of regulatory mechanisms important for Warburg effect and its role in tumor biology. Since several reviews have covered aspects of this topic in recent years, we focus on newest contributions to the field and reference other reviews where appropriate.
Highlights
► This review discusses regulatory mechanisms that contribute to the Warburg effect in cancer. ► We list cancers for which FDG-PET has established applications as well as those cancers for which FDG-PET has not been established. ► PKM2 is highlighted as an important integrator of diverse cellular stimuli to modulate metabolic flux and cancer cell proliferation. ► We discuss how cancer metabolism can directly influence gene expression programs. ► Contribution of aerobic glycolysis to the cancer microenvironment and chemotherapeutic resistance/susceptibility is also discussed.
Regulation of the Warburg effect
PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation
Fig. 1. PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation
Metabolism can directly influence gene expression programs
Fig. 2. Metabolism can directly influence gene expression programs. A schematic representation of how metabolism can intrinsically influence epigenetics resulting in durable and heritable gene expression programs in progeny.
2.1.2.4 Choosing between glycolysis and oxidative phosphorylation. A tumor’s dilemma
A considerable amount of knowledge has been produced during the last five years on the bioenergetics of cancer cells, leading to a better understanding of the regulation of energy metabolism during oncogenesis, or in adverse conditions of energy substrate intermittent deprivation. The general enhancement of the glycolytic machinery in various cancer cell lines is well described and recent analyses give a better view of the changes in mitochondrial oxidative phosphorylation during oncogenesis. While some studies demonstrate a reduction of oxidative phosphorylation (OXPHOS) capacity in different types of cancer cells, other investigations revealed contradictory modifications with the upregulation of OXPHOS components and a larger dependency of cancer cells on oxidative energy substrates for anabolism and energy production. This apparent conflictual picture is explained by differences in tumor size, hypoxia, and the sequence of oncogenes activated. The role of p53, C-MYC, Oct and RAS on the control of mitochondrial respiration and glutamine utilization has been explained recently on artificial models of tumorigenesis. Likewise, the generation of induced pluripotent stem cells from oncogene activation also showed the role of C-MYC and Oct in the regulation of mitochondrial biogenesis and ROS generation. In this review article we put emphasis on the description of various bioenergetic types of tumors, from exclusively glycolytic to mainly OXPHOS, and the modulation of both the metabolic apparatus and the modalities of energy substrate utilization according to tumor stage, serial oncogene activation and associated or not fluctuating microenvironmental substrate conditions. We conclude on the importance of a dynamic view of tumor bioenergetics.
Research Highlights
►The bioenergetics of cancer cells differs from normals. ►Warburg hypothesis is not verified in tumors using mitochondria to synthesize ATP. ►Different oncogenes can either switch on or switch off OXPHOS. ►Bioenergetic profiling is a prerequisite to metabolic therapy. ►Aerobic glycolysis and OXPHOS cooperate during cancer progression.
Cancer cell variable bioenergetics
Cancer cells exhibit profound genetic, bioenergetic and histological differences as compared to their non-transformed counterpart. All these modifications are associated with unlimited cell growth, inhibition of apoptosis and intense anabolism. Transformation from a normal cell to a malignant cancer cell is a multi-step pathogenic process which includes a permanent interaction between cancer gene activation (oncogenes and/or tumor-suppressor genes), metabolic reprogramming and tumor-induced changes in microenvironment. As for the individual genetic mapping of human tumors, their metabolic characterization (metabolic–bioenergetic profiling) has evidenced a cancer cell-type bioenergetic signature which depends on the history of the tumor, as composed by the sequence of oncogenes activated and the confrontation to intermittent changes in oxygen, glucose and amino-acid delivery.
In the last decade, bioenergetic studies have highlighted the variability among cancer types and even inside a cancer type as regards to the mechanisms and the substrates preferentially used for deriving the vital energy. The more popular metabolic remodeling described in tumor cells is an increase in glucose uptake, the enhancement of glycolytic capacity and a high lactate production, along with the absence of respiration despite the presence of high oxygen concentration (Warburg effect) [1]. To explain this abnormal bioenergetic phenotype pioneering hypotheses proposed the impairment of mitochondrial function in rapidly growing cancer cells [2].
Although the increased consumption of glucose by tumor cells was confirmed in vivo by positron emission tomography (PET) using the glucose analog 2-(18F)-fluoro-2-deoxy-d-glucose (FDG), the actual utilization of glycolysis and oxidative phosphorylation (OXPHOS) cannot be evaluated with this technique. Nowadays, Warburg’s “aerobic-glycolysis” hypothesis has been challenged by a growing number of studies showing that mitochondria in tumor cells are not inactive per se but operate at low capacity [3] or, in striking contrast, supply most of the ATP to the cancer cells [4]. Intense glycolysis is effectively not observed in all tumor types. Indeed not all cancer cells grow fast and intense anabolism is not mandatory for all cancer cells. Rapidly growing tumor cells rely more on glycolysis than slowly growing tumor cells. This is why a treatment with bromopyruvate, for example is very efficient only on rapidly growing cells and barely useful to decrease the growth rate of tumor cells when their normal proliferation is slow. Already in 1979, Reitzer and colleagues published an article entitled “Evidence that glutamine, not sugar, is the major energy source for cultured Hela cells”, which demonstrated that oxidative phosphorylation was used preferentially to produce ATP in cervical carcinoma cells [5]. Griguer et al. also identified several glioma cell lines that were highly dependent on mitochondrial OXPHOS pathway to produce ATP [6]. Furthermore, a subclass of glioma cells which utilize glycolysis preferentially (i.e., glycolytic gliomas) can also switch from aerobic glycolysis to OXPHOS under limiting glucose conditions [7] and [8], as observed in cervical cancer cells, breast carcinoma cells, hepatoma cells and pancreatic cancer cells [9], [10] and [11]. This flexibility shows the interplay between glycolysis and OXPHOS to adapt the mechanisms of energy production to microenvironmental changes as well as differences in tumor energy needs or biosynthetic activity. Herst and Berridge also demonstrated that a variety of human and mouse leukemic and tumor cell lines (HL60, HeLa, 143B, and U937) utilize mitochondrial respiration to support their growth [12]. Recently, the measurement of OXPHOS contribution to the cellular ATP supply revealed that mitochondria generate 79% of the cellular ATP in HeLa cells, and that upon hypoxia this contribution is reduced to 30% [4]. Again, metabolic flexibility is used to survive under hypoxia. All these studies demonstrate that mitochondria are efficient to synthesize ATP in a large variety of cancer cells, as reviewed by Moreno-Sanchez [13]. Despite the observed reduction of the mitochondrial content in tumors [3], [14], [15], [16], [17], [18] and [19], cancer cells maintain a significant level of OXPHOS capacity to rapidly switch from glycolysis to OXPHOS during carcinogenesis. This switch is also observed at the level of glutamine oxidation which can occur through two modes, “OXPHOS-linked” or “anoxic”, allowing to derive energy from glutamine or serine regardless of hypoxia or respiratory chain reduced activity [20].
While glutamine, glycine, alanine, glutamate, and proline are typically oxidized in normal and tumor mitochondria, alternative substrate oxidations may also contribute to ATP supply by OXPHOS. Those include for instance the oxidation of fatty-acids, ketone bodies, short-chain carboxylic acids, propionate, acetate and butyrate (as recently reviewed in [21]).
Varying degree of mitochondrial utilization during tumorigenesis
In vivo metabolomic analyses suggest the existence of a continuum of bioenergetic remodeling in rat tumors according to tumor size and its rate of growth [22]. Peter Vaupel’s group showed that small tumors were characterized by a low conversion of glucose to lactate whereas the conversion of glutamine to lactate was high. In medium sized tumors the flow of glucose to lactate as well as oxygen utilization was increased whereas glutamine and serine consumption were reduced. At this stage tumor cells started with glutamate and alanine production. Large tumors were characterized by a low oxygen and glucose supply but a high glucose and oxygen utilization rate. The conversion of glucose to glycine, alanine, glutamate, glutamine, and proline reached high values and the amino acids were released [22]. Certainly, in the inner layers constituting solid tumors, substrate and oxygen limitation is frequently observed. Experimental studies tried to reproduce these conditions in vitro and revealed that nutrients and oxygen limitation does not affect OXPHOS and cellular ATP levels in human cervix tumor [23]. Furthermore, the growth of HeLa cells, HepG2 cells and HTB126 (breast cancer) in aglycemia and/or hypoxia even triggered a compensatory increase in OXPHOS capacity, as discussed above. Yet, the impact of hypoxia might be variable depending on cell type and both the extent and the duration of oxygen limitation.
In two models of sequential oncogenesis, the successive activation of specific oncogenes in non-cancer cells evidenced the need for active OXPHOS to pursue tumorigenesis. Funes et al. showed that the transformation of human mesenchymal stem cells increases their dependency on OXPHOS for energy production [24], while Ferbeyre et al. showed that cells expressing oncogenic RAS display an increase in mitochondrial mass, mitochondrial DNA, and mitochondrial production of reactive oxygen species (ROS) prior to the senescent cell cycle arrest [25]. Such observations suggest that waves of gene regulation could suppress and then restore OXPHOS in cancer cells during tumorigenesis [20]. Therefore, the definition of cancer by Hanahan and Weinberg [26] restricted to six hallmarks (1—self-sufficiency in growth signals, 2—insensitivity to growth-inhibitory (antigrowth) signals, 3—evasion of programmed cell death (apoptosis), 4—limitless replicative potential, 5—sustained angiogenesis, and 6—tissue invasion and metastases) should also include metabolic reprogramming, as the seventh hallmark of cancer. This amendment was already proposed by Tennant et al. in 2009 [27]. In 2006, the review Science published a debate on the controversial views of Warburg theory [28], in support of a more realistic description of cancer cell’s variable bioenergetic profile. The pros think that high glycolysis is an obligatory feature of human tumors, while the cons propose that high glycolysis is not exclusive and that tumors can use OXPHOS to derive energy. A unifying theory closer to reality might consider that OXPHOS and glycolysis cooperate to sustain energy needs along tumorigenesis [20]. The concept of oxidative tumors, against Warburg’s proposal, was introduced by Guppy and colleagues, based on the observation that breast cancer cells can generate 80% of their ATP by the mitochondrion [29]. The comparison of different cancer cell lines and excised tumors revealed a variety of cancer cell’s bioenergetic signatures which raised the question of the mechanisms underlying tumor cell metabolic reprogramming, and the relative contribution of oncogenesis and microenvironment in this process. It is now widely accepted that rapidly growing cancer cells within solid tumors suffer from a lack of oxygen and nutrients as tumor grows. In such situation of compromised energy substrate delivery, cancer cell’s metabolic reprogramming is further used to sustain anabolism (Fig. 1), through the deviation of glycolysis, Krebs cycle truncation and OXPHOS redirection toward lipid and protein synthesis, as needed to support uncontrolled tumor growth and survival [30] and [31]. Again, these features are not exclusive to all tumors, as Krebs cycle truncation was only observed in some cancer cells, while other studies indicated that tumor cells can maintain a complete Krebs cycle [13] in parallel with an active citrate efflux. Likewise, generalizations should be avoided to prevent over-interpretations.
Fig. 1. Energy metabolism at the crossroad between catabolism and anabolism.
Energy metabolism at the crossroad between catabolism and anabolism.
The oncogene C-MYC participate to these changes via the stimulation of glutamine utilization through the coordinate expression of genes necessary for cells to engage in glutamine catabolism [30]. According to Newsholme EA and Board M [32] both glycolysis and glutaminolysis not only serve for ATP production, but also provide precious metabolic intermediates such as glucose-6-phosphate, ammonia and aspartate required for the synthesis of purine and pyrimidine nucleotides (Fig. 1). In this manner, the observed apparent excess in the rates of glycolysis and glutaminolysis as compared to the requirement for energy production could be explained by the need for biosynthetic processes. Yet, one should not reduce the shift from glycolysis to OXPHOS utilization to the sole activation of glutaminolysis, as several other energy substrates can be used by tumor mitochondria to generate ATP [21]. The contribution of these different fuels to ATP synthesis remains poorly investigated in human tumors.
The metabolism of pre-cancer cells and its ongoing modulation by carcinogenesis
At the beginning of cancer, there might have been a cancer stem cell hit by an oncogenic event, such as alterations in mitogen signaling to extracellular growth factor receptors (EGFR), oncogenic activation of these receptors, or oncogenic alterations of downstream targets in the pathways that leads to cell proliferation (RAS–Raf–ERK and PI3K–AKT, both leading to m-TOR activation stimulating cell growth). Alterations of checkpoint genes controlling the cell cycle progression like Rb also participate in cell proliferation (Fig. 2) and this re-entry in the cell cycle implies three major needs to fill in: 1) supplying enough energy to grow and 2) synthesize building blocks de novo and 3) keep vital oxygen and nutrients available. However, the bioenergetic status of the pre-cancer cell could determine in part the evolution of carcinogenesis, as shown on mouse embryonic stem cells. In this study, Schieke et al. showed that mitochondrial energy metabolism modulates both the differentiation and tumor formation capacity of mouse embryonic stem cells [37]. The idea that cancer derives from a single cell, known as the cancer stem cell hypothesis, was introduced by observations performed on leukemia which appeared to be organized as origination from a primitive hematopoietic cell [38]. Nowadays cancer stem cells were discovered for all types of tumors [39], [40], [41] and [42], but little is known of their bioenergetic properties and their metabolic adaptation to the microenvironment. This question is crucial as regards the understanding of what determines the wide variety of cancer cell’s metabolic profile.
Impact of different oncogenes on tumor progression and energy metabolism remodeling.
Fig. 2. Impact of different oncogenes on tumor progression and energy metabolism remodeling.
The analysis of the metabolic changes that occur during the transformation of adult mesenchymal stem cells revealed that these cells did not switch to aerobic glycolysis, but their dependency on OXPHOS was even increased [24]. Hence, mitochondrial energy metabolism could be critical for tumorigenesis, in contrast with Warburg’s hypothesis. As discussed above, the oncogene C-MYC also stimulates OXPHOS [30]. Furthermore, it was recently demonstrated that cells chronically treated with oligomycin repress OXPHOS and produce larger tumors with higher malignancy [19]. Likewise, alteration of OXPHOS by mutations in mtDNA increases tumorigenicity in different types of cancer cells [43], [44] and [45].
Recently, it was proposed that mitochondrial energy metabolism is required to generate reactive oxygen species used for the carcinogenetic process induced by the K-RAS mutation [46]. This could explain the large number of mitochondrial DNA mutations found in several tumors. The analysis of mitochondria in human embryonic cells which derive energy exclusively from anaerobic glycolysis have demonstrated an immature mitochondrial network characterized by few organelles with poorly developed cristae and peri-nuclear distribution [47] and [48]. The generation of human induced pluripotent stem cell by the introduction of different oncogenes as C-MYC and Oct4 reproduced this reduction of mitochondrial OXPHOS capacity[49] and [50]. This indicates again the impact of oncogenes on the control of OXPHOS and might explain the existence of pre-cancer stem cells with different bioenergetic backgrounds, as modeled by variable sequences of oncogene activation. Accordingly, the inhibition of mitochondrial respiratory chain has been recently found associated with enhancement of hESC pluripotency [51].
Based on the experimental evidence discussed above, one can argue that 1) glycolysis is indeed a feature of several tumors and associates with faster growth in high glucose environment, but 2) active OXPHOS is also an important feature of (other) tumors taken at a particular stage of carcinogenesis which might be more advantageous than a “glycolysis-only” type of metabolism in conditions of intermittent shortage in glucose delivery. The metabolic apparatus of cancer cells is not fixed during carcinogenesis and might depend both on the nature of the oncogenes activated and the microenvironment. It was indeed shown that cancer cells with predominant glycolytic metabolism present a higher malignancy when submitted to carcinogenetic induction and analysed under fixed experimental conditions of high glucose [19]. Yet, if one grows these cells in a glucose-deprived medium they shift their metabolism toward predominant OXPHOS, as shown in HeLa cells and other cell types [9]. Therefore, one might conclude that glycolytic cells have a higher propensity to generate aggressive tumors when glucose availability is high. However, these cells can become OXPHOS during tumor progression [24] and [52]. All these observations indicate again the importance of maintaining an active OXPHOS metabolism to permit evolution of both embryogenesis and carcinogenesis, which emphasizes the importance of targeting mitochondria to alter this malignant process.
Oncogenes and the modulation of energy metabolism
Several oncogenes and associated proteins such as HIF-1α, RAS, C-MYC, SRC, and p53 can influence energy substrate utilization by affecting cellular targets, leading to metabolic changes that favor cancer cell survival, independently of the control of cell proliferation. These oncogenes stimulate the enhancement of aerobic glycolysis, and an increasing number of studies demonstrate that at least some of them can also target directly the OXPHOS machinery, as discussed in this article (Fig. 2). For instance, C-MYC can concurrently drive aerobic glycolysis and/or OXPHOS according to the tumor cell microenvironment, via the expression of glycolytic genes or the activation of mitochondrial oxidation of glutamine [53]. The oncogene RAS has been shown to increase OXPHOS activity in early transformed cells [24], [52] and [54] and p53 modulates OXPHOS capacity via the regulation of cytochrome c oxidase assembly [55]. Hence, carcinogenic p53 deficiency results in a decreased level of COX2 and triggers a shift toward anaerobic metabolism. In this case, lactate synthesis is increased, but cellular ATP levels remain stable [56]. The p53-inducible isoform of phosphofructokinase, termed TP53-induced glycolysis and apoptotic regulator, TIGAR, a predominant phosphatase activity isoform of PFK-2, has also been identified as an important regulator of energy metabolism in tumors [57].
Tumor specific isoforms (or mutated forms) of energy genes
Tumors are generally characterized by a modification of the glycolytic system where the level of some glycolytic enzymes is increased, some fetal-like isozymes with different kinetic and regulatory properties are produced, and the reverse and back-reactions of the glycolysis are strongly reduced [60]. The GAPDH marker of the glycolytic pathway is also increased in breast, gastric, lung, kidney and colon tumors [18], and the expression of glucose transporter GLUT1 is elevated in most cancer cells. The group of Cuezva J.M. developed the concept of cancer bioenergetic signature and of bioenergetic index to describe the metabolic profile of cancer cells and tumors [18], [61], [64], [65]. This signature describes the changes in the expression level of proteins involved in glycolysis and OXPHOS, while the BEC index gives a ratio of OXPHOS protein content to glycolytic protein content, in good correlation with cancer prognostic[61]. Recently, this group showed that the beta-subunit of the mitochondrial F1F0-ATP synthase is downregulated in a large number of tumors, thus contributing to the Warburg effect [64] and [65]. It was also shown that IF1 expression levels were increased in hepatocellular carcinomas, possibly to prevent the hydrolysis of glytolytic ATP [66]. Numerous changes occur at the level of OXPHOS and mitochondrial biogenesis in human tumors, as we reviewed previously [67]. Yet the actual impact of these changes in OXPHOS protein expression level or catalytic activities remains to be evaluated on the overall fluxes of respiration and ATP synthesis. Indeed, the metabolic control analysis and its extension indicate that it is often required to inhibit activity beyond a threshold of 70–85% to affect the metabolic fluxes [68] and [69]. Another important feature of cancer cells is the higher level of hexokinase II bound to mitochondrial membrane (50% in tumor cells). A study performed on human gliomas (brain) estimated the mitochondrial bound HK fraction (mHK) at 69% of total, as compared to 9% for normal brain [70]. This is consistent with the 5-fold amplification of the type II HK gene observed by Rempel et al. in the rapidly growing rat AS-30D hepatoma cell line, relative to normal hepatocytes [71]. HKII subcellular fractionation in cancer cells was described in several studies [72], [73] and [74]. The group led by Pete Pedersen explained that mHK contributes to (i) the high glycolytic capacity by utilizing mitochondrially regenerated ATP rather than cytosolic ATP (nucleotide channelling) and (ii) the lowering of OXPHOS capacity by limiting Pi and ADP delivery to the organelle [75] and [76].
All these observations are consistent with the increased rate of FDG uptake observed by PET in living tumors which could result from both an increase in glucose transport, and/or an increase in hexokinase activity. However, FDG is not a complete substrate for glycolysis (it is only transformed into FDG-6P by hexokinase before to be eliminated) and cannot be used to evidence a general increase in the glycolytic flux. Moreover, FDG-PET scan also gives false positive and false negative results, indicating that some tumors do not depend on, or do not have, an increased glycolytic capacity. The fast glycolytic system described above is further accommodated in cancer cells by an increase in the lactate dehydrogenase isoform A (LDH-A) expression level. This isoform presents a higher Vmax useful to prevent the inhibition of high glycolysis by its end product (pyruvate) accumulation. Recently, Fantin et al. showed that inhibition of LDH-A in tumors diminishes tumorigenicity and was associated with the stimulation of mitochondrial respiration [79]. The preferential expression of the glycolytic pyruvate kinase isoenzyme M2 (PKM2) in tumor cells, determines whether glucose is converted to lactate for regeneration of energy (active tetrameric form, Warburg effect) or used for the synthesis of cell building blocks (nearly inactive dimeric form) [80]. In the last five years, mutations in proteins of the respiratory system (SDH, FH) and of the TCA cycle (IDH1,2) leading to the accumulation of metabolite and the subsequent activation of HIF-1α were reported in a variety of human tumors [81], [82] and [83].
Tumor microenvironment modulates cancer cell’s bioenergetics
It was extensively described how hypoxia activates HIF-1α which stimulates in turn the expression of several glycolytic enzymes such as HK2, PFK, PGM, enolase, PK, LDH-A, MCT4 and glucose transporters Glut 1 and Glut 3. It was also shown that HIF-1α can reduce OXPHOS capacity by inhibiting mitochondrial biogenesis [14] and [15], PDH activity [87] and respiratory chain activity [88]. The low efficiency and uneven distribution of the vascular system surrounding solid tumors can lead to abrupt changes in oxygen (intermittent hypoxia) but also energy substrate delivery. .. The removal of glucose, or the inhibition of glycolysis by iodoacetate led to a switch toward glutamine utilization without delay followed by a rapid decrease in acid release. This illustrates once again how tumors and human cancer cell lines can utilize alternative energy pathway such as glutaminolysis to deal with glucose limitation, provided the presence of oxygen. It was also observed that in situations of glucose limitation, tumor derived-cells can adapt to survive by using exclusively an oxidative energy substrate [9] and [10]. This is typically associated with an enhancement of the OXPHOS system. … In summary, cancer cells can survive by using exclusively OXPHOS for ATP production, by altering significantly mitochondrial composition and form to facilitate optimal use of the available substrate (Fig. 3). Yet, glucose is needed to feed the pentose phosphate pathway and generate ribose essential for nucleotide biosynthesis. This raises the question of how cancer cells can survive in the growth medium which do not contain glucose (so-called “galactose medium” with dialysed serum [9]). In the OXPHOS mode, pyruvate, glutamate and aspartate can be derived from glutamine, as glutaminolysis can replenish Krebs cycle metabolic pool and support the synthesis of alanine and NADPH [31]. Glutamine is a major source for oxaloacetate (OAA) essential for citrate synthesis. Moreover, the conversion of glutamine to pyruvate is associated with the reduction of NADP+ to NADPH by malic enzyme. Such NADPH is a required electron donor for reductive steps in lipid synthesis, nucleotide metabolism and GSH reduction. In glioblastoma cells the malic enzyme flux was estimated to be high enough to supply all of the reductive power needed for lipid synthesis [31].
Fig. 3. Interplay between energy metabolism, oncogenes and tumor microenvironment during tumorigenesis (the “metabolic wave model”).
Interplay between energy metabolism, oncogenes and tumor microenvironment
While the mechanisms leading to the enhancement of glycolytic capacity in tumors are well documented, less is known about the parallel OXPHOS changes. Both phenomena could result from a selection of pre-malignant cells forced to survive under hypoxia and limited glucose delivery, followed by an adaptation to intermittent hypoxia, pseudo-hypoxia, substrate limitation and acidic environment. This hypothesis was first proposed by Gatenby and Gillies to explain the high glycolytic phenotype of tumors [91], [92] and [93], but several lines of evidence suggest that it could also be used to explain the mitochondrial modifications observed in cancer cells.
Aerobic glycolysis and mitochondria cooperate during cancer progression
Metabolic flexibility considers the possibility for a given cell to alternate between glycolysis and OXPHOS in response to physiological needs. Louis Pasteur found that in most mammalian cells the rate of glycolysis decreases significantly in the presence of oxygen (Pasteur effect). Moreover, energy metabolism of normal cell can vary widely according to the tissue of origin, as we showed with the comparison of five rat tissues[94]. During stem cell differentiation, cell proliferation induces a switch from OXPHOS to aerobic glycolysis which might generate ATP more rapidly, as demonstrated in HepG2 cells [95] or in non-cancer cells[96] and [97]. Thus, normal cellular energy metabolism can adapt widely according to the activity of the cell and its surrounding microenvironment (energy substrate availability and diversity). Support for this view came from numerous studies showing that in vitro growth conditions can alter energy metabolism contributing to a dependency on glycolysis for ATP production [98].
Yet, Zu and Guppy analysed numerous studies and showed that aerobic glycolysis is not inherent to cancer but more a consequence of hypoxia[99].
Table 1. Impact of different oncogenes on energy metabolism
Impact of different oncogenes on energy metabolism.
For many years, mitochondria were viewed as semiautonomous organelles, required only for cellular energetics. This view has been largely supplanted by the concept that mitochondria are fully integrated into the cell and that mitochondrial stresses rapidly activate cytosolic signaling pathways that ultimately alter nuclear gene expression. Remarkably, this coordinated response to mild mitochondrial stress appears to leave the cell less susceptible to subsequent perturbations. This response, termed mitohormesis, is being rapidly dissected in many model organisms. A fuller understanding of mitohormesis promises to provide insight into our susceptibility for disease and potentially provide a unifying hypothesis for why we age.
Figure 1. The Basis of Mitohormesis. Any of a number of endogenous or exogenous stresses can perturb mitochondrial function. These perturbations are relayed to the cytosol through, at present, poorly understood mechanisms that may involve mitochondrial ROS as well as other mediators. These cytoplasmic signaling pathways and subsequent nuclear transcriptional changes induce various long-lasting cytoprotective pathways. This augmented stress resistance allows for protection from a wide array of subsequent stresses.
Figure 2. Potential Parallels between the Mitochondrial Unfolded Protein Response and Quorum Sensing in Gram-Positive Bacteria. In the C. elegans UPRmt response, mitochondrial proteins (indicated by blue swirls) are degraded by matrix proteases, and the oligopeptides that are generated are then exported through the ABC transporter family member HAF-1. Once in the cytosol, these peptides can influence the subcellular localization of the transcription factor ATFS-1. Nuclear ATFS-1 is capable of orchestrating a broad transcriptional response to mitochondrial stress. As such, this pathway establishes a method for mitochondrial and nuclear genomes to communicate. In some gram-positive bacteria, intracellularly generated peptides can be similarly exported through an ABC transporter protein. These peptides can be detected in the environment by a membrane-bound histidine kinases (HK) sensor. The activation of the HK sensor leads to phosphorylation of a response regulator (RR) protein that, in turn, can alter gene expression. This program allows communication between dispersed gram-positive bacteria and thus coordinated behavior of widely dispersed bacterial genomes.
Figure 3. The Complexity of Mitochondrial Stresses and Responses. A wide array of extrinsic and intrinsic mitochondrial perturbations can elicit cellular responses. As detailed in the text, genetic or pharmacological disruption of electron transport, incorrect folding of mitochondrial proteins, stalled mitochondrial ribosomes, alterations in signaling pathways, or exposure to toxins all appear to elicit specific cytoprotective programs within the cell. These adaptive responses include increased mitochondrial number (biogenesis), alterations in metabolism, increased antioxidant defenses, and augmented protein chaperone expression. The cumulative effect of these adaptive mechanisms might be an extension of lifespan and a decreased incidence of age-related pathologies.
2.1.2.6 Mitochondrial function and energy metabolism in cancer cells. Past overview and future perspectives
The involvements of energy metabolism aspects of mitochondrial dysfunction in cancer development, proliferation and possible therapy, have been investigated since Otto Warburg published his hypothesis. The main published material on cancer cell energy metabolism is overviewed and a new unique in vivo experimental approach that may have significant impact in this important field is suggested. The monitoring system provides real time data, reflecting mitochondrial NADH redox state and microcirculation function. This approach of in vivo monitoring of tissue viability could be used to test the efficacy and side effects of new anticancer drugs in animal models. Also, the same technology may enable differentiation between normal and tumor tissues in experimental animals and maybe also in patients.
Energy metabolism in mammalian cells
Fig. 1. Schematic representation of cellular energy metabolism and its relationship to microcirculatory blood flow and hemoglobin oxygenation.
Fig. 2. Schematic representation of the central role of the mitochondrion in the various processes involved in the pathology of cancer cells and tumors. Six issues marked as 1–6 are discussed in details in the text.
In vivo monitoring of tissue energy metabolism in mammalian cells
Fig. 3. Schematic presentation of the six parameters that could be monitored for the evaluation of tissue energy metabolism (see text for details).
Optical spectroscopy of tissue energy metabolism in vivo
Multiparametric monitoring system
Fig. 4. (A) Schematic representation of the Time Sharing Fluorometer Reflectometer (TSFR) combined with the laser Doppler flowmeter (D) for blood flow monitoring. The time sharing system includes a wheel that rotates at a speed of3000 rpm wit height filters: four for the measurements of mitochondrial NADH(366 nm and 450 nm)and four for oxy-hemoglobin measurements (585 nm and 577 nm) as seen in (C). The source of light is a mercury lamp. The probe includes optical fibers for NADH excitation (Ex) and emission (Em), laser Doppler excitation (LD in), laser Doppler emission (LD out) as seen in part E The absorption spectrum of Oxy- and Deoxy- Hemoglobin indicating the two wave length used (C).
Fig. 7. Comparison between mitochondrial metabolic states in vitro and the typical tissue metabolic states in vivo evaluated by NADH redox state, tissue blood flow and hemoglobin oxygenation as could be measured by the suggested monitoring system.
(very important)
2.1.2.7 Metabolic Reprogramming. Cancer Hallmark Even Warburg Did Not Anticipate
Cancer metabolism has long been equated with aerobic glycolysis, seen by early biochemists as primitive and inefficient. Despite these early beliefs, the metabolic signatures of cancer cells are not passive responses to damaged mitochondria but result from oncogene-directed metabolic reprogramming required to support anabolic growth. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation. No longer can cancer-associated alterations in metabolism be viewed as an indirect response to cell proliferation and survival signals. We contend that altered metabolism has attained the status of a core hallmark of cancer.
The propensity for proliferating cells to secrete a significant fraction of glucose carbon through fermentation was first elucidated in yeast. Otto Warburg extended these observations to mammalian cells, finding that proliferating ascites tumor cells converted the majority of their glucose carbon to lactate, even in oxygen-rich conditions. Warburg hypothesized that this altered metabolism was specific to cancer cells, and that it arose from mitochondrial defects that inhibited their ability to effectively oxidize glucose carbon to CO2. An extension of this hypothesis was that dysfunctional mitochondria caused cancer (Koppenol et al., 2011). Warburg’s seminal finding has been observed in a wide variety of cancers. These observations have been exploited clinically using 18F-deoxyglucose positron emission tomography (FDG-PET). However, in contrast to Warburg’s original hypothesis, damaged mitochondria are not at the root of the aerobic glycolysis exhibited by most tumor cells. Most tumor mitochondria are not defective in their ability to carry out oxidative phosphorylation. Instead, in proliferating cells mitochondrial metabolism is reprogrammed to meet the challenges of macromolecular synthesis. This possibility was never considered by Warburg and his contemporaries.
Advances in cancer metabolism research over the last decade have enhanced our understanding of how aerobic glycolysis and other metabolic alterations observed in cancer cells support the anabolic requirements associated with cell growth and proliferation. It has become clear that anabolic metabolism is under complex regulatory control directed by growth factor signal transduction in non-transformed cells. Yet despite these advances, the repeated refrain from traditional biochemists is that altered metabolism is merely an indirect phenomenon in cancer, a secondary effect that pales in importance to the activation of primary proliferation and survival signals (Hanahan and Weinberg, 2011). Most proto-oncogenes and tumor suppressor genes encode components of signal transduction pathways. Their roles in carcinogenesis have traditionally been attributed to their ability to regulate the cell cycle and sustain proliferative signaling while also helping cells evade growth suppression and/or cell death (Hanahan and Weinberg, 2011). But evidence for an alternative concept, that the primary functions of activated oncogenes and inactivated tumor suppressors are to reprogram cellular metabolism, has continued to build over the past several years. Evidence is also developing for the proposal that proto-oncogenes and tumor suppressors primarily evolved to regulate metabolism.
We begin this review by discussing how proliferative cell metabolism differs from quiescent cell metabolism on the basis of active metabolic reprogramming by oncogenes and tumor suppressors. Much of this reprogramming depends on utilizing mitochondria as functional biosynthetic organelles. We then further develop the idea that altered metabolism is a primary feature selected for during tumorigenesis. Recent advances have demonstrated that altered metabolism in cancer extends beyond adaptations to meet the increased anabolic requirements of a growing and dividing cell. Changes in cancer cell metabolism can also influence cellular differentiation status, and in some cases these changes arise from oncogenic alterations in metabolic enzymes themselves.
Metabolism in quiescent vs. proliferating cells: both use mitochondria.
(A) In the absence of instructional growth factor signaling, cells in multicellular organisms lack the ability to take up sufficient nutrients to maintain themselves. Neglected cells will undergo autophagy and catabolize amino acids and lipids through the TCA cycle, assuming sufficient oxygen is available. This oxidative metabolism maximizes ATP production. (B) Cells that receive instructional growth factor signaling are directed to increase their uptake of nutrients, most notably glucose and glutamine. The increased nutrient uptake can then support the anabolic requirements of cell growth: mainly lipid, protein, and nucleotide synthesis (biomass). Excess carbon is secreted as lactate. Proliferating cells may also use strategies to decrease their ATP production while increasing their ATP consumption. These strategies maintain the ADP:ATP ratio necessary to maintain glycolytic flux. Green arrows represent metabolic pathways, while black arrows represent signaling.
Metabolism is a direct, not indirect, response to growth factor signaling nihms-360138-f0002
Metabolism is a direct, not indirect, response to growth factor signaling.
(A) The traditional demand-based model of how metabolism is altered in proliferating cells. In response to growth factor signaling, increased transcription and translation consume free energy and decrease the ADP:ATP ratio. This leads to enhanced flux of glucose carbon through glycolysis and the TCA cycle for the purpose of producing more ATP. (B) Supply-based model of how metabolism changes in proliferating cells. Growth factor signaling directly reprograms nutrient uptake and metabolism. Increased nutrient flux through glycolysis and the mitochondria in response to growth factor signaling is used for biomass production. Metabolism also impacts transcription and translation through mechanisms independent of ATP availability.
Alterations in classic oncogenes directly reprogram cell metabolism to increase nutrient uptake and biosynthesis. PI3K/Akt signaling downstream of receptor tyrosine kinase (RTK) activation increases glucose uptake through the transporter GLUT1, and increases flux through glycolysis. Branches of glycolytic metabolism contribute to nucleotide and amino acid synthesis. Akt also activates ATP-citrate lyase (ACL), promoting the conversion of mitochondria-derived citrate to acetyl-CoA for lipid synthesis. Mitochondrial citrate can be synthesized when glucose-derived acetyl-CoA, generated by pyruvate dehydrogenase (PDH), condenses with glutamine-derived oxaloacetate (OAA) via the activity of citrate synthase (CS). mTORC1 promotes protein synthesis and mitochondrial metabolism. Myc increases glutamine uptake and the conversion of glutamine into a mitochondrial carbon source by promoting the expression of the enzyme glutaminase (GLS). Myc also promotes mitochondrial biogenesis. In addition, Myc promotes nucleotide and amino acid synthesis, both through direct transcriptional regulation and through increasing the synthesis of mitochondrial metabolite precursors.
Pyruvate kinase M2 (PKM2) expression in proliferating cells is regulated by signaling and mitochondrial metabolism to facilitate macromolecular synthesis. PKM2 is a less active isoform of the terminal glycolytic enzyme pyruvate kinase. It is also uniquely inhibited downstream of tyrosine kinase signaling. The decreased enzymatic activity of PKM2 in the cytoplasm promotes the accumulation of upstream glycolytic intermediates and their shunting into anabolic pathways. These pathways include the serine synthetic pathway that contributes to nucleotide and amino acid production. When mitochondrial metabolism is excessive, reactive oxygen species (ROS) from the mitochondria can feedback to inhibit PKM2 activity. Acetylation of PKM2, dependent on acetyl-CoA availability, may also promote PKM2 degradation and further contribute to increased flux through anabolic synthesis pathways branching off glycolysis.
IDH1 and IDH2 mutants convert glutamine carbon to the oncometabolite 2-hydroxyglutarate to dysregulate epigenetics and cell differentiation. (A) α-ketoglutarate, produced in part by wild-type isocitrate dehydrogenase (IDH), can enter the nucleus and be used as a substrate for dioxygenase enzymes that modify epigenetic marks. These enzymes include the TET2 DNA hydroxylase enzyme which converts 5-methylcytosine to 5-hydroxymethylcytosine, typically at CpG dinucleotides. 5-hydroxymethylcytosine may be an intermediate in either active or passive DNA demethylation. α-ketoglutarate is also a substrate for JmjC domain histone demethylase enzymes that demethylate lysine residues on histone tails. (B) The common feature of cancer-associated mutations in cytosolic IDH1 and mitochondrial IDH2 is the acquisition of a neomorphic enzymatic activity. This activity converts glutamine-derived α-ketoglutarate to the oncometabolite 2HG. 2HG can competitively inhibit α-ketoglutarate-dependent enzymes like TET2 and the JmjC histone demethylases, thereby impairing normal epigenetic regulation. This results in altered histone methylation marks, in some cases DNA hypermethylation at CpG islands, and dysregulated cellular differentiation.
Hypoxia and HIF-1 activation promote an alternative pathway for citrate synthesis through reductive metabolism of glutamine. (A) In proliferating cells under normoxic conditions, citrate is synthesized from both glucose and glutamine. Glucose carbon provides acetyl-CoA through the activity of PDH. Glutamine carbon provides oxaloacetate through oxidative mitochondrial metabolism dependent on NAD+. Glucose-derived acetyl-CoA and glutamine-derived oxaloacetate condense to form citrate via the activity of citrate synthase (CS). Citrate can be exported to the cytosol for lipid synthesis. (B) In cells proliferating in hypoxia and/or with HIF-1 activation, glucose is diverted away from mitochondrial acetyl-CoA and citrate production. Citrate can be maintained through an alternative pathway of reductive carboxylation, which we propose to rely on reverse flux of glutamine-derived α-ketoglutarate through IDH2. This reverse flux in the mitochondria would promote electron export from the mitochondria when the activity of the electron transport chain is inhibited because of the lack of oxygen as an electron acceptor. Mitochondrial reverse flux can be accomplished by NADH conversion to NADPH by mitochondrial transhydrogenase and the resulting NADPH use in α-ketoglutarate carboxylation. When citrate/isocitrate is exported to the cytosol, some may be metabolized in the oxidative direction by IDH1 and contribute to a shuttle that produces cytosolic NADPH.
A major paradox remaining with PKM2 is that cells expressing PKM2 produce more glucose-derived pyruvate than PKM1-expressing cells, despite having a form of the pyruvate kinase enzyme that is less active and more sensitive to inhibition. One way to get around the PKM2 bottleneck and maintain/enhance pyruvate production may be through an proposed alternative glycolytic pathway, involving an enzymatic activity not yet purified, that dephosphorylates PEP to pyruvate without the generation of ATP (Vander Heiden et al., 2010). Another answer to this paradox may emanate from the serine synthetic pathway. The decreased enzymatic activity of PKM2 can promote the accumulation of the 3-phosphoglycerate glycolytic intermediate that serves as the entry point for the serine synthetic pathway branch off glycolysis. The little studied enzyme serine dehydratase can then directly convert serine to pyruvate. A third explanation may lie in the oscillatory activity of PKM2 from the inactive dimer to active tetramer form. Regulatory inputs into PKM2 like tyrosine phosphorylation and ROS destabilize the tetrameric form of PKM2 (Anastasiou et al., 2011; Christofk et al., 2008b; Hitosugi et al., 2009), but other inputs present in glycolytic cancer cells like fructose-1,6-bisphosphate and serine can continually allosterically activate and/or promote reformation of the PKM2 tetramer (Ashizawa et al., 1991; Eigenbrodt et al., 1983). Thus, PKM2 may be continually switching from inactive to active forms in cells, resulting in an apparent upregulation of flux through anabolic glycolytic branching pathways while also maintaining reasonable net flux of glucose carbon through PEP to pyruvate. With such an oscillatory system, small changes in the levels of any of the above-mentioned PKM2 regulatory inputs can cause exquisite, rapid, adjustments to glycolytic flux. This would be predicted to be advantageous for proliferating cells in the setting of variable extracellular nutrient availability. The capability for oscillatory regulation of PKM2 could also provide an explanation for why tumor cells do not select for altered glycolytic metabolism upstream of PKM2 through deletions and/or loss of function mutations of other glycolytic enzymes.
IDH1 mutations at R132 are not simply loss-of-function for isocitrate and α-ketoglutarate interconversion, but also acquire a novel reductive activity to convert α-ketoglutarate to 2-hydroxyglutarate (2HG), a rare metabolite found at only trace amounts in mammalian cells under normal conditions (Dang et al., 2009). However, it still remained unclear if 2HG was truly a pathogenic “oncometabolite” resulting from IDH1 mutation, or if it was just the byproduct of a loss of function mutation. Whether 2HG production or the loss of IDH1 normal function played a more important role in tumorigenesis remained uncertain.
A potential answer to whether 2HG production was relevant to tumorigenesis arrived with the study of mutations in IDH2, the mitochondrial homolog of IDH1. Up to this point a small fraction of gliomas lacking IDH1 mutations were known to harbor mutations at IDH2 R172, the analogous residue to IDH1 R132 (Yan et al., 2009). However, given the rarity of these IDH2 mutations, they had not been characterized for 2HG production. The discovery of IDH2 R172 mutations in AML as well as glioma samples prompted the study of whether these mutations also conferred the reductive enzymatic activity to produce 2HG. Enzymatic assays and measurement of 2HG levels in primary AML samples confirmed that these IDH2 R172 mutations result in 2HG elevation (Gross et al., 2010; Ward et al., 2010).
It was then investigated if the measurement of 2HG levels in primary tumor samples with unknown IDH mutation status could serve as a metabolite screening test for both cytosolic IDH1 and mitochondrial IDH2 mutations. AML samples with low to undetectable 2HG were subsequently sequenced and determined to be IDH1 and IDH2 wild-type, and several samples with elevated 2HG were found to have neomorphic mutations at either IDH1 R132 or IDH2 R172 (Gross et al., 2010). However, some 2HG-elevated AML samples lacked IDH1 R132 or IDH2 R172 mutations. When more comprehensive sequencing of IDH1 and IDH2 was performed, it was found that the common feature of this remaining subset of 2HG-elevated AMLs was another mutation in IDH2, occurring at R140 (Ward et al., 2010). This discovery provided additional evidence that 2HG production was the primary feature being selected for in tumors.
In addition to intensifying efforts to find the cellular targets of 2HG, the discovery of the 2HG-producing IDH1 and IDH2 mutations suggested that 2HG measurement might have clinical utility in diagnosis and disease monitoring. While much work is still needed in this area, serum 2HG levels have successfully correlated with IDH1 R132 mutations in AML, and recent data have suggested that 1H magnetic resonance spectroscopy can be applied for 2HG detection in vivo for glioma (Andronesi et al., 2012; Choi et al., 2012; Gross et al., 2010; Pope et al., 2012). These methods may have advantages over relying on invasive solid tumor biopsies or isolating leukemic blast cells to obtain material for sequencing of IDH1 and IDH2. Screening tumors and body fluids by 2HG status also has potentially increased applicability given the recent report that additional IDH mutations can produce 2HG (Ward et al., 2011). These additional alleles may account for the recently described subset of 2HG-elevated chondrosarcoma samples that lacked the most common IDH1 or IDH2 mutations but were not examined for other IDH alterations (Amary et al., 2011). Metabolite screening approaches can also distinguish neomorphic IDH mutations from SNPs and sequencing artifacts with no effect on IDH enzyme activity, as well as from an apparently rare subset of loss-of-function, non 2HG-producing IDH mutations that may play a secondary tumorigenic role in altering cellular redox (Ward et al., 2011).
Will we find other novel oncometabolites like 2HG? We should consider basing the search for new oncometabolites on those metabolites already known to cause disease in pediatric inborn errors of metabolism (IEMs). 2HG exemplifies how advances in research on IEMs can inform research on cancer metabolism, and vice versa. Methods developed by those studying 2HG aciduria were used to demonstrate that R(-)-2HG (also known as D-2HG) is the exclusive 2HG stereoisomer produced by IDH1 and IDH2 mutants (Dang et al., 2009; Ward et al., 2010). Likewise, following the discovery of 2HG-producing IDH2 R140 mutations in leukemia, researchers looked for and successfully found germline IDH2 R140 mutations in D-2HG aciduria. IDH2 R140 mutations now account for nearly half of all cases of this devastating disease (Kranendijk et al., 2010). While interest has surrounded 2HG due to its apparent novelty as a metabolite not found in normal non-diseased cells, there are situations where 2HG appears in the absence of metabolic enzyme mutations. For example, in human cells proliferating in hypoxia, α-ketoglutarate can accumulate and be metabolized through an enhanced reductive activity of wild-type IDH2 in the mitochondria, leading to 2HG accumulation in the absence of IDH mutation (Wise et al., 2011). The ability of 2HG to alter epigenetics may reflect its evolutionary ancient status as a signal for elevated glutamine/glutamate metabolism and/or oxygen deficiency.
With this broadened view of what constitutes an oncometabolite, one could argue that the discoveries of two other oncometabolites, succinate and fumarate, preceded that of 2HG. Loss of function mutations in the TCA cycle enzymes succinate dehydrogenase (SDH) and fumarate hydratase (FH) have been known for several years to occur in pheochromocytoma, paraganglioma, leiomoyoma, and renal carcinoma. It was initially hypothesized that these mutations contribute to cancer through mitochondrial damage producing elevated ROS (Eng et al., 2003). However, potential tumorigenic effects were soon linked to the elevated levels of succinate and fumarate arising from loss of SDH and FH function, respectively. Succinate was initially found to impair PHD2, the α-ketoglutarate-dependent enzyme regulating HIF stability, through product inhibition (Selak et al., 2005). Subsequent work confirmed that fumarate could inhibit PHD2 (Isaacs et al., 2005), and that succinate could also inhibit the related enzyme PHD3 (Lee et al., 2005). These observations linked the elevated HIF levels observed in SDH and FH deficient tumors to the activity of the succinate and fumarate metabolites. Recent work has suggested that fumarate may have other important roles that predominate in FH deficiency. For example, fumarate can modify cysteine residues to inhibit a negative regulator of the Nrf2 transcription factor. This post-translational modification leads to the upregulation of antioxidant response genes (Adam et al., 2011; Ooi et al., 2011).
There are still many unanswered questions regarding the biology of SDH and FH deficient tumors. In light of the emerging epigenetic effects of 2HG, it is intriguing that succinate has been shown to alter histone demethylase activity in yeast (Smith et al., 2007). Perhaps elevated succinate and fumarate resulting from SDH and FH mutations can promote tumorigenesis in part through epigenetic modulation.
Despite rapid technological advances in studying cell metabolism, we remain unable to reliably distinguish cytosolic metabolites from those in the mitochondria and other compartments. Current fractionation methods often lead to metabolite leakage. Even within one subcellular compartment, there may be distinct pools of metabolites resulting from channeling between metabolic enzymes. A related challenge lies in the quantitative measurement of metabolic flux; i.e., measuring the movement of carbon, nitrogen, and other atoms through metabolic pathways rather than simply measuring the steady-state levels of individual metabolites. While critical fluxes have been quantified in cultured cancer cells and methods for these analyses continue to improve (DeBerardinis et al., 2007; Mancuso et al., 2004; Yuan et al., 2008), many obstacles remain such as cellular compartmentalization and the reliance of most cell culture on complex, incompletely defined media.
Over the past decade, the study of metabolism has returned to its rightful place at the forefront of cancer research. Although Warburg was wrong about mitochondria, he was prescient in his focus on metabolism. Data now support the concepts that altered metabolism results from active reprogramming by altered oncogenes and tumor suppressors, and that metabolic adaptations can be clonally selected during tumorigenesis. Altered metabolism should now be considered a core hallmark of cancer. There is much work to be done.
2.1.2.8 A Role for the Mitochondrial Pyruvate Carrier as a Repressor of the Warburg Effect and Colon Cancer Cell Growth
Cancer cells are typically subject to profound metabolic alterations, including the Warburg effect wherein cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis. We show herein that 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. Cancer cells re-expressing MPC1 and MPC2 display increased mitochondrial pyruvate oxidation, with no changes in cell growth in adherent culture. MPC re-expression exerted profound effects in anchorage-independent growth conditions, however, including impaired colony formation in soft agar, spheroid formation, and xenograft growth. We also observed a decrease in markers of stemness and traced the growth effects of MPC expression to the stem cell compartment. We propose that reduced MPC activity is an important aspect of cancer metabolism, perhaps through altering the maintenance and fate of stem cells.
Figure 2. Re-Expressed MPC1 and MPC2 Form a Mitochondrial Complex (A and B) (A) Western blot and (B) qRT-PCR analysis of the indicated colon cancer cell lines with retroviral expression of MPC1 (or MPC1-R97W) and/or MPC2. (C) Western blots of human heart tissue, hematologic cancer cells, and colon cancer cell lines with and without MPC1 and MPC2 re-expression. (D) Fluorescence microscopy of MPC1-GFP and MPC2-GFP overlaid with Mitotracker Red in HCT15 cells. Scale bar: 10 mm. (E) Blue-native PAGE analysis of mitochondria from control and MPC1/2-expressing cells. (F) Western blots of metabolic and mitochondrial proteins across four colon cancer cell lines with or without MPC1/2 expression
Figure 3. MPC Re-Expression Alters Mitochondrial Pyruvate Metabolism (A) OCR at baseline and maximal respiration in HCT15 (n = 7) and HT29 (n = 13) with pyruvate as the sole carbon source (mean ± SEM). (B and C) Schematic and citrate mass isotopomer quantification in cells cultured with D-[U-13C]glucose and unlabeled glutamine for 6 hr (mean ± SD, n = 2). (D) Glucose uptake and lactate secretion normalized to protein concentration (mean ± SD, n = 3). (E–G) (E) Western blots of PDH, phospho-PDH, and PDK1; (F) PDH activity assay and (G) CS activity assay with or without MPC1 and MPC2 expression (mean ± SD, n = 4). (H and I) Effects of MPC1/2 re-expression on mitochondrial membrane potential and ROS production (mean ± SD, n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 4. MPC Re-Expression Alters Growth under Low-Attachment Conditions (A) Cell number of control and MPC1/2 re-expressing cell lines in adherent culture (mean ± SD, n = 7). (B) Cell viability determined by trypan blue exclusion and Annexin V/PI staining (mean ± SD, n = 3). (C–F) (C) EdU incorporation of MPC re-expressing cell lines at 3 hr post EdU pulse. Growth in 3D culture evaluated by (D) soft agar colony formation (mean ± SD, n = 12, see also Table S1) and by ([E] and [F]) spheroid formation ± MPC inhibitor UK5099 (mean ± SEM, n = 12). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 7. MPC Re-Expression Alters the Cancer Initiating Cell Population (A) Western blot quantification of ALDHA and Lin28A from control or MPC re-expressing HT29 xenografts (mean ± SEM, n = 10). (B and C) Percentage of ALDHhi (n = 3) and CD44hi (n = 5) cells as determined by flow cytometry (mean ± SEM). (D) Western blot analysis of stem cell markers in control and MPC re-expressing cell lines. (E) Relative MPC1 and MPC2 mRNA levels in ALDH sorted HCT15 cells (n = 4,mean ± SEM). 2D growth of (F) whole-population HCT15 cells and (G) ALDH sorted cells. Area determined by ImageJ after crystal violet staining (mean ± SD, n = 6). (H and I) (H) Adherent and (I) spheroid growth of main population (MP) versus side population (SP) HCT15 cells. (mean ± SD, n = 6). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Our demonstration that the MPC is lost or underexpressed in many cancers might provide clarifying context for earlier attempts to exploit metabolic regulation for cancer therapeutics. The PDH kinase inhibitor dichloroacetate, which impairs PDH phosphorylation and increases pyruvate oxidation, has been explored extensively as a cancer therapy (Bonnet et al., 2007; Olszewski et al., 2010). It has met with mixed results, however, and has typically failed to dramatically decrease tumor burden as a monotherapy (Garon et al., 2014;
Sanchez-Arago et al., 2010; Shahrzadetal.,2010). Is one possible reason for these failures that the MPC has been lost or inactivated, thereby limiting the metabolic effects of PDH activity? The inclusion of the MPC adds additional complexity to targeting cancer metabolism for therapy but has the potential to explain why treatments may be more effective in some studies than in others (Fulda et al., 2010; Hamanaka and Chandel, 2012; Tennant et al., 2010; Vander Heiden, 2011). The redundant measures to limit pyruvate oxidation make it easy to understand why expression of the MPC leads to relatively modest metabolic changes in cells grown in adherent culture conditions. While subtle, we observed a number of changes in metabolic parameters, all of which are consistent with enhanced mitochondrial pyruvate entry and oxidation. There are at least two possible explanations for the discrepancy that we observed between the impact on adherent and nonadherent cell proliferation. One hypothesis is that the stress of nutrient deprivation and detachment combines with these subtle metabolic effects to impair survival and proliferation.
2.1.2.9 ECM1 promotes the Warburg effect through EGF-mediated activation of PKM2
The Warburg effect is an oncogenic metabolic switch that allows cancer cells to take up more glucose than normal cells and favors anaerobic glycolysis. Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein that is overexpressed in various types of carcinoma. Using two-dimensional digest-liquid chromatography-mass spectrometry (LC-MS)/MS, we showed that the expression of proteins associated with the Warburg effect was upregulated in trastuzumab-resistant BT-474 cells that overexpressed ECM1 compared to control cells. We further demonstrated that ECM1 induced the expression of genes that promote the Warburg effect, such as glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and hypoxia-inducible factor 1 α (HIF-1α). The phosphorylation status of pyruvate kinase M2 (PKM-2) at Ser37, which is responsible for the expression of genes that promote the Warburg effect, was affected by the modulation of ECM1 expression. Moreover, EGF-dependent ERK activation that was regulated by ECM1 induced not only PKM2 phosphorylation but also gene expression of GLUT1 and LDHA. These findings provide evidence that ECM1 plays an important role in promoting the Warburg effect mediated by PKM2.
Fig. 1.ECM1 induces a metabolic shift toward promoting Warburg effect. (A) The levels of glucose uptake were examined with a cell-based assay. (B) Levels of lactate production were measured using a lactate assay kit. (C) Cellular ATP content was determined with a Cell Titer-Glo luminescent cell viability assay. Error bars represent mean ± SD of triplicate experiments (*p b 0.05, ***p b 0.0005).
Fig.2. ECM1 up-regulates expression of gene sassociated with the Warburg effect. (A) Cell lysates were analyzed by western blotting using antibodies specific for ECM1, LDHA, GLUT1,and actin (as a loading control). The intensities of the bands were quantified using 1D Scan software and plotted. (BandC) mRNA levels of each gene were determined by real-time PCR using specific primers. (D) HIF-1α-dependent transcriptional activities were examined using a hypoxia response element (HRE) reporter indual luciferase assays. Error bars represent mean ± SD of triplicate experiments (*p b 0.05, **p b 0.005, ***p b 0.0005).
Fig.3. ECM1-dependent upregulation of gene expression is not mediated byEgr-1.
Fig.4. ECM1 activates PKM2 via EGF-mediated ERK activation
Fig. 5. TheWarburg effect is attenuated by silencing of PKM2 in breast cancer cells
Recently, a non-glycolytic function of PKM2 was reported. Phosphorylated PKM2 at Ser37 is translocated into the nucleus after EGFR and ERK activation and regulates the expression of cyclin D1, c-Myc, LDHA, and GLUT1[19,37]. Here, we showed that ECM1 regulates the phosphorylation level and translocation of PKM2 via the EGFR/ ERK pathway. As we previously showed that ECM1 enhances the EGF response and increases EGFR expression through MUC1-dependent stabilization [17], it seemed likely that activation of the EGFR/ERK pathway by ECM1 is linked to PKM2 phosphorylation. Indeed, we show here that ECM1 regulates the phosphorylation of PKM2 at Ser37 and enhances the Warburg effect through the EGFR/ERK pathway. HIF-1α is known to be responsible for alterations in cancer cell metabolism [38] and our current studies showed that the expression level of HIF-1α is up-regulated by ECM1 (Fig. 2C and D). To determine the mechanism by which ECM1 upregulated HIF-1α expression, we focused on the induction of Egr-1 by EGFR/ERK signaling [39]. However, although Egr-1 expression was regulated by ECM1 we failed to find evidence that Egr-1 affected the expression of genes involved in the Warburg effect (Fig. 3C). Moreover, ERK-dependent PKM2 activation did not regulate HIF-1α expression in BT-474 cells (Fig. 4D and5B). These results suggested that the upregulation of HIF-1α by ECM1 is not mediated by the EGFR/ERK pathway.
Conclusions
In the current study we showed that ECM1 altered metabolic phenotypes of breast cancer cells toward promoting the Warburg effect.
Phosphorylation and nuclear translocation of PKM2 were induced by ECM1 through the EGFR/ERK pathway. Moreover, phosphorylated PKM2 increased the expression of metabolic genes such as LDHA and GLUT1, and promoted glucose uptake and lactate production. These findings provide a new perspective on the distinct functions of ECM1 in cancer cell metabolism. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.cellsig.2014.11.004
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2.1.2.10 Glutamine Oxidation Maintains the TCA Cycle and Cell Survival during impaired Mitochondrial Pyruvate Transport
Mitochondria produce acetyl-CoA from glutamine during MPC inhibition
•Alanine synthesis is suppressed during MPC inhibition
•MPC inhibition activates GDH to supply pools of TCA cycle intermediates
•GDH supports cell survival during periods of MPC inhibition
Summary
Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.
Yang et al, Graphical Abstract
Graphical abstract
Figure 1. Pyruvate Depletion Redirects Glutamine Metabolism to Produce AcetylCoA and Citrate (A) Top: Anaplerosis supplied by [U-13C]glutamine. Glutamine supplies OAA via a-KG, while acetylCoA is predominantly supplied by other nutrients, particularly glucose. Bottom: Glutamine is converted to acetyl-CoA in the absence of glucosederived pyruvate. Red circles represent carbons arising from [U-13C]glutamine, and gray circles are unlabeled. Reductive carboxylation is indicated by the green dashed line. (B) Fraction of succinate, fumarate, malate, and aspartate containing four 13C carbons after culture of SFxL cells for 6 hr with [U-13C]glutamine in the presence or absence of 10 mM unlabeled glucose (Glc). (C) Mass isotopologues of citrate after culture of SFxL cells for 6 hr with [U-13C]glutamine and 10 mM unlabeled glucose, no glucose, or no glucose plus 6 mM unlabeled pyruvate (Pyr). (D) Citrate m+5 and m+6 after culture of HeLa or Huh-7 cells for 6 hr with [U-13C]glutamine and 10 mM unlabeled glucose, no glucose, or no glucose plus 6 mM unlabeled pyruvate. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 2. Isolated Mitochondria Convert Glutamine to Citrate (A) Western blot of whole-cell lysates (Cell) and preparations of isolated mitochondria (Mito) or cytosol from SFxL cells. (B) Oxygen consumption in a representative mitochondrial sample. Rates before and after addition of ADP/GDP are indicated. (C) Mass isotopologues of citrate produced by mitochondria cultured for 30 min with [U-13C] glutamine and with or without pyruvate.
Figure 3. Blockade of Mitochondrial Pyruvate Transport Activates Glutamine-Dependent Citrate Formation (A) Dose-dependent effects of UK5099 on citrate labeling from [U-13C]glucose and [U-13C]glutamine in SFxL cells. (B) Time course of citrate labeling from [U-13C] glutamine with or without 200 mM UK5099. (C) Abundance of total citrate and citrate m+6 in cells cultured in [U-13C]glutamine with or without 200 mM UK5099. (D) Mass isotopologues of citrate in cells cultured for 6 hr in [U-13C]glutamine with or without 10 mM CHC or 200 mM UK5099. (E) Effect of silencing ME2 on citrate m+6 after 6 hr of culture in [U-13C]glutamine. Relative abundances of citrate isotopologues were determined by normalizing total citrate abundance measured by mass spectrometry against cellular protein for each sample then multiplying by the fractional abundance of each isotopologue. (F) Effect of silencing MPC1 or MPC2 on formation of citrate m+6 after 6 hr of culture in [U-13C]glutamine. (G) Citrate isotopologues in primary human fibroblasts of varying MPC1 genotypes after culture in [U-13C]glutamine. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S1.
Figure 4. Kinetic Analysis of the Metabolic Effects of Blocking Mitochondrial Pyruvate Transport (A) Summation of 13C spectra acquired over 2 min of exposure of SFxL cells to hyperpolarized [1-13C] pyruvate. Resonances are indicated for [1-13C] pyruvate (Pyr1), the hydrate of [1-13C]pyruvate (Pyr1-Hydr), [1-13C]lactate (Lac1), [1-13C]alanine (Ala1), and H[13C]O3 (Bicarbonate). (B) Time evolution of appearance of Lac1, Ala1, and bicarbonate in control and UK5099-treated cells. (C) Relative 13C NMR signals for Lac1, Ala1, and bicarbonate. Each signal is summed over the entire acquisition and expressed as a fraction of total 13C signal. (D) Quantity of intracellular and secreted alanine in control and UK5099-treated cells. Data are the average and SD of three independent cultures. *p < 0.05; ***p < 0.001. See also Figure S2.
Figure 5. Inhibiting Mitochondrial Pyruvate Transport Enhances the Contribution of Glutamine to Fatty Acid Synthesis (A) Mass isotopologues of palmitate extracted from cells cultured with [U-13C] glucose or [U-13C]glutamine, with or without 200 mM UK5099. For simplicity, only even-labeled isotopologues (m+2, m+4, etc.) are shown. (B) Fraction of lipogenic acetyl-CoA derived from glucose or glutamine with or without 200 mM UK5099. Data are the average and SD of three independent cultures. ***p < 0.001. See also Figure S3.
Figure 6. Blockade of Mitochondrial Pyruvate Transport Induces GDH (A) Two routes by which glutamate can be converted to AKG. Blue and green symbols are the amide (g) and amino (a) nitrogens of glutamine, respectively. (B) Utilization and secretion of glutamine (Gln), glutamate (Glu), and ammonia (NH4+) by SFxL cells with and without 200 mM UK5099. (C) Secretion of 15N-alanine and 15NH4+ derived from [a-15N]glutamine in SFxL cells expressing a control shRNA (shCtrl) or either of two shRNAs directed against GLUD1 (shGLUD1-A and shGLUD1-B). (D) Left: Phosphorylation of AMPK (T172) and acetyl-CoA carboxylase (ACC, S79) during treatment with 200 mM UK5099. Right: Steady-state levels of ATP 24 hr after addition of vehicle or 200 mM UK5099. (E) Fractional contribution of the m+6 isotopologue to total citrate in shCtrl, shGLUD1-A, and shGLUD1-B SFxL cells cultured in [U-13C]glutamine with or without 200 mM UK5099. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S4.
Figure 7. GDH Sustains Growth and Viability during Suppression of Mitochondrial Pyruvate Transport (A) Relative growth inhibition of shCtrl, shGLUD1A, and shGLUD1-B SFxL cells treated with 50 mM UK5099 for 3 days. (B) Relative growth inhibition of SFxL cells treated with combinations of 50 mM of the GDH inhibitor EGCG, 10 mM of the GLS inhibitor BPTES, and 200 mM UK5099 for 3 days. (C) Relative cell death assessed by trypan blue staining in SFxL cells treated as in (B). (D) Relative cell death assessed by trypan blue staining in SF188 cells treated as in (B) for 2 days. (E) (Left) Growth of A549-derived subcutaneous xenografts treated with vehicle (saline), EGCG, CHC, or EGCG plus CHC (n = 4 for each group). Data are the average and SEM. Right: Lactate abundance in extracts of each tumor harvested at the end of the experiment. Data in (A)–(D) are the average and SD of three independent cultures. NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S5.
Mitochondrial metabolism complements glycolysis as a source of energy and biosynthetic precursors. Precursors for lipids, proteins, and nucleic acids are derived from the TCA cycle. Maintaining pools of these intermediates is essential, even under circumstances of nutrient limitation or impaired supply of glucose-derived pyruvate to the mitochondria. Glutamine’s ability to produce both acetyl-CoA and OAA allows it to support TCA cycle activity as a sole carbon source and imposes a greater cellular dependence on glutamine metabolism when MPC function or pyruvate supply is impaired. Other anaplerotic amino acids could also supply both OAA and acetyl-CoA, providing flexible support for the TCA cycle when glucose is limiting. Although fatty acids are an important fuel in some cancer cells (Caro et al., 2012), and fatty acid oxidation is induced upon MPC inhibition, this pathway produces acetyl-CoA but not OAA. Thus, fatty acids would need to be oxidized along with an anaplerotic nutrient in order to enable the cycle to function as a biosynthetic hub. Notably, enforced MPC overexpression also impairs growth of some tumors (Schell et al., 2014), suggesting that maximal growth may require MPC activity to be maintained within a narrow window. After decades of research on mitochondrial pyruvate transport, molecular components of the MPC were recently reported (Halestrap, 2012; Schell and Rutter, 2013). MPC1 and MPC2 form a heterocomplex in the inner mitochondrial membrane, and loss of either component impairs pyruvate import, leading to citrate depletion (Bricker et al., 2012; Herzig et al., 2012). Mammalian cells lacking functional MPC1 display normal glutamine-supported respiration (Bricker et al., 2012), consistent with our observation that glutamine supplies the TCA cycle in absence of pyruvate import. We also observed that isolated mitochondria produce fully labeled citrate from glutamine, indicating that this pathway operates as a self-contained mechanism to maintain TCA cycle function. Recently, two well-known classes of drugs have unexpectedly been shown to inhibit MPC. First, thiazolidinediones, commonly used as insulin sensitizers, impair MPC function in myoblasts (Divakaruni et al.,2013). Second, the phosphodiesterase inhibitor Zaprinast inhibits MPC in the retina and brain (Du et al., 2013b). Zaprinast also induced accumulation of aspartate, suggesting that depletion of acetyl-CoA impaired the ability of a new turn of the TCA cycle to be initiated from OAA; as a consequence, OAA was transaminated to aspartate. We noted a similar phenomenon in cancer cells, suggesting that UK5099 elicits a state in which acetyl-CoA supply is insufficient to avoid OAA accumulation. Unlike UK5099, Zaprinast did not induce glutamine-dependent acetyl-CoA formation. This may be related to the reliance of isolated retinas on glucose rather than glutamine to supply TCA cycle intermediates or the exquisite system used by retinas to protect glutamate from oxidation (Du et al., 2013a). Zaprinast was also recently shown to inhibit glutaminase (Elhammali et al., 2014), which would further reduce the contribution of glutamine to the acetyl-CoA pool.
Comment by reader –
The results from these studies served as a good
reason to attempt the vaccination of patients using p53-
derived peptides, and a several clinical trials are currently
in progress. The most advanced work used a long
synthetic peptide mixture derived from p53 (p53-SLP; ISA
Pharmaceuticals, Bilthoven, the Netherlands) (Speetjens
et al., 2009; Shangary et al., 2008; Van der Burg et al.,
2001). The vaccine is delivered in the adjuvant setting
and induces T helper type cells.
A Synthesis of the Beauty and Complexity of How We View Cancer
Author: Larry H. Bernstein, MD, FCAP
Cancer Volume One – Summary
A Synthesis of the Beauty and Complexity of How We View Cancer
This document has covered a broad spectrum of the research, translational biology, diagnostics (both laboratory and imaging methodologies), and treatments for a variety of cancers, mainly by organs, and selectively by the most common cancers seen in human populations. A number of observations stand out on review of all the material presented. 1. The most common cancers affecting humans is spread worldwide, with some variation by region. 2. Cancers within geographic regions may be expressed differently in relationship to population migrations, the incidence of specific environmental pollutants, occurrence of insect transmitted and sexually transmitted diseases (HIV, HCV, HPV), and possibly according to age, or relationship to ultraviolet or high dose radiation exposure. 3. Cancers are expressed within generally recognized age timelines. For example, acute lymphocytic leukemia and neuroblastoma in children under 10 years age; malignant giant cell tumor and osteosarcoma in the third and fourth decade; prostate cancer and breast cancer over age 40, and are more aggressive at an earlier age, both having a strong sex hormone dependence. 4. There is dispute about the effectiveness of screening for cancer with respect to what age, excessive risk in treatment modality, and the duration of progression free survival. Despite the evidence of several years potential life extension, a long term survival of 10 years is not the expected outcome. However, the quality of life in the remaining years is a valid point in favor of progress. 5. There has been a significant reduction in toxicity of treatment, but attention has been focused on a patient-centric decision process. 6. There has been a dramatic improvement in surgical approaches, post-surgical surveillance, and in diagnosis by invasive and noninvasive methods, especially in the combination of needle biopsy and imaging techniques. 7. There is significant variation within cancer cell types with respect to disease-free survival.
The work presented has several main components: First, there is the biology and mechanisms involved in carcinogenesis related to (1) mutations; (2) carcinogenesis; (3) cell regulatory mechanisms; (4) cell signaling pathways; (5) apoptosis (6) ubitination (7) mitochondrial dysfunction; (8) cell-cell interactions; (9) cell migration; (10) metastasis. Then there are large portions covering (1) imaging; (2) specific targeted therapy; (3) nanotechology-based therapy; (4) specific organ-type cancers; (5) genomics-based testing; (6) circulating cancer cells; (7) miRNAs; (8) siRNAs; (9) cancer immunology and (10) immunotherapy.
Classically, we refer to cancer development in terms of the germ cell layers – ectoderm, mesoderm, and endoderm. These are formative in embryonic development. The most active development occurs during embryonic development, with a high growth rate of cells and also a high utilization of energy. The cells utilize oxidation for energy in this period characterized by movement of cells in differentiation and organogenesis. This was observed to be unlike the cell metabolism in carcinogenesis, which is characterized by impaired mitochondrial function and reliance on lactate production for energy – termed anaerobic glycolysis, as investigated by Meyerhof, Embden, Warburg, Szent-Gyorgy, H. Krebs, Theorell, AV Hill, B Chance, P Mitchell, P Boyer, F Lippman, and others.
In addition, the body economy has been divided into two major metabolic compartments: fat and lean body mass (LBM), which is further denoted as visceral and structural. This denotes the gut, kidneys, liver, lung, pancreas, sexual organs, endocrines, brain and fat cells in one compartment, and skeletal muscle, bone and cardiovascular in another. LBM is calculated as fat free mass. Further, brown fat is distinguished from white fat. But this was a first layer of construction of the human body. One peels away this layer to find a second layer. For example, the gut viscera have an inner (outer) epithelial layer, a muscularis, and a deep epithelium, which has circulation and fat. There is also an interstitium between the gut epithelium and muscularis. The lung has an epithelium exposed to the airspaces, then capillaries, and then epithelium, designed for exchange of O2 and CO2, the source of heat generation. The pancreas has an endocrine portion in the islets that are embedded in an exocrine secretory organ. The sexual organs have a combination of glandular structures embedded in a mesothelium.
The structural compartment is entirely accounted for by the force of contraction. If this is purely anatomical, that is not really the case when one goes into the functioning substructures of these tissues – cytoplasm, endoplasmic reticulum (ribosomal), mitochondria, liposomes, chromatin apparatus, cell membrane and vesicles. Within and between these structures are the working and interacting mechanisms of the cell in its unique role. What ties these together was first thought to be found in the dogma following the discovery of the genetic code in 1953 that begat DNA to RNA to protein.
This led to many other discoveries that made it clear that it was only a first approximation. It did not account for noncoding DNA, which became unmasked with the culmination of the Human Genome Project and concurrent advances in genomics (mtDNA, mtRNA, siRNA, exosomes, proteomics, synthetic biology, predictive analytics, and regulatory pathways directed by signaling molecules. Here is a list of signaling pathways: 1. JAK-STAT 2. GPCR 3. Endocrine 4. Cytochemical 5. RTK 6. P13K 7. NF-KB 8. MAPK 9. Ubiquitin 10. TGF-beta 11. Stem cell These signaling pathways have become the basis for the discovery of inhibitors of signaling pathways (suppressors), as well as activators, as these have been considered as specific targets for selective therapy. (.See Figure below) Of course, extensive examination of these pathways has required that all such findings are validated based on the STRENGTH of their effect on the target and in the impact of suppression.
Let us continue this discussion elucidating several major points. While the early observations that drove the interest in biochemical behavior of cancer cells has been displaced, it has not faded from view.
Bioenergetics of Cancer cells
Michael J. Gonzalez (Bioenergetic_Theory_of_Carcinigenesis. http://www.academia.edu/2224071/ Bioenergetic_Theory_of_Carcinigenesis) maintains that the altered energy metabolism of tumor cells provides a viable target for a non-toxic chemotherapeutic approach. An increased glucose consumption rate has been observed in malignant cells. Warburg (NobelLaureate in medicine) postulated that the respiratory process of malignant cells was impaired in the malignant transformation. Szent-Györgyi (Nobel in medicine) also viewed cancer as originating from insufficient oxygen utilization. Oxygen inhibits anaerobic metabolism (fermentation and lactic acid production). Interestingly, during cell differentiation (where cell energy level is high) there is an increased cellular production of oxidation products that appear to provide physiological stimulation for changes in gene expression that may lead to a terminal differentiated state. The failure to maintain high ATP production (high cell energy levels) may be a consequence of inactivation of key enzymes, especially those related to the Krebs cycle and the electron transport system. A distorted mitochondrial function (transmembrane potential) may result. This aspect could be suggestive of an important mitochondrial involvement in the carcinogenic process in addition to presenting it as a possible therapeutic target for cancer. Intermediate metabolic correction of the mitochondria is postulated as a possible non-toxic therapeutic approach for cancer.
Fermentation is the anaerobic metabolic breakdown of glucose without net oxidation. Fermentation does not release all the available energy of glucose or need oxygen as part of its biochemical reactions ; it merely allows glycolysis (a process that yields two ATP per mole of glucose) to continue by replenishing reduced coenzymes and yields lactate as its final product. The first step in aerobic and anaerobic energy producing pathways, it occurs in the cytoplasm of cells, not in specialized organelles, and is found in all living organisms. Cancer cells have a fundamentally different energy metabolism compared to normal cells, that are obligate aerobes (oxygen-requiring cells) meeting their energy needs with oxidative metabolic processes., while cancer cells do not require oxygen for their survival. This increase in glycolytic flux is a metabolic strategy of tumor cells to ensure growth and survival in environments with low oxygen concentrations.
Radoslav Bozov has commented that the process of genomic evolution cannot be fully revealed through comparative genomicsHe states that DNA would be entropic- favorable stable state going towards absolute ZERO temp. Themodynamics measurement in subnano discrete space would go negative towards negativity. DNA is like a cold melting/growing crystal, quite stable as it appears not due to hydrogen bonding , but due to interference of C-N-O. That force is contradicted via proteins onto which we now know large amount of negative quantum redox state carbon attaches. The more locally one attempts to observe, the more hidden variables would emerge as a consequence of discrete energy spaces opposing continuity of matter/time. But stability emerges out of non-stable states, and never reaches absolute stability, for there would be neither feelings nor freedom.
Membrane potential(Vm)
Membrane potential (Vm), the voltage across the plasma membrane, arises because of the presence of differention channels/transporters with specific ion selectivity and permeability. Vm is a key biophysical signal in non-excitable cells, modulating important cellular activities, such as proliferation and differentiation. Therefore, the multiplicities of various ion channels/transporters expressed on different cells are finely tuned in order to regulate the Vm. (M Yang and WJ Brackenbury.
It is well-established that cancer cells possess distinct bioelectrical properties. Notably, electrophysiological analyses in many cancer cell types have revealed a depolarized Vm that favors cell proliferation. Ion channels/transporters control cell volume and migration, and emerging data also suggest that the level of Vm has functional roles in cancer cell migration. In addition, yperpolarization is necessary for stem cell differentiation. For example, both osteogenesis and adipogenesis are hindered in human mesenchymal stem cells (hMSCs) under depolarizing conditions. Therefore, in the context of cancer, membrane depolarization might be important for the emergence and maintenance of cancer stem cells (CSCs), giving rise to sustained tumor growth. This review aims to provide a broad understanding of the Vm as a bioelectrical signal in cancer cells by examining several key types of ion channels that contribute to its regulation. The mechanisms by which Vm regulates cancer cell proliferation, migration, and differentiation will be discussed. In the long term, Vm might be avaluable clinical marker for tumor detection with prognostic value, and could even be artificially modified in order to inhibit tumor growth and metastasis.
Perspective beyond Cancer Genomics: Bioenergetics of Cancer Stem Cells
Although the notion that cancer is a disease caused by genetic and epigenetic alterations is now widely accepted, perhaps more emphasis has been given to the fact that cancr is a genetic disease. It should be noted that in the post-genome sequencing project period of the 21st century, the underlined phenomenon nevertheless could not be discarded towards the complete control of cancer disaster as the whole strategy, and in depth investigation of the factors associated with tumorigenesis is required for achieving it. Otto Warburg has won a Nobel Prize in 1931 for the discovery of tumor bioenergetics, which is now commonly used as the basis of positron emission tomography (PET), a highly sensitive noninvasive technique used in cancer diagnosis. Furthermore, the importance of the cancer stem cell (CSC) hypothesis in therapy-related resistance and metastasis has been recognized during the past 2 decades. Accumulating evidence suggests that tumor bioenergetics plays a critical role in CSC regulation; this finding has opened up a new era of cancer medicine, which goes beyond cancer genomics.
Efficient execution of cell death in non-glycolytic cells requires the generation of ROS controlled by the activity of mitochondrial H+-ATP synthase.
There is a large body of clinical data documenting that most human carcinomas contain reduced levels of the catalytic subunit of the mitochondrial H+-ATP synthase. In colon and lung cancer this alteration correlates with a poor patient prognosis. Furthermore, recent findings in colon cancer cells indicate that down-regulation of the H+-ATP synthase is linked to the resistance of the cells to chemotherapy. However, the mechanism by which the H+-ATP synthase participates in cancer progression is unknown. In this work, we show that inhibitors of the H+-ATP synthase delay
staurosporine-induced cell death in liver cells that are dependent on oxidative phosphorylation for energy provision whereas it has no effect on glycolytic cells. Efficient execution of cell death requires the generation of reactive oxygen species (ROS) controlled by the activity of the H+-ATP synthase in a process that is concurrent with the rapid disorganization of the cellular mitochondrial network. The generation of ROS after staurosporine treatment is highly dependent on the mitochondrial membrane potential and most likely caused by reverse electron flow to Complex I. The generated ROS promote the carbonylation and covalent modification of cellular and mitochondrial proteins. Inhibition of the activity of the H+-ATP synthase blunted ROS production, prevented the oxidation of cellular proteins and the modification of mitochondrial proteins, delaying the release of cyt c and the execution of cell death. The results in this work establish the down-regulation of the H+-ATP synthase, and thus of oxidative phosphorylation, as part of the molecular strategy adapted by cancer cells to avoid reactive oxygen species-mediated cell death. Furthermore, the results provide a mechanistic explanation to understand chemotherapeutic resistance of cancer cells that rely on glycolysis as main energy provision pathway.
see also –
The tumor suppressor function of mitochondria: Translation into the clinics
Recently, the inevitable metabolic reprogramming experienced by cancer cells as a result of the onset of cellular proliferation has been added to the list of hallmarks of the cancer cell phenotype. Proliferation is bound to the synchronous fluctuation of cycles of an increased glycolysis concurrent with a restrained oxidative phosphorylation. Mitochondria are key players in the metabolic cycling experienced during proliferation because of their essential roles in the transduction of biological energy and in defining the life–death fate of the cell. These two activities are molecularly and functionally integrated and are both targets of commonly altered cancer genes. Moreover, energetic metabolism of the cancer cell also affords a target to develop new therapies because the activity of mitochondria has an unquestionable tumor suppressor function. In this review, we summarize most of these findings paying special attention to the opportunity that translation of energetic metabolism into the clinics could afford for the management of cancer patients. More specifically, we emphasize the role that mitochondrial β-F1-ATPase has as a marker for the prognosis of different cancer patients as well as in predicting the tumor response to therapy.
Self-Destructive Behavior in Cells May Hold Key to a Longer Life
Carl Zimmer, MY Times October 5, 2009
In recent years, scientists have found evidence of autophagy in preventing a much wider range of diseases. Many disorders, like Alzheimer’s disease, are the result of certain kinds of proteins forming clumps. Lysosomes can devour these clumps before they cause damage, slowing the onset of diseases.
Lysosomes may also protect against cancer. As mitochondria get old, they cast off charged molecules that can wreak havoc in a cell and lead to potentially cancerous mutations. By gobbling up defective mitochondria, lysosomes may make cells less likely to damage their DNA. Many scientists suspect it is no coincidence that breast cancer cells are often missing autophagy-related genes. The genes may have been deleted by mistake as a breast cell divided. Unable to clear away defective mitochondria, the cell’s descendants become more vulnerable to mutations.
Unfortunately, as we get older, our cells lose their cannibalistic prowess. The decline of autophagy may be an important factor in the rise of cancer, Alzheimer’s disease and other disorders that become common in old age. Unable to clear away the cellular garbage, our bodies start to fail.
If this hypothesis turns out to be right, then it may be possible to slow the aging process by raising autophagy. It has long been known, for example, that animals that are put on a strict low-calorie diet can live much longer than animals that eat all they can. Recent research has shown that caloric restriction raises autophagy in animals and keeps it high. The animals seem to be responding to their low-calorie diet by feeding on their own cells, as they do during famines. In the process, their cells may also be clearing away more defective molecules, so that the animals age more slowly.
Some scientists are investigating how to manipulate autophagy directly. Dr. Cuervo and her colleagues, for example, have observed that in the livers of old mice, lysosomes produce fewer portals on their surface for taking in defective proteins. So they engineered mice to produce lysosomes with more portals. They found that the altered lysosomes of the old experimental mice could clear away more defective proteins. This change allowed the livers to work better.
Essentiality of pyruvate kinase, oxidation, and phosphorylation
We can move to the next level with greater clarity. Yu et al. reported an important relationship between Pyruvate kinase M2 (PKM2) and the Warburg effect of cancer cells ( M Yu, et al. PIM2 phosphorylates PKM2 and promotes Glycolysis in Cancer Cells. J Biol Chem (PMID: 24142698) http://dx.doi.org10.1074/jbc.M113.508226 ). They found that PIM2 could directly phosphorylate PKM2 on the Thr454 residue, which resulted in an increase of PKM2 protein levels. PKM2 with a phosphorylation-defective mutation displayed a reduced effect on glycolysis compared to the wild-type, thereby co-activating HIF-1α and β-catenin, and enhanced mitochondria respiration and chemotherapeutic sensitivity of cancer cells. This indicated that PIM2-dependent phosphorylation of PKM2 is critical for regulating the Warburg effect in cancer, highlighting PIM2 as a potential therapeutic target.
In another study of the effect of 3 homoplastic mtDNA mutations on oxidative metabolism of osteosarcoma cells, there was a difference proportional to the magnitude of the defect. (Iommarini L, et al. Different mtDNA mutations modify tumor progression in dependence of the degree of respiratory complex I impairment. Hum Mol Genet. 2013 Nov 11. [Epub ahead of print]; PMID: 24163135 ). Osteosarcoma cells carrying the most marked impairment of the gene encoding mitochondrial complex I (CI) of oxidative phosphorylation displayed a reduced tumorigenic potential both in vitro and in vivo, when compared with cells with mild CI dysfunction. The severe CI dysfunction was an energetic defect associated with a compensatory increase in glycolytic metabolism and AMP-activated protein kinase activation. The result suggested that mtDNA mutations may display diverse impact on tumorigenic potential depending on the type and severity of the resulting oxidative phosphorylation dysfunction. The modulation of tumor growth was independent from reactive oxygen species production but correlated with hypoxia-inducible factor 1α stabilization, indicating that structural and functional integrity of CI and oxidative phosphorylation are required for hypoxic adaptation and tumor progression.
An unrelated finding shares some agreement with what has been identified (Systematic isolation of context-dependent vulnerabilities in NSCLC. Cell, 24 Oct 2013; 155 (3): 552-566, http://dx.doi.org/10.1016/ j.cell.2013.09.041). They report three distinct target/response-indicator pairings that are represented with significant frequencies (6%–16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature.This is depicted in the Figure below. The authors noted a frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets.
The forging of a cancer-metabolism link and twists in the chain (Biome 19th April 2013)
Ten years ago, Grahame Hardie and Dario Alessi discovered that the elusive upstream kinase required for the activation of AMP-activated protein kinase (AMPK) by metabolic stress that the Hardie lab had been pursuing in their research on the metabolic regulator AMPK was the tumor suppressor, LKB1, that the neighbouring Alessi lab was working on at the time. This finding represented the first clear link between AMPK and cancer.
The resulting paper [1], published in 2003 in what was then Journal of Biology (now BMC Biology), was one [1] of three [2, 3] connecting these two kinases and that helped to swell of a surge of interest in the metabolism of tumor cells that was just beginning at about that time and is still growing. (LKB1 and AMPK and the cancer-metabolism link – ten years after. D Grahame Hardie, and Dario R Alessi. BMC Biology 2013, 11:36. http://dx doi.org.10.1186/1741-7007-11-36.)
In September 2003, both groups published a joint paper [1] in Journal of Biology (now BMC Biology) that identified the long-sought and elusive upstream kinase acting on AMP-activated protein kinase (AMPK) as a complex containing LKB1, a known tumor suppressor. Similar findings were reported at about the same time by David Carling and Marian Carlson [2] and by Reuben Shaw and Lew Cantley [3]; at the time of writing these three papers have received between them a total of over 2,000 citations. These findings provided a direct link between a protein kinase, AMPK, which at the time was mainly associated with regulation of metabolism, and another protein kinase, LKB1, which was known from genetic studies to be a tumor suppressor. While the idea that cancer is in part a metabolic disorder (first suggested by Warburg in the 1920s [4]) is well recognized today [5], this was not the case in 2003, and our paper perhaps contributed towards its renaissance.
The distinctive metabolic feature of tumor cells that enables them to meet the demands of unrestrained growth is the switch from oxidative generation of ATP to aerobic glycolysis – a phenomenon now well known as the Warburg effect. Operating this switch is one of the central functions of the AMP-activated protein kinase (AMPK) that has long been the focus of research in the Hardie lab. AMPK is an energy sensor that is allosterically tuned by competitive binding of ATP, ADP and AMP to sites on its g regulatory subunit (its portrait here, with AMP bound at two sites, was kindly provided by Bing Xiao and Stephen Gamblin). When phosphorylated by LKB1, AMPK responds to depletion of ATP by turning off anabolic reactions required for growth, and turning on catabolic reactions and oxidative phosphorylation – the reverse of the Warburg effect. In this light, it is not surprising that LKB1 is inactivated in some proportion of many different types of tumors.
AMPK as an energy sensor and metabolic switch
AMPK was discovered as a protein kinase activity that phosphorylated and inactivated two key enzymes of fatty acid and sterol biosynthesis: acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). The ACC kinase activity was reported to be activated by 5’-AMP, and the HMGR kinase activity by reversible phosphorylation, but for many years the two activities were thought to be due to distinct enzymes. However, in 1987 the DGH laboratory showed that both were functions of a single protein kinase, which we renamed AMPK after its allosteric activator, 5’-AMP. It was subsequently found that AMPK regulated not only lipid biosynthesis, but also many other metabolic pathways, both by direct phosphorylation of metabolic enzymes, and through longer-term effects mediated by phosphorylation of transcription factors and co-activators. In general, AMPK switches off anabolic pathways that consume ATP and NADPH, while switching on catabolic pathways that generate ATP (Figure 1).
Summary of a selection of target proteins and metabolic pathways regulated by AMPK. Anabolic pathways switched off by AMPK are shown in the top half of the ‘wheel’ and catabolic pathways switched on by AMPK in the bottom half. Where a protein target for AMPK responsible for the effect is known, it is shown in the inner wheel; a question mark indicates that it is not yet certain that the protein is directly phosphorylated. For original references see [54].
Regulation of AMPK. AMPK can be activated by increases in cellular AMP:ATP or ADP:ATP ratio, or Ca2+ concentration. AMPK is activated >100-fold on conversion from a dephosphorylated form (AMPK) to a form phosphorylated at Thr172 (AMPK-P) catalyzed by at least two upstream kinases: LKB1, which appears to be constitutively active, and CaMKKβ, which is only active when intracellular Ca2+ increases. Increases in AMP or ADP activate AMPK by three mechanisms: (1) binding of AMP or ADP to AMPK, causing a conformational change that promotes phosphorylation by upstream kinases (usually this will be LKB1, unless [Ca2+] is elevated); (2) binding of AMP or ADP, causing a conformational change that inhibits dephosphorylation by protein phosphatases; (3) binding of AMP (and not ADP), causing allosteric activation of AMPK-P. All three effects are antagonized by ATP, allowing AMPK to act as an energy sensor.
Members of the AMPK and AMPK-related kinase (ARK) family. All the kinases named in the figure are phosphorylated and activated by LKB1, although what regulates this phosphorylation is known only for AMPK. Alternative names are shown, where applicable.
Three possible mechanisms to explain how the AMPK-activating drugs metformin or phenformin might provide protection against cancer. (a) Metformin acts on the liver and other insulin target tissues by activating AMPK (and probably via other targets), normalizing blood glucose; this reduces insulin secretion from pancreatic β cells, reducing the growth-promoting effects of insulin (and high glucose) on tumor cells. Since metformin does not reduce glucose levels in normoglycemic individuals, this mechanism would only operate in insulin-resistant subjects. (b) Metformin or phenformin activates AMPK in pre-neoplastic cells, restraining their growth and proliferation and thus delaying the onset of tumorigenesis; this mechanism would only operate in cells where the LKB1-AMPK pathway was intact. (c) Metformin or phenformin inhibits mitochondrial ATP synthesis in tumor cells, promoting cell death. If the LKB1-AMPK pathway was down-regulated in the tumor cells, they would be more sensitive to cell death induced by the biguanides than surrounding normal cells.
Metformin and phenformin are biguanides that inhibit mitochondrial function and so deplete ATP by inhibiting its production . AMPK is activated by any metabolic stress that depletes ATP, either by inhibiting its production (as do hypoxia, glucose deprivation, and treatment with biguanides) or by accelerating its consumption (as does muscle contraction). By switching off anabolism and other ATP-consuming processes and switching on alternative ATP-producing catabolic pathways, AMPK acts to restore cellular energy homeostasis.
Findings that AMPK is activated in skeletal muscle during exercise and that it increases muscle glucose uptake and fatty acid oxidation led to the suggestion that AMPK-activating drugs might be useful for treating type 2 diabetes. Indeed, it turned out that AMPK is activated by metformin, a drug that had at that time been used to treat type 2 diabetes for over 40 years, and by phenformin , a closely related drug that had been withdrawn for treatment of diabetes due to side effects of lactic acidosis.
If only it were so simple. Effects of metformin on cancer in type 2 diabetics could be secondary to reduction in insulin levels, and although there is evidence for direct effects of AMPK activation on the development of tumors in mice, there is also recent evidence that tumors that become established without down-regulating LKB1 survive metformin better than those that have lost it – probably because metformin poisons the mitochondrial respiratory chain, depressing ATP levels, and cells in which AMPK can still be activated in response to the challenge do better than those in which it can’t.
In their review, Hardie and Alessi chart these twists and turns, and point to the explosion of further possibilities opened up by the discovery, since their 2003 publication, of at least one other class of kinase upstream of AMPK (the CaM kinases), and at least a dozen other downstream targets of LKB1 (AMPK-related kinases, or ARKs) – not to mention the innumerable downstream targets of AMPK; all which make half their schematic illustrations look like hedgehogs.
Analysis of respiration in human cancer
Bioenergetic profiling of cancer cells is of great potential because it can bring forward new and effective
Therapeutic strategies along with early diagnosis. Metabolic Control Analysis (MCA) is a methodology that enables quantification of the flux control exerted by different enzymatic steps in a metabolic network thus assessing their contribution to the system‘s function.
(T Kaambre,V Chekulayev, I Shevchuk, et al. Metabolic control analysis of respiration in human cancer tissue. Frontiers Physiol 2013 (4); 151: 1. http://dx.doi.org/10.3389/fphys.2013.00151)
Our main goal is to demonstrate the applicability of MCA for in situ studies of energy
Metabolism in human breast and colorectal cancer cells as well as in normal tissues .We seek to determine the metabolic conditions leading to energy flux redirection in cancer cells. A main result obtained is that the adenine nucleotide translocator exhibits the highest control of respiration in human breast cancer thus becoming a prospective therapeutic target. Additionally, we present evidence suggesting the existence of mitochondrial respiratory supercomplexes that may represent a way by which cancer cells avoid apoptosis. The data obtained show that MCA applied in situ can be insightful in cancer cell energetic research.
Representative traces of change in the rate of oxygen consumption by permeabilized human colorectal cancer (HCC) fibers after their titration with increasing concentrations of mersalyl, an inhibitor of inorganic phosphate carrier (panel A). The values of respiration rate obtained were plotted vs. mersalyl concentration (panel B) and from the plot the corresponding flux control coefficient was calculated. Bars are ±SEM.
Oncologic diseases such as breast and colorectal cancers are still one of the main causes of premature death. The low efficiency of contemporary medicine in the treatment of these malignancies is largely mediated by a poor understanding of the processes involved in metastatic dissemination of cancer cells as well as the unique energetic properties of mitochondria from tumors. Current knowledge supports the idea that human breast and colorectal cancer cells exhibit increased rates of glucose consumption displaying Warburg phenotype,i.e.,elevated glycolysis even in the presence of oxygen (Warburg and Dickens, 1930; Warburg, 1956 ;Izuishietal., 2012). Notwithstanding, there are some evidences that in these malignancies mitochondrial oxidative phosphorylation (OXPHOS) is the main source of ATP rather than glycolysis. Cancer cells have been classified according to their pattern of metabolic remodeling depending of the relative balance between aerobic glycolysis and OXPHOS (Bellanceetal.,2012). The first type of tumor cells is highly glycolytic, the second OXPHOS deficient and the third type of tumors dislay enhanced OXPHOS. Recent studies strongly sug gest that cancer cells can utilize lactate, free fatty acids, ketone bodies, butyrate and glutamine as key respiratory substrate selic iting metabolic remodeling of normal surrounding cells toward aerobic glycolysis—“reverse Warburg”effect (Whitaker-Menezes et al.,2011;Salem et al.,2012;Sotgia et al.,2012;Witkiewicz et al., 2012).
In normal cells,the OXPHOS system is usually closely linked to phosphotransfer systems, including various creatine kinase(CK) isotypes,which ensure a safe operation of energetics over a broad functional range of cellular activities (Dzejaand Terzic,2003). However, our current knowledge about the function of CK/creatine (Cr) system in human breast and colorectal cancer is insufficient. In some malignancies, for example sarcomas the CK/Cr system was shown to be strongly downregulated (Beraetal.,2008;Patraetal.,2008). Our previous studies showed that the mitochondrial-bound CK (MtCK) activity was significantly decreased in HL-1 tumor cells (Mongeetal.,2009), as compared to normal parent cardiac cells where the OXPHOS is the main ATP source of and the CK system is a main energy carrier. In the present study,we estimated the role of MtCK in maintaining energy homeostasis in human colorectal cancer cells. Understanding the control and regulation of energy metabolism requires analytical tools that take into account the existing interactions between individual network components and their impact on systemic network function. Metabolic Control Analysis(MCA) is a theoretical framework relating the properties of metabolic systems to the kinetic characteristics of their individual enzymatic components (Fell,2005). An experimental approach of MCA has been already successfully applied to the studies of OXPHOS in isolated mitochondria (Tageretal.,1983; Kunzetal.,1999; Rossignoletal.,2000) and in skinned muscle fibers (Kuznetsovetal.,1997;Teppetal.,2010).
Since Otto Warburg discovered that most cancer cells predominantly produce energy by glycolysis rather than by oxidative phosphorylation in mitochondria, much interest has been focused on the alterations of these organelles in cancer cells. Mitochondria have been shown to be key players in numerous cellular events tightly related with the biology of cancer. Although energy production relies on the glycolytic pathway in cancer cells, these organelles also participate in many other processes essential for cell survival and proliferation such as ROS production, apoptotic and necrotic cell death, modulation of oxygen concentration, calcium and iron homeostasis, and certain metabolic and biosynthetic pathways. Many of these mitochondrial-dependent processes are altered in cancer cells, leading to a phenotype characterized, among others, by higher oxidative stress, inhibition of apoptosis, enhanced cell proliferation, chemoresistance, induction of angiogenic genes and aggressive fatty acid oxidation. Uncoupling proteins, a family of inner mitochondrial membrane proteins specialized in energy-dissipation, has aroused enormous interest in cancer due to their relevant impact on such processes and their potential for the development of novel therapeutic strategies.
Uncoupling proteins (UCPs) are a family of inner mitochondrial membrane proteins whose function is to allow the re-entry of protons to the mitochondrial matrix, by dissipating the proton gradient and, subsequently, decreasing membrane potential and production of reactive oxygen species (ROS). Due to their pivotal role in the intersection between energy efficiency and oxidative stress UCPs are being investigated for a potential role in cancer. In this review we compile the latest evidence showing a link between uncoupling and the carcinogenic process, paying special attention to their involvement in cancer initiation, progression and drug chemoresistance.
The Warburg Effect
Uncoupling the Warburg effect from cancer
A Najafov and DR Alessi
Proc Nat Acad Sci www.pnas.org/cgi/doi/10.1073/pnas.1014047107
A remarkable trademark of most tumors is their ability to break down glucose by glycolysis at a vastly higher rate than in normal tissues, even when oxygen is copious. This phenomenon, known as the Warburg effect, enables rapidly dividing tumor cells to generate essential biosynthetic building blocks such as nucleic acids, amino acids, and lipids from glycolytic intermediates to permit growth and duplication of cellular components during division (1). An assumption dominating research in this area is that the Warburg effect is specific to cancer. Thus, much of the focus has been on uncovering mechanisms by which cancer-causing mutations influence metabolism to stimulate glycolysis.
This has lead to many exciting discoveries. For example, the p53 tumor suppressor can suppress glycolysis through its ability to control expression of key metabolic genes, such as phosphoglycerate mutase (2), synthesis of cytochrome C oxidase-2 (3), and TP53-induced glycolysis and apoptosis regulator (TIGAR) (4). Many cancer-causing mutations lead to activation of the Akt and mammalian target of rapamycin (mTOR) pathway that profoundly influences metabolism and expression of metabolic enzymes to promoteglycolysis (5).
Strikingly, all cancer cells but not nontransformed cells express a specific splice variant of pyruvate kinase, termed M2-PK, that is less active, leading to the build up of phosphoenolpyruvate (6). Recent work has revealed that reduced activity of M2-PK promotes a unique glycolytic pathway in which phosphoenolpyruvate is converted to pyruvate by a histidine-dependent phosphorylation of phosphoglycerate mutase, promoting assimilation of glycolytic products into biomass (7). However, despite these observations, one might imagine that the Warburg effect need not be specific for cancer and that any normal cell would need to stimulate glycolysis to generate sufficient biosynthetic materials to fuel expansion and division.
Recent work by Salvador Moncada’s group published in PNAS (8) and other recent work from the same group (9, 10) provides exciting evidence supporting the idea that the Warburg effect is also required for the proliferation of noncancer cells.
The key discovery was that the anaphase promoting complex/cyclosome-Cdh1(APC/C-Cdh1), a master regulator of the transition of G1 to S phase of the cell cycle, inhibits glycolysis in proliferating noncancer cells by mediating the degradation of two key metabolic enzymes, namely 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase isoform3 (PFKFB3) (9, 10) and glutaminase-(Fig. 1) (8).
Fig. Mechanism by which APC/C-Cdh1 inhibits glycolysis and glutaminolysis to suppress cell proliferation.
APC/C-Cdh1 E3 ligase recognizes KEN-box–containing metabolic enzymes, such as PFKFB3 and glutaminase-1 (GLS1), and ubiquitinates and targets them for proteasomal degradation. This inhibits glycolysis and glutaminolysis, leading to decrease in metabolites that can be assimilated into biomass, thereby suppressing proliferation.
PFKFB3 potently stimulates glycolysis by catalyzing the formation of fructose-2,6-bisphosphate, the allosteric activatorof 6-phosphofructo-1-kinase (11). Glutaminase-1 is the first enzyme in glutaminolysis, converting glutamine to lactate, yielding biosyntheticintermediates required for cell proliferation (12).
APC/C is a cell cycle-regulated E3 ubiquitin ligase that promotes ubiquitination of a distinct set of cell cycle proteins containing either a D-box (destruction box) or a KEN-box, named after the essential Lys-Glu-Asn motif required for APC recognition (13). Among its well-known substrates are crucial cell cycle proteins, such as cyclin B1, securin, and Plk1. By ubiquitinating and targeting its substrates to 26S proteasome-mediated degradation, APC/C regulates processes in late mitotic stage, exit from mitosis, and several events in G1 (14). The Cdh1 subunit is the KENbox binding adaptor of the APC/C ligase and is essential for G1/S transition.
Importantly, APC/C-Cdh1 is inactivated at the initiation of the S-phase of the cell cycle when DNA and cellular organelles are replicated at the time of the greatest need for generation of biosynthetic materials. APC/C-Cdh1 is reactivated later at the mitosis/G1 phase of the cell cycle when there is a lower requirement for biomassgeneration.
Both PFKFB3 (9, 10) and glutaminase-1 (8) possess a KEN-box and are rapidly degraded in nonneoplastic lymphocytes during the cell cycle when APC/C-Cdh1 is active. Consistent with destruction being mediated by APC-C-Cdh1, ablation of the KEN-box prevents degradation of PFKFB3 (9, 10) and glutaminase-1 (8). Inhibiting the proteasomal-dependent degradation with the MG132 inhibitor
markedly increases levels of ubiquitinated PFKFB3 and glutaminase-1 (8). Moreover, overexpression of Cdh1 to activate APC/C-Cdh1 decreases levels of PFKFB3 as well as glutmaninase-1 and concomitantly inhibited glycolysis, as judged by decrease in lactate production. This effect is also observed when cells were treated with a glutaminase-1 inhibitor (6-diazo-5- oxo-L-norleucine) (8). The final evidence supporting the authors’ hypothesis is that proliferation and glycolysis is inhibited after shRNA-mediated silencing of either PFKFB3 or glutaminase-1 (8).
These results are interesting, because unlike most recent work in this area, Colombo et al. (8) link the Warburg effect to the machinery of the cell cycle that is present in all cells rather than to cancer driving mutations. Further work is required to properly define the overall importance of this pathway, which has thus far only been studied in a limited number of cells. It would also be of value to undertake a more detailed analysis of how the rate of glycolysis and other metabolic pathways vary during the cell cycle of normal and cancer cells…(see full 2 page article) at PNAS.
The Warburg Effect Suppresses Oxidative Stress Induced Apoptosis in a Yeast Model for Cancer
Colonies of Saccharomyces cerevisiae, suitable for manipulation of mitochondrial respiration and shows mitochondria-mediated cell death, were used as a model. Repression of respiration as well as ROS-scavenging via glutathione inhibited apoptosis, conferred a survival advantage during seeding and early development of this fast proliferating solid cell population. In contrast, enhancement of respiration triggered cell death.
Conclusion/Significance: The Warburg effect might directly contribute to the initiation of cancer formation – not only by enhanced glycolysis – but also via decreased respiration in the presence of oxygen, which suppresses apoptosis.
Serine/threonine protein kinase PIM2, a known oncogene is a binding partner of pyruvate kinase M2 (PKM2), a key player in the Warburg effect of cancer cells. PIM2 interacts with PKM2 and phosphorylates PKM2 on the Thr454 residue.
The phosphorylation of PKM2 increases glycolysis and proliferation in cancer cells.
The PIM2-dependent phosphoirylation of ZPKM2 is critical for regulating the Warburg effect in cancer.
Genome-Scale Metabolic Modeling Elucidates the Role of Proliferative Adaptation in Causing the Warburg Effect
Shlomi T, Benyamini T, Gottlieb E, Sharan R, Ruppin E
PLoS Comput Biol 2011; 7(3): e1002018. http://dx.doi.org/10.1371/journal.pcbi.1002018
The Warburg effect – a classical hallmark of cancer metabolism – is a counter-intuitive phenomenon in which rapidly proliferating cancer cells resort to inefficient ATP production via glycolysis leading to lactate secretion, instead of relying primarily on more efficient energy production through mitochondrial oxidative phosphorylation, as most normal cells do.
The causes for the Warburg effect have remained a subject of considerable controversy since its discovery over 80 years ago, with several competing hypotheses. Here, utilizing a genome-scale human metabolic network model accounting for stoichiometric and enzyme solvent capacity considerations, we show that the Warburg effect is a direct consequence of the metabolic adaptation of cancer cells to increase biomass production rate. The analysis is shown to accurately capture a three phase metabolic behavior that is observed experimentally during oncogenic progression, as well as a prominent characteristic of cancer cells involving their preference for glutamine uptake over other amino acids.
1- Oncogenes express proteins of “Tyrosine kinase receptor pathways”, a receptor family including insulin or IGF-Growth Hormone receptors. Other oncogenes alter the PP2A phosphatase brake over these kinases.
2- Experiments on pancreatectomized animals; treated with pure insulin or total pancreatic extracts, showed that choline in the extract, preserved them from hepatomas.
Since choline is a methyle donor, and since methylation regulates PP2A, the choline protection may result from PP2A methylation, which then attenuates kinases.
3- Moreover, kinases activated by the boosted signaling pathway inactivate pyruvate kinase and pyruvate dehydrogenase. In addition, demethylated PP2A would no longer dephosphorylate these enzymes. A “bottleneck” between glycolysis and the oxidative-citrate cycle interrupts the glycolytic pyruvate supply now provided via proteolysis and alanine transamination. This pyruvate forms lactate (Warburg effect) and NAD+ for glycolysis. Lipolysis and fatty acids provide acetyl CoA; the citrate condensation increases, unusual oxaloacetate sources are available. ATP citrate lyase follows, supporting aberrant transaminations with glutaminolysis and tumor lipogenesis. Truncated urea cycles, increased polyamine synthesis, consume the methyl donor SAM favoring carcinogenesis.
4- The decrease of butyrate, a histone deacetylase inhibitor, elicits epigenic changes (PETEN, P53, IGFBP decrease; hexokinase, fetal-genes-M2, increase)
5- IGFBP stops binding the IGF – IGFR complex, it is perhaps no longer inherited by a single mitotic daughter cell; leading to two daughter cells with a mitotic capability.
6- An excess of IGF induces a decrease of the major histocompatibility complex MHC1, Natural killer lymphocytes should eliminate such cells that start the tumor, unless the fever prostaglandin PGE2 or inflammation, inhibit them…
Introduction
The metabolic network of biochemical pathways forms a system controlled by a few switches, changing the finality of this system. Specific substrates and hormones control such switches. If for example, glycemia is elevated, the pancreas releases insulin, activating anabolism and oxidative glycolysis, energy being required to form new substance or refill stores. If starvation decreases glycemia, glucagon and epinephrine activate gluconeogenesis and ketogenesis to form nutriments, mobilizing body stores. The different finalities of the system are or oriented by switches sensing the NADH/NAD+, the ATP/AMP, the cAMP/AMP ratios or the O2 supply… We will not describe here these metabolic finalities and their controls found in biochemistry books.
Many of the switches depend of the phosphorylation of key enzymes that are active or not. Evidently, there is some coordination closing or opening the different pathways. Take for example gluconeogenesis, the citrate condensation slows down, sparing OAA, which starts the gluconeogenic pathway. In parallel, one also has to close pyruvate kinase (PK); if not, phosphoenolpyruvate would give back pyruvate, interrupting the pathway. Hence, the properties of key enzymes acting like switches on the pathway specify the finality of the system. Our aim is to show that tumor cells invent a new specific finality, with mixed glycolysis and gluconeogenesis features. This very special metabolism gives to tumor cells a selective advantage over normal cells, helping the tumor to develop at the detriment of the rest of the body.
I Abnormal metabolism of tumors, a selective advantage
The initial observation of Warburg 1956 on tumor glycolysis with lactate production is still a crucial observation [1]. Two fundamental findings complete the metabolic picture: the discovery of the M2 pyruvate kinase (PK) typical of tumors [2] and the implication of tyrosine kinase signals and subsequent phosphorylations in the M2 PK blockade [3–5].
A typical feature of tumor cells is a glycolysis associated to an inhibition of apoptosis. Tumors over-express the high affinity hexokinase 2, which strongly interacts with the mitochondrial ANT-VDAC-PTP complex. In this position, close to the ATP/ADP exchanger (ANT), the hexokinase receives efficiently its ATP substrate [6,7]. As long as hexokinase occupies this mitochondria site, glycolysis is efficient. However, this has another consequence, hexokinase pushes away from the mitochondria site the permeability transition pore (PTP), which inhibits the release of cytochrome C, the apoptotic trigger [8]. The site also contains a voltage dependent anion channel (VDAC) and other proteins. The repulsion of PTP by hexokinase would reduce the pore size and the release of cytochrome C. Thus, the apoptosome-caspase proteolytic structure does not assemble in the cytoplasm. The liver hexokinase or glucokinase, is different it has less interaction with the site, has a lower affinity for glucose; because of this difference, glucose goes preferentially to the brain.
Further, phosphofructokinase gives fructose 1-6 bis phosphate; glycolysis is stimulated if an allosteric analogue, fructose 2-6 bis phosphate increases in response to a decrease of cAMP. The activation of insulin receptors in tumors has multiple effects, among them; a decrease of cAMP, which will stimulate glycolysis.
Another control point is glyceraldehyde P dehydrogenase that requires NAD+ in the glycolytic direction. If the oxygen supply is normal, the mitochondria malate/aspartate (MAL/ASP) shuttle forms the required NAD+ in the cytosol and NADH in the mitochondria. In hypoxic conditions, the NAD+ will essentially come via lactate dehydrogenase converting pyruvate into lactate. This reaction is prominent in tumor cells; it is the first discovery of Warburg on cancer.
At the last step of glycolysis, pyruvate kinase (PK) converts phosphoenolpyruvate (PEP) into pyruvate, which enters in the mitochondria as acetyl CoA, starting the citric acid cycle and oxidative metabolism. To explain the PK situation in tumors we must recall that PK only works in the glycolytic direction, from PEP to pyruvate, which implies that gluconeogenesis uses other enzymes for converting pyruvate into PEP. In starvation, when cells need glucose, one switches from glycolysis to gluconeogenesis and ketogenesis; PK and pyruvate dehydrogenase (PDH) are off, in a phosphorylated form, presumably following a cAMP-glucagon-adrenergic signal. In parallel, pyruvate carboxylase (Pcarb) becomes active. Moreover, in starvation, much alanine comes from muscle protein proteolysis, and is transaminated into pyruvate. Pyruvate carboxylase first converts pyruvate to OAA and then, PEP carboxykinase converts OAA to PEP etc…, until glucose. The inhibition of PK is necessary, if not one would go back to pyruvate. Phosphorylation of PK, and alanine, inhibit the enzyme.
Well, tumors have a PK and a PDH inhibited by phosphorylation and alanine, like for gluconeogenesis, in spite of an increased glycolysis! Moreover, in tumors, one finds a particular PK, the M2 embryonic enzyme [2,9,10] the dimeric, phosphorylated form is inactive, leading to a “bottleneck “. The M2 PK has to be activated by fructose 1-6 bis P its allosteric activator, whereas the M1 adult enzyme is a constitutive active form. The M2 PK bottleneck between glycolysis and the citric acid cycle is a typical feature of tumor cell glycolysis.
We also know that starvation mobilizes lipid stores from adipocyte to form ketone bodies, they are like glucose, nutriments for cells. Growth hormone, cAMP, AMP, activate a lipase, which provides fatty acids; their β oxidation cuts them into acetyl CoA in mitochondria and in peroxisomes for very long fatty acids; forming ketone bodies. Normally, citrate synthase slows down, to spare acetyl CoA for the ketogenic route, and OAA for the gluconeogenic pathway. Like for starvation, tumors mobilize lipid stores. But here, citrate synthase activity is elevated, condensing acetyl CoA and OAA [11–13]; citrate increases, ketone bodies decrease. Consequently, ketone bodies will stop stimulating Pcarb. In tumors, the OAA needed for citrate synthase will presumably come from PEP, via reversible PEP carboxykinase or other sources. The quiescent Pcarb will not process the pyruvate produced by alanine transamination after proteolysis, leaving even more pyruvate to lactate dehydrogenase, increasing the lactate released by the tumor, and the NAD+ required for glycolysis.
Above the bottleneck, the massive entry of glucose accumulates PEP, which converts to OAA via mitochondria PEP carboxykinase, an enzyme requiring biotine-CO2-GDP. This source of OAA is abnormal, since Pcarb, another biotin-requiring enzyme, should have provided OAA. Tumors may indeed contain “morule inclusions” of biotin-enzyme [14] suggesting an inhibition of Pcarb, presumably a consequence of the maintained citrate synthase activity, and decrease of ketone bodies that normally stimulate Pcarb. The OAA coming via PEP carboxykinase and OAA coming from aspartate transamination or via malate dehydrogenase condenses with acetyl CoA, feeding the elevated tumoral citric acid condensation starting the Krebs cycle. Thus, tumors have to find large amounts of acetyl CoA for their condensation reaction; it comes essentially from lipolysis and β oxidation of fatty acids, and enters in the mitochondria via the carnitine transporter. This is the major source of acetyl CoA; since PDH that might have provided acetyl CoA remains in tumors, like PK, in the inactive phosphorylated form. The blockade of PDH [15] was recently reversed by inhibiting its kinase [16,17].
The key question is then to find out why NADH, a natural citrate synthase inhibitor did not switch off the enzyme in tumor cells. Probably, the synthesis of NADH by the dehydrogenases of the Krebs cycle and malate/aspartate shuttle, was too low, or the oxidation of NADH via the respiratory electron transport chain and mitochondrial complex1 (NADH dehydrogenase) was abnormally elevated. Another important point concerns PDH and α ketoglutarate dehydrogenase that are homologous enzymes, they might be regulated in a concerted way; when PDH is off, α ketoglutarate dehydrogenase might be also be slowed. Moreover, this could be associated to an upstream inhibition of aconinase by NO, or more probably to a blockade of isocitrate dehydrogenase, which favors in tumor cells, the citrate efflux from mitochondria, and the ATP citrate lyase route.
Normally, an increase of NADH inhibits the citrate condensation, favoring the ketogenic route associated to gluconeogenesis, which turns off glycolysis. Apparently, this regulation does not occur in tumors, since citrate synthase remains active. Moreover, in tumor cells, the α ketoglutarate not processed by
α ketoglutarate dehydrogenase converts to glutamate, via glutamate dehydrogenase, in this direction the reaction forms NAD+, backing up the LDH production. Other sources of glutamate are glutaminolysis, which increases in tumors [2].
The Figure Figure11 shows how tumors bypass the PK and PDH bottlenecks and evidently, the increase of glucose influx above the bottleneck, favors the supply of substrates to the pentose shunt, as pentose is needed for synthesizing ribonucleotides, RNA and DNA. The Figure Figure11 represents the stop below the citrate condensation. Hence, citrate quits the mitochondria to give via ATP citrate lyase, acetyl CoA and OAA in the cytosol of tumor cells. Acetyl CoA supports the synthesis of fatty acids and the formation of triglycerides. The other product of the ATP citrate lyase reaction, OAA, drives the transaminase cascade (ALAT and GOT transaminases) in a direction that consumes GLU and glutamine and converts in fine alanine into pyruvate and lactate plus NAD+. This consumes protein body stores that provide amino acids and much alanine (like in starvation).
The Figure Figure11 indicates that malate dehydrogenase is a source of NAD+ converting OAA into malate, which backs-up LDH. Part of the malate converts to pyruvate (malic enzyme) and processed by LDH. Moreover, malate enters in mitochondria via the shuttle and gives back OAA to feed the citrate condensation. Glutamine will also provide amino groups for the “de novo” synthesis of purine and pyrimidine bases particularly needed by tumor cells. The Figure Figure11 indicates that ASP shuttled out of the mitochondrial, joins the ASP formed by cytosolic transaminases, to feed the synthesis of pyrimidine bases via ASP transcarbamylase, a process also enhanced in tumor cells. In tumors, this silences the argininosuccinate synthetase step of the urea cycle [18–20].
This blockade also limits the supply of fumarate to the Krebs cycle. The latter, utilizes the α ketoglutarate provided by the transaminase reaction, since α ketoglutarate coming via aconitase slows down. Indeed, NO and peroxynitrite increase in tumors and probably block aconitase. The Figure Figure11 indicates the cleavage of arginine into urea and ornithine. In tumors, the ornithine production increases, following the polyamine pathway. Ornithine is decarboxylated into putrescine by ornithine decarboxylase, then it captures the backbone of S adenosyl methionine (SAM) to form polyamines spermine then spermidine, the enzyme controlling the process is SAM decarboxylase. The other reaction product, 5-methlthioribose is then decomposed into methylthioribose and adenine, providing purine bases to the tumor. We shall analyze below the role of SAM in the carcinogenic mechanism, its destruction aggravates the process.
Cancer metabolism. Glycolysis is elevated in tumors, but a pyruvate kinase (PK) “bottleneck” interrupts phosphoenol pyruvate (PEP) to pyruvate conversion. Thus, alanine following muscle proteolysis transaminates to pyruvate, feeding lactate dehydrogenase, …
In summary, it is like if the mechanism switching from gluconeogenesis to glycolysis was jammed in tumors, PK and PDH are at rest, like for gluconeogenesis, but citrate synthase is on. Thus, citric acid condensation pulls the glucose flux in the glycolytic direction, which needs NAD+; it will come from the pyruvate to lactate conversion by lactate dehydrogenase (LDH) no longer in competition with a quiescent Pcarb. Since the citrate condensation consumes acetyl CoA, ketone bodies do not form; while citrate will support the synthesis of triglycerides via ATP citrate lyase and fatty acid synthesis… The cytosolic OAA drives the transaminases in a direction consuming amino acid. The result of these metabolic changes is that tumors burn glucose while consuming muscle protein and lipid stores of the organism. In a normal physiological situation, one mobilizes stores for making glucose or ketone bodies, but not while burning glucose! Tumor cell metabolism gives them a selective advantage over normal cells. However, one may attack some vulnerable points.
Cancer metabolism. Glycolysis is elevated in tumors, but a pyruvate kinase (PK) “bottleneck” interrupts phosphoenol pyruvate (PEP) to pyruvate conversion. Thus, alanine following muscle proteolysis transaminates to pyruvate, feeding lactate dehydrogenase, converting pyruvate to lactate, (Warburg effect) and NAD+ required for glycolysis. Cytosolic malate dehydrogenase also provides NAD+ (in OAA to MAL direction). Malate moves through the shuttle giving back OAA in the mitochondria. Below the PK-bottleneck, pyruvate dehydrogenase (PDH) is phosphorylated (second bottleneck). However, citrate condensation increases: acetyl-CoA, will thus come from fatty acids β-oxydation and lipolysis, while OAA sources are via PEP carboxy kinase, and malate dehydrogenase, (pyruvate carboxylase is inactive). Citrate quits the mitochondria, (note interrupted Krebs cycle). In the cytosol, ATPcitrate lyase cleaves citrate into acetyl CoA and OAA. Acetyl CoA will make fatty acids-triglycerides. Above all, OAA pushes transaminases in a direction usually associated to gluconeogenesis! This consumes protein stores, providing alanine (ALA); like glutamine, it is essential for tumors. The transaminases output is aspartate (ASP) it joins with ASP from the shuttle and feeds ASP transcarbamylase, starting pyrimidine synthesis. ASP in not processed by argininosuccinate synthetase, which is blocked, interrupting the urea cycle. Arginine gives ornithine via arginase, ornithine is decarboxylated into putrescine by ornithine decarboxylase. Putrescine and SAM form polyamines (spermine spermidine) via SAM decarboxylase. The other product 5-methylthioadenosine provides adenine. Arginine deprivation should affect tumors. The SAM destruction impairs methylations, particularly of PP2A, removing the “signaling kinase brake”, PP2A also fails to dephosphorylate PK and PDH, forming the “bottlenecks”. (Black arrows = interrupted pathways).
II Starters for cancer metabolic anomaly
1. Lessons from oncogenes
Following the discovery of Rous sarcoma virus transmitting cancer [21], we have to wait the work of Stehelin [22] to realize that this retrovirus only transmitted a gene captured from a previous host. When one finds that the transmitted gene encodes the Src tyrosine kinase, we are back again to the tyrosine kinase signals, similar to those activated by insulin or IGF, which control carbohydrate metabolism, anabolism and mitosis.
An up regulation of the gene product, now under viral control causes tumors. However, the captured viral oncogene (v-oncogene) derives from a normal host gene the proto-oncogene. The virus only perturbs the expression of a cellular gene the proto-oncogene. It may modify its expression, or its regulation, or transmit a mutated form of the proto-oncogene. Independently of any viral infection, a similar tumorigenic process takes place, if the proto-oncogene is translocated in another chromosome; and transcribed under the control of stronger promoters. In this case, the proto-oncogene becomes an oncogene of cellular origin (c-oncogene). The third mode for converting a prot-oncogene into an oncogene occurs if a retrovirus simply inserts its strong promoters in front of the proto-oncogene enhancing its expression.
It is impressive to find that retroviral oncogenes and cellular oncogenes disturb this major signaling pathway: the MAP kinases mitogenic pathways. At the ligand level we find tumors such Wilm’s kidney cancer, resulting from an increased expression of insulin like growth factor; we have also the erbB or V-int-2 oncogenes expressing respectively NGF and FGF growth factor receptors. The receptors for these ligands activate tyrosine kinase signals, similarly to insulin receptors. The Rous sarcoma virus transmits the src tyrosine kinase, which activates these signals, leading to a chicken leukemia. Similarly, in murine leukemia, a virus captures and retransmits the tyrosine kinase abl. Moreover, abl is also stimulated if translocated and expressed with the bcr gene of chromosome 22, as a fusion protein (Philadelphia chromosome). Further, ahead Ras exchanging protein for GTP/GDP, and then the Raf serine-threonine kinases proto-oncogenes are known targets for oncogenes. Finally, at the level of transcription factors activated by MAP kinases, one finds cjun, cfos or cmyc. An avian leucosis virus stimulates cmyc, by inserting its strong viral promoter. The retroviral attacks boost the mitogenic MAP kinases similarly to inflammatory cytokins, or to insulin signals, that control glucose transport and gycolysis.
In addition to the MAP kinase mitogenic pathway, tyrosine kinase receptors activate PI3 kinase pathways; PTEN phosphatase counteracts this effect, thus acting as a tumor suppressor. Recall that a DNA virus, the Epstein-Barr virus of infectious mononucleose, gives also the Burkitt lymphoma; the effect of the virus is to enhance PI3 kinase. Down stream, we find mTOR (the target of rapamycine, an immune-suppressor) mTOR, inhibits PP2A phosphatase, which is also a target for the simian SV40 and Polyoma viruses. Schematically, one may consider that the different steps of MAP kinase pathways are targets for retroviruses, while the different steps of PI3 kinase pathway are targets for DNA viruses. The viral-driven enhanced function of these pathways mimics the effects of their prolonged activation by their usual triggers, such as insulin or IGF; one then expects to find an associated increase of glycolysis. The insulin or IGF actions boost the cellular influx of glucose and glycolysis. However, if the signaling pathway gets out of control, the tyrosine kinase phosphorylations may lead to a parallel PK blockade [3–5] explaining the tumor bottleneck at the end of glycolysis. Since an activation of enyme kinases may indeed block essential enzymes (PK, PDH and others); in principle, the inactivation of phosphatases may also keep these enzymes in a phosphorylated form and lead to a similar bottleneck and we do know that oncogenes bind and affect PP2A phosphatase. In sum, a perturbed MAP kinase pathway, elicits metabolic features that would give to tumor cells their metabolic advantage.
2. The methylation hypothesis and the role of PP2A phosphatase
In a remarkable comment, Newberne [23] highlights interesting observations on the carcinogenicity of diethanolamine [24] showing that diethanolamine decreased choline derivatives and methyl donors in the liver, like does a choline deficient diet. Such conditions trigger tumors in mice, particularly in the B6C3F1 strain. Again, the historical perspective recalled by Newberne’s comment brings us back to insulin. Indeed, after the discovery of insulin in 1922, Banting and Best were able to keep alive for several months depancreatized dogs, treated with pure insulin. However, these dogs developed a fatty liver and died. Unlike pure insulin, the total pancreatic extract contained a substance that prevented fatty liver: a lipotropic substance identified later as being choline [25]. Like other lipotropes, (methionine, folate, B12) choline supports transmethylation reactions, of a variety of substrates, that would change their cellular fate, or action, after methylation. In the particular case concerned here, the removal of triglycerides from the liver, as very low-density lipoprotein particles (VLDL), requires the synthesis of lecithin, which might decrease if choline and S-adenosyl methionine (SAM) are missing. Hence, a choline deficient diet decreases the removal of triglycerides from the liver; a fatty liver and tumors may then form. In sum, we have seen that pathways exemplified by the insulin-tyrosine kinase signaling pathway, which control anabolic processes, mitosis, growth and cell death, are at each step targets for oncogenes; we now find that insulin may also provoke fatty liver and cancer, when choline is not associated to insulin.
We must now find how the lipotropic methyl donor controls the signaling pathway. We know that after the tyrosine kinase reaction, serine-threonine kinases take over along the signaling route. It is thus highly probable that serine-threonine phosphatases will counteract the kinases and limit the intensity of the insulin or insulin like signals. One of the phosphatases involved is PP2A, itself the target of DNA viral oncogenes (Polyoma or SV40 antigens react with PP2A subunits and cause tumors). We found a possible link between the PP2A phosphatase brake and choline in works on Alzheimer’s disease [26]. Indeed, the catalytic C subunit of PP2A is associated to a structural subunit A. When C receives a methyle, the dimer recruits a regulatory subunit B. The trimer then targets specific proteins that are dephosphorylated [27].
In Alzheimer’s disease, the poor methylation of PP2A is associated to an increase of homocysteine in the blood [26]. The result of the PP2A methylation failure is a hyperphosphorylation of Tau protein and the formation of tangles in the brain. Tau protein is involved in tubulin polymerization, controlling axonal flow but also the mitotic spindle. It is thus possible that choline, via SAM, methylates PP2A, which is targeted toward the serine-threonine kinases that are counteracted along the insulin-signaling pathway. The choline dependent methylation of PP2A is the brake, the “antidote”, which limits “the poison” resulting from an excess of insulin signaling. Moreover, it seems that choline deficiency is involved in the L to M2 transition of PK isoenzymes [28].
3. Cellular distribution of PP2A
In fact, the negative regulation of Ras/MAP kinase signals mediated by PP2A phosphatase seems to be complex. The serine-threonine phosphatase does more than simply counteracting kinases; it binds to the intermediate Shc protein on the signaling cascade, which is inhibited [29]. The targeting of PP2A towards proteins of the signaling pathway depends of the assembly of the different holoenzymes. The carboxyl methylation of C-terminal leucine 309 of the catalytic C unit, permits to a dimeric form made of C and a structural unit A, to recruit one of the many regulatory units B, giving a great diversity of possible enzymes and effects. The different methylated ABC trimers would then find specific targets. It is consequently essential to have more information on methyl transferases and methyl esterases that control the assembly or disassembly of PP2A trimeric forms.
A specific carboxyl methyltransferase for PP2A [30] was purified and shown to be essential for normal progression through mitosis [31]. In addition, a specific methylesterase that demethylates PP2A has been purified [32]. Is seems that the methyl esterase cancels the action of PP2A, on signaling kinases that increase in glioma [33]. Evidently, the cellular localization of the methyl transferase (LCMT-1) and the phosphatase methyl esterase (PME-1) are crucial for controlling PP2A methylation and targeting. Apparently, LCMT-1 mainly localizes to the cytoplasm and not in the nucleus, where PME-1 is present, and the latter harbors a nuclear localization signal [34]. From these observations, one may suggest that PP2A gets its methyles in the cytoplasm and regulates the tyrosine kinase-signaling pathway, attenuating its effects.
A methylation deficit should then decrease the methylation of PP2A and boost the mitotic insulin signals as discussed above for choline deficiency, steatosis and hepatoma. At the nucleus, where PME-1 is present, it will remove the methyl, from PP2A, favoring the formation of dimeric AC species that have different targets, presumably proteins involved in the cell cycle. It is interesting to quote here the structural mechanism associated to the demethylation of PP2A. The crystal structures of PME-1 alone or in complex with PP2A dimeric core was reported [35] PME-1 binds directly to the active site of PP2A and this rearranges the catalytic triad of PME-1 into an active conformation that should demethylate PP2A, but this also seems to evict a manganese required for the phosphatase activity. Hence, demethylation and inactivation would take place in parallel, blocking mitotic actions.
However, another player is here involved, the so-called PTPA protein, which is a PP2A phosphatase activator. Apparently, this activator is a new type of cis/trans of prolyl isomerase, acting on Pro190 of the catalytic C unit isomerized in presence of Mg-ATP [36], which would then cancel the inactivation mediated by PME-1. Following the PTPA action, the demethylated phosphatase would become active again in the nucleus, and stimulate cell cycle proteins [37,38] inducing mitosis. Unfortunately, the ligand of this new prolyl isomerase is still unknown. Moreover, we have to consider that other enzymes such as cytochrome P450 have also demethylation properties.
In spite of deficient methylations and choline dehydrogenase pathway, tumor cells display an enhanced choline kinase activity, associated to a parallel synthesis of lecithin and triglycerides.
The hypothesis to consider is that triglycerides change the fate of methylated PP2A, by targeting it to the nucleus, there a methylesterase demethylates it; the phosphatase attacks new targets such as cell cycle proteins, inducing mitosis. Moreover, the phosphatase action on nuclear membrane proteins may render the nuclear membrane permeable to SAM the general methyl donor; promoters get methylated inducing epigenetic changes.
The relative decrease of methylated PP2A in the cytosol, not only cancels the brake over the signaling kinases, but also favors the inactivation of PK and PDH, which remain phosphorylated, contributing to the metabolic anomaly of tumor cells.
In order to prevent tumors, one should then favor the methylation route rather than the phosphorylation route for choline metabolism. This would decrease triglycerides, promote the methylation of PP2A and keep it in the cytosol, reestablishing the brake over signaling kinases.
Hypoxia is an essential issue to discuss
Many adequate “adult proteins” replace their fetal isoform: muscle proteins utrophine, switches to dystrophine; enzymes such as embryonic M2 PK [39] is replaced by M1. Hypoxic conditions seem to trigger back the expression of the fetal gene packet via HIF1-Von-Hippel signals. The mechanism would depend of a double switch since not all fetal genes become active after hypoxia. First, the histones have to be in an acetylated form, opening the way to transcription factors, this depends either of histone deacetylase (HDAC) inhibition or of histone acetyltransferase (HAT) activation, and represents the main switch. Second, a more specific switch must be open, indicating the adult/fetal gene couple concerned, or more generally the isoform of a given gene that is more adapted to the specific situation. When the adult gene mutates, an unbound ligand may indeed indicate, directly or indirectly, the particular fetal copy gene to reactivate [40]. In anoxia, lactate is more difficult to release against its external gradient, leading to a cytosolic increase of up-stream glycolytic products, 3P glycerate or others. These products may then be a second signal controlling the specific switch for triggering the expression of fetal genes, such as fetal hemoglobin or the embryonic M2 PK; this takes place if histones (main switch) are in an acetylated form.
Growth hormone-IGF actions, the control of asymmetrical mitosis
When IGF – Growth hormone operate, the fatty acid source of acetyl CoA takes over. Indeed, GH stimulates a triglyceride lipase in adipocytes, increasing the release of fatty acids and their β oxidation. In parallel, GH would close the glycolytic source of acetyl CoA, perhaps inhibiting the hexokinase interaction with the mitochondrial ANT site. This effect, which renders apoptosis possible, does not occur in tumor cells. GH mobilizes the fatty acid source of acetyl CoA from adipocytes, which should help the formation of ketone bodies, but since citrate synthase activity is elevated in tumors, ketone bodies do not form.
Compounds for correcting tumor metabolism
The figure figure1 indicates interrupted and enhanced metabolic pathways in tumor cells.
In table table1,1, the numbered pathways represent possible therapeutic targets; they cover several enzymes. When the activity of the pathway is increased, one may give inhibitors; when the activity of the pathway decreases, we propose possible activators
Table 1 Mol Cancer. 2011; 10 70. Published online Jun 7, 2011. doi 10.1186_1476-4598-10-70
The origin of Cancers by means of metabolic selection
The disruption of cells by internal or external compounds, releases substrates stimulating the tyrosine kinase signals for anabolism proliferation and stem cell repair, like for most oncogenes. If such signals are not limited, there is a parallel blockade of key metabolic enzymes by activated kinases or inhibited phosphatases. The result is a metabolism typical of tumor cells, which gives them a selective advantage; stabilized by epigenetic changes. A proliferation process, in which the two daughter cells divide, increases the tumor mass at the detriment of the body. Inevitable mutations follow.
Maurice Israël, et al. Mol Cancer. 2011;10:70-70.
Transcriptomics and Regulatory Processes
What are lncRNAs?
It was traditionally thought that the transcriptome would be mostly comprised of mRNAs, however advances in high-throughput RNA sequencing technologies have revealed the complexity of our genome. Non-coding RNA is now known to make up the majority of transcribed RNAs and in addition to those that carry out well-known housekeeping functions (e.g. tRNA, rRNA etc), many different types of regulatory RNAs have been and continue to be discovered.
Long noncoding RNAs (lncRNAs) are a large and diverse class of transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins. Their expression is developmentally regulated and lncRNAs can be tissue- and cell-type specific. A significant proportion of lncRNAs are located exclusively in the nucleus. They are comprised of many types of transcripts that can structurally resemble mRNAs, and are sometimes transcribed as whole or partial antisense transcripts to coding genes. LncRNAs are thought to carry out important regulatory functions, adding yet another layer of complexity to our understanding of genomic regulation.
The evolution of genome-scale models of cancer metabolism
The importance of metabolism in cancer is becoming increasingly apparent with the identification of metabolic enzyme mutations and the growing awareness of the influence of metabolism on signaling, epigenetic markers, and transcription. However, the complexity of these processes has challenged our ability to make sense of the metabolic changes in cancer. Fortunately, constraint-based modeling, a systems biology approach, now enables one to study the entirety of cancer metabolism and simulate basic phenotypes. With the newness of this field, there has been a rapid evolution of both the scope of these models and their applications. (NE Lewis and AM.Abdel-Haleem. frontiers physiol 2013;4(237): 1 http://dx.doi.org/10.3389/fphys.2013.00237)
Here we review the various constraint-based models built for cancer metabolism and how their predictions are shedding new light on basic cancer phenotypes, elucidating pathway differences between tumors, and discovering putative anti-cancer targets. As the field continues to evolve, the scope of these genome-scale cancer models must expand beyond central metabolism to address questions related to the diverse processes contributing to tumor development and metastasis.
“One of the goals of cancer research is to ascertain the mechanisms of cancer.”These words, penned by Dulbecco (1986), began a treatise on how a mechanistic understanding of cancer requires a sequenced human genome. Now with the abundance of sequence data, we are finding diverse genetic changes among different cancers (Vogelstein et al.,2013). While we are cataloging these mutations, the associated mechanisms leading to phenotypic changes are often unclear since mutations occur in the context of complex biological networks. For example, mutations to isocitrate dehydrogenase lead to oncometabolite synthesis, which alters DNA methylation and ultimately changes gene expression and the balance of normal cell processes (Sasakietal.,2012). Furthermore, many different combinations of mutations can lead to cancer. Since the genetic heterogeneity between tumors can be large, the biomolecular mechanisms underlying tumor physiology can vary substantially.
This is apparent in metabolism, where tumors can differ in serine metabolism dependence (Possematoetal., 2011) or TCA cycle function (Frezzaetal., 2011b). In addition, diverse mutations can alter NADPH synthesis by differentially regulat ing signaling pathways, such as the AMPK pathway (Cairnsetal., 2011; Jeonetal., 2012). The challenges regarding complexity and heterogeneity in cancer metabolism are beginning to be addressed with the COnstraint-Based Reconstruction and Analysis (COBRA) approach (Hernández Patiñoetal., 2012; Sharma and König, 2013), an emerging field in systems biology.Specifically, it accounts for the complexity of the perturbed biochemical processes by using genome-scale metabolic network reconstructions (Duarteetal., 2007; Maetal., 2007;Thieleetal., 2013).
In a reconstruction, the stoichiometric chemical reactions in a cell are carefully annotated and stitched together into a large network, often containing thousands of reactions. Genes and enzymes associated with each reaction are also delineated. The networks are converted into computational models and analyzed using many algorithms (Lewisetal., 2012). COBRA approaches are also beginning to address heterogeneity in cancer by integrating experimental data with the reconstructions (Blazier and Papin, 2012; Hydukeetal., 2013) to tailor the models to the unique gene expression profiles of general cancer tissue, and even individual cell lines and tumors. Here we describe the recent conceptual evolution that has occurred for constraint-based cancer modeling.
Targeting of gene expression
Tumor Suppressor Genes and its Implications in Human Cancer
Gain-of-function mutations in oncogenes and loss-of-function mutations in tumor suppressor genes (TSG) lead to cancer. In most human cancers, these mutations occur in somatic tissues. However, hereditary forms of cancer exist for which individuals are heterozygous for a germline mutation in a TSG locus at birth. The second allele is frequently inactivated by gene deletion, point mutation, or promoter methylation in classical TSGs that meet Knudson’s two-hit hypothesis. Conversely, the second allele remains as wild-type, even in tumors in which the gene is haplo-insufficient for tumor suppression. (K Inoue, EA Fry and Pj Taneja. Recent Progress in Mouse Models for Tumor Suppressor Genes and its Implications in Human Cancer. Clinical Medicine Insights: Oncology2013:7 103–122). This article highlights the importance of PTEN, APC, and other tumor suppressors for counteracting aberrant PI3K, β-catenin, and other oncogenic signaling pathways. We discuss the use of gene-engineered mouse models (GEMM) of human cancer focusing on Pten and Apc knockout mice that recapitulate key genetic events involved in initiation and progression of human neoplasia.
Targeting cancer metabolism – aiming at a tumour’s sweet-spot
Neil P. Jones and Almut Schulze
Drug Discovery Today January 2012
Targeting cancer metabolism has emerged as a hot topic for drug discovery. Most cancers have a high demand for metabolic inputs (i.e. glucose/glutamine), which aid proliferation and survival. Interest in targeting cancer metabolism has been renewed in recent years with the discovery that many cancer related (e.g. oncogenic and tumor suppressor) pathways have a profound effect on metabolism and that many tumors become dependent on specific metabolic processes. Considering the recent increase in our understanding of cancer metabolism and the increasing knowledge of the enzymes and pathways involved, the question arises: could metabolism be cancer’s Achilles heel?
During recent years, interest into the possible therapeutic benefit of targeting metabolic pathways in cancer has increased dramatically with academic and pharmaceutical groups actively pursuing this aspect of tumor physiology. Therefore, what has fuelled this revived interest in targeting cancer metabolism and what are the major advances and potential challenges faced in the race to develop new therapeutics in this area? This review will attempt to answer these questions and illustrate why we, and others, believe that targeting metabolism in cancer presents such a promising therapeutic rationale.
Oncogenes and cancer metabolism
Glycolysis TCA cycle Pentose phosphate pathway
FIGURE 1
Schematic representation of the regulation of cancer metabolism pathways. Metabolic enzymes are regulated by signaling pathways involving oncogenes and tumor suppressors. Complex regulatory mechanisms, key pathway interactions and enzymes are shown along with key metabolic endpoints (shown in purple) necessary for proliferation and survival (biosynthetic intermediates and NADPH). Key oncogenic pathways are shown in green and key tumor suppressor pathways are shown in red. Mutant IDH (mIDH) pathway is listed but is only functional in cancers containing mIDH.
FIGURE 2
Schematic representation of key components of the pentose phosphate pathway (PPP). Key enzymes are shown in blue boxes and key intermediates in purple text/box outline. DNA damage can activate ATM which in turn activates G6PDH to upregulate nucleotide synthesis for DNA repair and NAPDH to combat reactive oxygen species. PPP is also regulated by the tumour suppressor p53. The PPP can function as two separate branches (oxidative and non-oxidative) or be coupled into a recycling pathway – the pentose phosphate shunt – for maximum NADPH production.
Serine biosynthesis
Another branch diverting from glycolysis recently implicated in cancer is the serine biosynthesis pathway which converts the glycolytic intermediate 3-phosphoglycerate into serine (Fig. 3). Serine is an amino acid and an important neurotransmitter but can also provide fuel for the synthesis of other amino acids and nucleotides. The serine biosynthesis pathway also provides another key metabolic intermediate, a-KG, from glutamate breakdown via the action of phosphoserine aminotransferase (PSAT1). This pathway couples glycolysis (via 3-phosphoglycerate) with glutaminolysis (via glutamate), thereby linking two metabolic pathways known to be activated in many cancers.
FIGURE 3
Schematic representation of the serine biosynthesis pathway. Synthesis of serine involves integration of metabolites from glycolysis and glutaminolysis pathways and generates a-ketoglutarate, a key biosynthetic intermediate, and serine. Serine has many essential uses in the cell including amino acid, phospholipid and nucleotide synthesis.
Silencing of tumor suppressor genes by recruiting DNA methyltransferase 1 (DNMT1)
Ubiquitin-like containing PHD and Ring finger 1 (UHRF1) contributes to silencing of tumor suppressorgenes by recruiting DNA methyltransferase 1 (DNMT1) to their hemi-methylated promoters. Conversely,demethylation of these promoters has been ascribed to the natural anti-cancer drug, epigallocatechin-3-gallate (EGCG). The aim of the present study was to investigate whether the UHRF1/DNMT1 pair is an important target of EGCG action. (Mayada Achour, et al. Epigallocatechin-3-gallate up-regulates tumor suppressor gene expression via a reactive oxygen species-dependent down-regulation of UHRF1. Biochemical and Biophysical Research Communications 430 (2013) 208–212. http://dx.doi.org/10.1016/j.bbrc.2012.11.087)
Here, we show that EGCG down-regulates UHRF1 and DNMT1 expression in Jurkat cells, with subsequent up-regulation of p73 and p16INK4A genes. The down-regulation of UHRF1 is dependent upon the generation of reactive oxygen species by EGCG. Up-regulation of p16INK4A is strongly correlated with decreased promoter binding by UHRF1. UHRF1 over-expression counteracted EGCG-induced G1-arrested cells, apoptosis, and up-regulation of p16INK4A and p73. Mutants of the Set and Ring Associated (SRA) domain of UHRF1 were unable to down-regulate p16INK4A and p73, either in the presence or absence of EGCG. Our results show that down-regulation of UHRF1 is upstream to many cellular events, including G1 cell arrest, up-regulation of tumor suppressor genes and apoptosis.
Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant
ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, which includes the Epidermal Growth Factor Receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Mounting evidence indicates that ErbB4, unlike EGFR or ErbB2, functions as a tumor suppressor in many human malignancies. Previous analyses of the constitutively-dimerized and –active ErbB4 Q646C mutant indicate that ErbB4 kinase activity and phosphorylation of ErbB4 Tyr1056 are both required for the tumor suppressor activity of this mutant in human breast, prostate, and pancreatic cancer cell lines. However, the cytoplasmic region of ErbB4 possesses additional putative functional motifs, and the contributions of these functional motifs to ErbB4 tumor suppressor activity have been largely underexplored. (Citation: Richard M. Gallo, et al. (2013) Multiple Functional Motifs Are Required for the Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant. J Cancer Res Therap Oncol 1: 1-10)
Here we demonstrate that ErbB4 BH3 and LXXLL motifs, which are thought to mediate interactions with Bcl family proteins and steroid hormone receptors, respectively, are required for the tumor suppressor activity of the ErbB4 Q646C mutant. Furthermore, abrogation of the site of ErbB4 cleavage by gamma-secretase also disrupts the tumor suppressor activity of the ErbB4 Q646C mutant. This last result suggests that ErbB4 cleavage and subcellular trafficking of the ErbB4 cytoplasmic domain may be required for the tumor suppressor activity of the ErbB4 Q646C mutant. Indeed, here we demonstrate that mutants that disrupt ErbB4 kinase activity, ErbB4 phosphorylation at Tyr1056, or ErbB4 cleavage by gamma-secretase also disrupt ErbB4 trafficking away from the plasma membrane and to the cytoplasm. This supports a model for ErbB4 function in which ErbB4 tumor suppressor activity is dependent on ErbB4 trafficking away from the plasma membrane and to the cytoplasm, mitochondria, and/or the nucleus.
EGF Receptor
Initiation of pancreatic ductal adenocarcinoma (PDA) is definitively linked to activating mutations in the KRAS oncogene. However, PDA mouse models show that mutant Kras expression early in development gives rise to a normal pancreas, with tumors forming only after a long latency or pancreatitis induction.
Here, we show that oncogenic KRAS upregulates endogenous EGFR expression and activation, the latter being dependent on the EGFR ligand sheddase, ADAM17. Genetic ablation or pharmacological inhibition of EGFR or ADAM17 effectively eliminates KRAS-driven tumorigenesis in vivo. Without EGFR activity, active RAS levels are not sufficient to induce robust MEK/ERK activity, a requirement for epithelial transformation
The almost universal lethality of PDA has led to the intense study of genetic mutations responsible for its formation and progression. The most common oncogenic mutations associated with all PDA stages are found in the KRAS gene, suggesting it as the primary initiator of pancreatic neoplasia. However, mutant Kras expression throughout the mouse pancreatic parenchyma shows that the oncogene remains largely indolent until secondary events, such as pancreatitis, unlock its transforming potential. We find KRAS requires an inside-outside-in signaling axis that involves ligand-dependent EGFR activation to initiate the signal transduction and cell biological changes that link PDA and pancreatitis. (Cancer Cell (2012); 22: 304–317).
HER4 (EGFR/ErbB, HER2/Neu, HER3)
ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, which includes the Epidermal Growth Factor Receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Mounting evidence indicates that ErbB4, unlike EGFR or ErbB2, functions as a tumor suppressor in many human malignancies. Previous analyses of the constitutively-dimerized and –active ErbB4 Q646C mutant indicate that ErbB4 kinase activity and phosphorylation of ErbB4 Tyr1056 are both required for the tumor suppressor activity of this mutant in human breast, prostate, and pancreatic cancer cell lines. However, the cytoplasmic region of ErbB4 possesses additional putative functional motifs, and the contributions of these functional motifs to ErbB4 tumor suppressor activity have been largely underexplored.
ErbB4 Possesses Multiple Functional Motifs and Mutations Have Been Engineered to Target These Motifs.
The organization of ErbB4 is as indicated in this schematic. The extracellular ligand-binding motifs reside in the amino-terminal region upstream of amino acid residue 651. The singlepass transmembrane domain consists of amino acid residues 652-675. The cytoplasmic tyrosine kinase domain consists of amino acid residues 713-989. The majority of cytoplasmic sites of tyrosine phosphorylation reside in amino acid residues 990-1308, most notably Tyr1056. Additional putative functional motifs include a TACE cleavage site, a gamma-secretase cleavage site, two LXXLL (steroid hormone receptor binding) motifs, a BH3 domain, three WW domain binding motifs, and a PDZ domain binding motif. Mutations that disrupt these motifs are noted. Finally, note the two locations of alternative transcriptional splicing, resulting in a total of four different splicing isoforms.
Here we demonstrate that ErbB4 BH3 and LXXLL motifs, which are thought to mediate interactions with Bcl family proteins and steroid hormone receptors, respectively, are required for the tumor suppressor activity of the ErbB4 Q646C mutant. Furthermore, abrogation of the site of ErbB4 cleavageby gamma-secretase also disrupts the tumor suppressor activity of the ErbB4 Q646C mutant. This last result suggests that ErbB4 cleavage and subcellular trafficking of the ErbB4 cytoplasmic domain may be required for the tumor suppressor activity of the ErbB4 Q646C mutant. Indeed, here we demonstrate that mutants that disrupt ErbB4 kinase activity, ErbB4 phosphorylation at Tyr1056, or ErbB4 cleavage by gamma-secretase also disrupt ErbB4 trafficking away from the plasma membrane and to the cytoplasm. This supports a model for ErbB4 function in which ErbB4 tumor suppressor activity is dependent on ErbB4 trafficking away from the plasma membrane and to the cytoplasm, mitochondria, and/or the nucleus.
(Richard M. Gallo, et al. (2013) Multiple Functional Motifs Are Required for the Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant. J Cancer Res Therap Oncol 1: 1-10)
Resistance to Receptor Tyrosine Kinase Inhibition
Receptor tyrosine kinases (RTKs) are activated by somatic genetic alterations in a subset of cancers, and such cancers are often sensitive to specific inhibitors of the activated kinase. Two well-established examples of this paradigm include lung cancers with either EGFR mutations or ALK translocations. In these cancers, inhibition of the corresponding RTK leads to suppression of key downstream signaling pathways, such as the PI3K (phosphatidylinositol 3-kinase)/AKT and MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal–regulated kinase) pathways, resulting in cell growth arrest and death. Despite the initial clinical efficacy of ALK (anaplastic lymphoma kinase) and EGFR (epidermal growth factor receptor) inhibitors in these cancers, resistance invariably develops, typically within 1 to 2 years. (MJ Niederst and JA Engelman. Sci Signal, 24 Sep 2013; 6(294), p. re6 . http://dx.doi.org/10.1126/scisignal.2004652)
Over the past several years, multiple molecular mechanisms of resistance have been identified, and some common themes have emerged. One is the development of resistance mutations in the drug target that prevent the drug from effectively inhibiting the respective RTK. A second is activation of alternative RTKs that maintain the signaling of key downstream pathways despite sustained inhibition of the original drug target. Indeed, several different RTKs have been implicated in promoting resistance to EGFR and ALK inhibitors in both laboratory studies and patient samples. In this mini-review, we summarize the concepts underlying RTK-mediated resistance, the specific examples known to date, and the challenges of applying this knowledge to develop improved therapeutic strategies to prevent or overcome resistance.
The TGF-β Pathway
Aberrations in the enzymes that modify ubiquitin moieties have been observed to cause a myriad of diseases, including cancer. Therefore a better understanding of these enzymes and their substrates will lead to the identification of prospective druggable targets. Here we discuss the role of ubiquitin modifying enzymes in the canonical TGF-β pathway highlighting the ubiquitin regulating enzymes, which may potentially be targeted by small molecule inhibitors. (Pieter Eichhorn. (DE) -Ubiquitination in The TGF-β Pathway. J Cancer Res Therap Oncol 2013; 1: 1-6).
TGF-β is a multifunctional cytokine that plays a key role in embryogenesis and adult tissue homoeostasis. TGF-β is secreted by a myriad of cell types triggering a varied array of cellular functions including apoptosis, proliferation, migration, endothelial and mesenchymal transition, and extracellular matrix production. Downstream TGFβ responses can also be modulated by other signalling pathways (i.e. PI3K, ERK, WNT, etc.) resulting in a complex web of TGF-β pathway activation or repression depending on the nature of the signal and cellular context. Apart from TGF-β mediated cell autonomous effects TGF-β can further play an important function in regulating tumour microenvironments effecting the interaction between stromal fibroblasts and tumour cells.
Due to the central role of TGF-β in cellular processes it is therefore unsurprising that loss of TGF-β pathway integrity is frequently observed in a variety of human diseases, including cancer. However, the TGF-β pathway plays a complex dual role in cancer. In normal epithelial cells and premalignant cells TGF-β acts a potent tumor suppressor eliciting a cytostatic response inhibiting tumor progression. Supporting this notion, inactivating mutations in members of the TGF-βpathway have been observed in a variety of cancers including pancreatic, colorectal, and head and neck cancer.
In contrast, during tumor progression the TGF-β antiproliferative function is lost, and in certain advanced cancers TGF-β becomes an oncogenic factor inducing cellular proliferation, invasion, angiogenesis, and immune suppression. As a consequence, the TGFβ pathway is currently considered a therapeutic target in advanced cancers and several anti- TGF-β agents in clinical trials have shown promising results. However, due to the complex dichotomous role of TGF-β in oncogenesis a detailed understanding of TGF-β biology is required in order to design successful therapeutic strategies to identify patient populations that will benefit most from these compounds.
G protein receptor
G protein-coupled receptors (GPCRs) modulate a vast array of cellular processes. The current review gives an overview of the general characteristics of GPCRs and their role in physiological conditions. In addition, it describes the current knowledge of the physiological and pathophysiological functions of GPR55, an orphan GPCR, and how it can be exploited as a therapeutic target to combat various cancers.
(D Leyva-Illades, S DeMorrow . Orphan G protein receptor GPR55 as an emerging target in cancer therapy and management. Cancer Management and Research 2013:5 147–155)
Signal transduction is essential for maintaining cellular homeostasis and to coordinate the activity of cells in all organisms. Proteins localized in the cell membrane serve as the interface between the outside and inside of the cell. G protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and are encoded by at least 800 genes in the human genome. GPCRs are also known as seven-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptors, and G protein-linked receptors. GPCRs can detect an expansive array of extracellular signals or ligands that include photons, ions, odors, pheromones, hormones, and neurotransmitters. Nonsensory GPCRs (excluding light, odor, and taste receptors) have been classified into four families: class A rhodopsin-like, class B secretin-like, class C metabotropic glutamate/pheromone, and frizzled receptors. They have a peculiar structure that has been highly conserved over the course of evolution and are made up of an amino acid chain, the N-terminal of which is localized outside of the cellular membrane and the C-terminal in the cytoplasm. The amino acid chain spans the cellular membrane seven times and has three intracellular and three extracellular loops.
GPCRs are called that because they exert their actions by associating with a family of heterotrimeric proteins (made up of α, β, and γ subunits) that are capable of binding and hydrolyzing guanosine triphosphate (GTP).To date, 16 different α subunits, five β subunits, and 11 γ subunits have been described in mammalian tissues. When activated, these receptors undergo conformational changes that are mechanically transduced to the G proteins, which then initiate a cycle of activation and inactivationassociated with the binding and hydrolysis of GTP. Activated G proteins can then positively or negatively modulate ion channels (mainly potassium and calcium) or the second messenger generating enzymes (ie, adenylate cyclase and phospholipase C [PLC]) that allow the signal to be propagated to the interior of the cell to ultimately affect cell function.
Matrix Metalloproteinases
Degradation of extracellular matrix is crucial for malignant tumour growth, invasion, metastasis and angiogenesis. Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases collectively capable of degrading essentially all components of the ECM. Elevated levels of distinct MMPs can be detected in tumour tissue or serumof patients with advanced cancer and their role as prognostic indicators in cancer is studied. In addition, therapeutic intervention of tumour growth and invasion based on inhibition of MMP activity is under intensive investigation and several MMP inhibitors are in clinical trials in cancer. In this review, we discuss the current view on the feasibility of MMPs as prognostic markers and as targets for therapeutic intervention in cancer.
(MATRIX METALLOPROTEINASES IN CANCER: PROGNOSTIC MARKERS AND THERAPEUTIC TARGETS.
Common properties of the MMPs include the requirement of zinc in their catalytic site for activity and their synthesis as inactive zymogens that generally need to be proteolytically cleaved to be active. Normally the MMPs are expressed only when and where needed for tissue remodeling accompanies various processes such as during embryonic development, wound healing, uterine and mammary involution, cartilage-to-bone transition during ossification, and trophoblast invasion into the endometrial stoma during placenta development. However, aberrant expression of various MMPs has been correlated with pathological conditions, such as periodontitis, rheumatoid arthritis, and tumor cell invasion and metastasis .
There are now over 20 members of the MMP family, and they can be subgrouped based on their structures. The minimal domain structure consists of a signal peptide, prodomain, and catalytic domain. The propeptide domain contains a conserved cysteine residue (the “cysteine switch”) that coordinates to the catalytic zinc to maintain inactivity. MMPs with only the minimal domain are referred to as matrilysins (MMP-7 and -26). The most common structures for secreted MMPs, including collagenases and stromelysins, have an additional hemopexin-like domain connected by a hinge region to the catalytic domain (MMP-1, -3, -8, -10, -12, -13, -19, and -20).
FIGURE 1 – Structure of human matrix metalloproteinases. The signal peptide directs the proenzyme for secretion. The propeptide contains a conserved sequence (PRCGxPD), in which the cysteine forms a covalent bond (cysteine switch), with the catalytic zinc (Zn2_) to maintain the latency of proMMPs. Catalytic domain contains the highly conserved zinc binding site (HExGHxxGxxHS) in which Zn2_is coordinated by 3 histidines. The proline-rich hinge region links the catalytic domain to the hemopexin domain, which determines the substrate specificity of specific MMPs. The hemopexin domain is absent in matrilysin (MMP-7) and matrilysin-2 (endometase, MMP-26). Gelatinases A and B (MMP-2 and MMP-9, respectively) contain 3 repeats of the fibronectin-type II domain inserted in the catalytic domain. MT1-, MT2-, MT3- and MT5-MMP contain a transmembrane domain and MT4- and MT6-MMPs contain a glycosylphosphatidylinositol (GPI) anchor in the C-terminus of the molecule, which attach these MMPs to the cell surface. MT-MMPs, MMP-11, MMP-23 and MMP-28 contain a furin cleavage site (RxKR) between the propeptide and catalytic domain, making these proenzymes susceptible to activation by intracellular furin convertases. MMP-23 contains an N-terminal signal anchor, which anchors proMMP-23 to the Golgi complex and has a different C-terminal domain instead of hemopexin-like domain.
The physiologic expression of MMP-13 in vivo is limited to situations, such as fetal bone development and fetal wound repair, in which rapid remodeling of collagenous ECM is required. MMP-13 is expressed in pathologic conditions, such as arthritis, chronic dermal and intestinal ulcers, chronic periodontal inflammation and atherosclerotic plaques. The expression of MMP-13 is detected in vivo in invasive malignant tumours, breast carcinomas, squamous cell carcinomas (SCCs) of the head and neck and vulva, malignant melanomas, chondrosarcomas and urinary bladder carcinomas.
Table I. Human MMPS, their chromosomal localization, substrates, exogenous activators, and activating capacity1
MMP expression and activity are regulated at several levels. In most cases, MMPs are not synthesized until needed. Transcription can be induced by various signals including cytokines, growth factors, and mechanical stress. In certain cases, regulation of mRNA stability and translational efficiencyhave been reported. Because most MMPs are secreted as inactive zymogens, they need to be activated, usually by proteolytic cleavage of their NH2-terminal prodomains. Some MMPs are activated by other serine proteases such as plasmin and furin, whereas some of the MMPs can activate other members of their family. The most well characterized is the activation of pro-MMP-2 by MT1-MMP.
A number of MMPs have been strongly implicated in multiple stages of cancer progression including the acquisition of invasive and metastatic properties. Thus, efforts have been made for the past 20 years to develop MMPIs that can be used to halt the spread of cancer, which is what ultimately kills the person. However, initial clinical trials using first generation MMPIs proved to be disappointing . In the ensuing years, much has been learned about the roles of specific MMPs in the different processes of carcinogenesis and more specific MMPIs are being developed and brought to clinical trials.
However, the dosing and scheduling for optimal efficacy is not the same as required for conventional cytotoxic drugs because the MMPIs do not directly kill cancer cells, but instead target such processes as angiogenesis (the development of new blood vessels), invasion, and metastatic spread. (Matrix Metalloproteinases, Angiogenesis, and Cancer. Joyce E. Rundhaug. Commentary re: A. C. Lockhart et al., Reduction of Wound Angiogenesis in Patients Treated with BMS-275291, a Broad Spectrum Matrix Metalloproteinase Inhibitor. Clin. Cancer Res., 2003; 9551–554).
Role of p38 MAP Kinase Signal Transduction in Solid Tumors
Mitogen-activated protein kinases (MAPKs) mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the main subgroups, the p38 MAP kinases, has been implicated in a wide range of complex biologic processes, such as cell proliferation, cell differentiation, cell death, cell migration, and invasion. Dysregulation of p38 MAPK levels in patients are associated with advanced stages and short survival in cancer patients (e.g., prostate, breast, bladder, liver, and lung cancer). p38 MAPK plays a dual role as a regulator of cell death, and it can either mediate cell survival or cell death depending not only on the type of stimulus but also in a cell type specific manner. In addition to modulating cell survival, an essential role of p38 MAPK in modulation of cell migration and invasion offers a distinct opportunity to target this pathway with respect to tumor metastasis. The specific function of p38 MAPK appears to depend not only on the cell type but also on the stimuli and/or the isoform that is activated.
Mitogen-activated protein kinase (MAPK) signal transduction pathways are evolutionarily conserved among eukaryotes and have been implicated to play key roles in a number of biological processes, including cell growth, differentiation, apoptosis, inflammation, and responses to environmental stresses.
They are typically organized in 3-tiered architecture consisting of a MAPK, a MAPK activator (MAPK kinase), and a MAPKK activator (MAPKK kinase). The MAPK pathways can be regulated at multiple levels as well as via multiple mechanisms, of which the regulation of mitogen-activated protein kinase kinase kinase (MAPKKK/MAP3K) has been proved to be the most challenging due to the great diversity and versatility between different modules at this level. The complex array of growth factors and other ligands that can initiate intracellular cell signaling requires a very high level of coordination among the different proteins involved.
GTP cyclohydrolase (GCH1)
GTP cyclohydrolase (GCH1) is the key-enzyme to produce the essential enzyme cofactor, tetrahydrobiopterin. The byproduct, neopterin is increased in advanced human cancer and used as cancer-biomarker, suggesting that pathologically increased GCH1 activity may promote tumor growth.
(G Picker, Hee-Young Lim, et al. Inhibition of GTP cyclohydrolase attenuates tumor growth by reducing angiogenesis and M2-like polarization of tumor associated macrophages. Int. J. Cancer 2003; 132: 591–604 (2013) http://dx.doi.org/10.1002/ijc.27706 )
We found that inhibition or silencing of GCH1 reduced tumor cell proliferation and survival and the tube formation of human umbilical vein endothelial cells, which upon hypoxia increased GCH1 and
endothelial NOS expression, the latter prevented by inhibition of GCH1. In nude mice xenografted with HT29-Luc colon cancer cells GCH1 inhibition reduced tumor growth and angiogenesis, determined by in vivo luciferase and near-infrared imaging of newly formed blood vessels. The treatment with the GCH1 inhibitor shifted the phenotype of tumor associated macrophages from the proangiogenic M2 towards M1, accompanied with a shift of plasma chemokine profiles towards tumor-attacking chemokines including CXCL10 and RANTES. GCH1 expression was increased in mouse AOM/DSS-induced colon tumors and in high grade human colon and skin cancer and oppositely, the growth of GCH1-deficient HT29-Luc tumor cells in mice was strongly reduced. The data suggest that GCH1 inhibition reduces tumor growth by (i) direct killing of tumor cells, (ii) by inhibiting angiogenesis, and (iii) by enhancing the antitumoral immune response.
The Role of Stroma in Tumour-Host Co-Existence
Molnár et al., The Role of Stroma in Tumour-Host Co-Existence: Some Perspectives in Stroma-Targeted Therapy of Cancer Biochem Pharmacol 2013, 2:1 http://dx.doi.org/10.4172/2167-0501.1000107
Cancer grows at the expense of the host as a parasite or superparasite following the second law of thermodynamics (conservation of energy). When the cancer cell progresses via replication to the special state called “spheroid”, a new phase begins with its intimate interaction and development of responses from the stroma which together assist in the formation of a full blown cancer. Among the processes involved are the development of blood vessels and lymphatic channels which are essential for maintenance and further growth of the cancer mass. In this way the condition of “parasitism” is completed with simultaneous suppression of the immune response of the host to the histo-incompatability of the tumor mass. Stroma/parenchyma promotes cancer invasion by feeding cancer cells and inducing immune tolerance. The dynamic changes in composition of stroma and biological consequences as feeder of cancer cells and immune tolerance can give a perspective for rational drug design in anti-stromal therapy. There are differences between normal and cancer cells at subcellular level such as compartmentalzation and structure of cytoskeleton and energy distribution (that is low generally, but locally high in normal cells). In cancer cannibalism of normal cells, the growing cancer mass is a factor for progression and invasion.
Cancer cells have been shown to kill normal cells and the products of cell death used for progression of growth of the cancer cell. Serum and growth factors produced by tumor stroma also provide the needed nutrients and conditions for further tumor growth. Cancer cannot feed off other cancer cells and therefore grow poorly. Probably, although not yet proven, the inability of cancer to “parasitise” other cancer cell types is probably due to some kind of competition or interference. The tumor is in charge of its own development due to its induction proteinases, lipid mobilization factors and angiogenetic factors as well as its ability to negate immune responses of the host response to what is in essence a foreign body.
In our review co-existence of normal and cancer cells in tumor with the growth promoting factors, and the immune tolerance mediating factors produced in the stromal and cancer cells/tissues will be discussed with perspective of stroma targeted therapy.
The clinical significance of cell cannibalism is well defined and described in a large number of publications. The direction of process of cancer development is defined as the tumor invades the normal tissue which never occurs in the reverse direction. This suggests that the cancer cell strives to achieve the lowest energy level possible. Therefore the first of the development of a full blown cancer can be considered as the 2nd Thermodynamic principle that explains, describes and drives the invading cancer into normal surrounding tissue.
From the normal living state, under particular conditions such as hypoxia, where ATP synthesis is decreased resulting in a switch to glycolytic pathways, cancer cells are selected from a fraction of the population [4]. Energetically, in the presence of electron transfer, by using high energy from respiration, the proliferating state is more stable than resting cells where a higher degree of protein stabilization occurs such as that needed for maintainance of the cytoskeleton of the cell. It was proposed that tumor-promotion might be controlled or modulated by small electronic currents originating from reactive oxygen species and transported through the cytoskeletal microfilament network of the cancer cell.
Aerobic glycolysis is the main energy producing process in cancer cells. Among many other aspects, recently the mitochondria have also been regarded as potential targets in the therapy of cancer. Several small molecules have been tested to restore their dysfunctional functions either by direct or indirect effects. Because of poorly functioning mitochondria, the electron transfer component of the respiration cycle is inefficient; therefore, cancer cells have smaller Gibbs energy than healthy cells. This means, that these cancer cells exists in a metastable state and are not able maintain normal cell structure.
Therefore, the cytoskeleton system is collapsed and dielectric bilayers are formed as a lower grade of cellular structure with decreased electron conductivity. Consequently, to halt cancer growth, one has to evaluate the process of cancer cell development in situ, where the primary tumor is growing as well as that of the metastatic cell that is invading surrounding or distal tissues. This affords one to suggest that the stroma is formed first during long term repeated oxidative stress, a process that is initially accompanied with inflammation due to an active immune response to the histoincompatability antigens present on the surface of the cancer cell. If the cancer cell evades the activity of killer T cells (Treg cells) by either secreting agents that reduce the response of the Treg cells or the immune system for whatever reason is ineffective (immunosuppressed states such as HIV/AIDS, pregnancy, transplantation therapy, etc.), the formed cancer cells have the opportunity to initiate tumor development. Because of the limited capacity of its electron transfer cycle, cancer cells are essentially starving cells that require glycolytically useful substrates. These substrates are obtained from the killing of normal cells by agents secreted by the cancer cell and the products yielded from dead normal cells “eaten” (phagocytosed) by the starving cancer cell which is digested by the cancer cells lysosomal system. This autophagic process of cannibalism keeps the cancer cell alive and thriving and is known as cytophagy, i.e., cannibalism of normal cells. This type of autophagocytosis results in a parasitic co-existence of tumor cells with normal cells and will determine the main pathway of interaction between the growing cancer tissue (tumor) and normal tissue where the cancer tissue gradually destroys normal tissues. This process obeys the second law of thermodynamics-conservation of energy within a defined system.
Treatments for Cancer
Bosutinib: a SRC–ABL tyrosine kinase inhibitor for treatment of chronic myeloid leukemia.
FE Rassi, HJ Khoury. Pharmacogenomics and Personalized Medicine 2013:6 57–62.
Bosutinib is one of five tyrosine kinase inhibitors commercially available in the United States for the treatment of chronic myeloid leukemia. This review of bosutinib summarizes the mode of action, pharmacokinetics, efficacy and safety data, as well as the patient-focused perspective through quality-of-life data. Bosutinib has shown considerable and sustained efficacy in chronic myeloid leukemia, especially in the chronic phase, with resistance or intolerance to prior tyrosine kinase inhibitors. Bosutinib has distinct but manageable adverse events. In the absence of T315I and V299L mutations, there are no absolute contraindications for the use of bosutinib in this patient population
Chronic myeloid leukemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the presence of a signature hybrid oncogene, the BCR–ABL. The Philadelphia chromosome (Ph+) results from a reciprocal translocation between chromosome 9 and chromosome 22 that juxtaposes the two genes BCR and ABL and drives the leukemogenesis in CML. The ABL gene encodes for a nonreceptor tyrosine kinase that becomes deregulated and constitutively active after the juxtaposition of BCR. BCR–ABL is central in controlling downstream pathways involved in cell proliferation, regulation of cellular adhesion, and apoptosis.The understanding of the importance of this kinase activity in the pathophysiology of CML led to the development of tyrosine kinase inhibitors (TKI) that specifically target BCR–ABL. These agents became the mainstay of modern therapy in CML. CML has a triphasic clinical course, and the majority of patients (∼80%) are diagnosed during the early phase or the chronic phase (CP). However, and without effective treatment, CML invariably progresses to the advanced phases of the disease – the accelerated phase (AP) and the blast phase (BP). BP CML is a lethal refractory secondary leukemia with a short predicted survival.
Comprehensive molecular portraits of human breast tumors
We analyzed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, mRNA arrays, microRNA sequencing and reverse phase protein arrays. Our ability to integrate information across platforms provided key insights into previously-defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity.
Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at > 10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the Luminal A subtype. We identified two novel protein expression-defined subgroups, possibly contributed by stromal/microenvironmental elements, and integrated analyses identified specific signaling pathways dominant in each molecular subtype including a HER2/p-HER2/HER1/p-HER1 signature within the HER2-Enriched expression subtype. Comparison of Basal-like breast tumors with high-grade Serous Ovarian tumors showed many molecular commonalities, suggesting a related etiology and similar therapeutic opportunities. The biologic finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biologic subtypes of breast cancer.
Most molecular studies of breast cancer have focused on just one or two high information content platforms, most frequently mRNA expression profiling or DNA copy number analysis, and more recently massively parallel sequencing. Supervised clustering of mRNA expression data has reproducibly established that breast cancers encompass several distinct disease entities, often referred to as the intrinsic subtypes of breast cancer. The recent development of additional high information content assays focused on abnormalities in DNA methylation, microRNA expression and protein expression, provide further opportunities to more completely characterize the molecular architecture of breast cancer.
Synbiology contribution and Nanotechnology
Synthetic RNAs Designed to Fight Cancer
Xiaowei Wang and his colleagues at Washington University School of Medicine in St. Louis have designed synthetic molecules that combine the advantages of two experimental RNA therapies against cancer. They have designed synthetic molecules that combine the advantages of two experimental RNA therapies against cancer. RNA plays an important role in how genes are turned on and off in the body. Both siRNAs and microRNAs are snippets of RNA known to modulate a gene’s signal or shut it down entirely. Separately, siRNA and microRNA treatment strategies are in early clinical trials against cancer, but few groups have attempted to marry the two.
“We are trying to merge two largely separate fields of RNA research and harness the advantages of both,” said Xiaowei Wang, assistant professor of radiation oncology and a research member of the Siteman Cancer Center. The study appears in the December issue of the journal RNA.
“We designed an artificial RNA that is a combination of siRNA and microRNA,” Wang said “our artificial RNA simultaneously inhibits both cell migration and proliferation.” For therapeutic purposes, “small interfering” RNAs, or siRNAs, are designed and assembled in a lab and can be made to shut down– or interfere with– a single specific gene that drives cancer. The siRNA molecules work extremely well at silencing a gene target because the siRNA sequence is made to perfectly complement the target sequence, thereby silencing a gene’s expression.
Though siRNAs are great at turning off the gene target, they also have potentially dangerous side effects: siRNAs inadvertently can shut down other genes that need to be expressed to carry out tasks that keep the body healthy. The siRNAs interfere with off-target genesthat closely complement their “seed region,” a section of the siRNA that governs binding to a gene target. “In the past, we tried to block the seed region in an attempt to reduce the side effects. Until now, we never tried to replace the seed region completely.”
Wang and his colleagues asked whether they could replace the siRNA’s seed region with the seed region from microRNA. Unlike siRNA, microRNA is a natural part of the body’s gene expression. And it can also shut down genes. As such, the microRNA seed region (with its natural targets) might reduce the toxic side effects caused by the artificial siRNA seed region. Plus, the microRNA seed region would add a new tool to shut down other genes that also may be driving cancer.
Wang’s group started with a bioinformatics approach, using a computer algorithm to design siRNA sequences against a common driver of cancer, a gene called AKT1 that encourages uncontrolled cell division. The program also selected siRNAs against AKT1 that had a seed region highly similar to the seed region of a microRNA known to inhibit a cell’s ability to move, thus potentially reducing the cancer’s ability to spread.
A Neutralizing RNA Aptamer
Nucleic acid aptamers have been developed as high-affinity ligands that may act as antagonists of disease-associated proteins. Aptamers are non immunogenic and characterised by high specificity and low toxicity thus representing a valid alternative to antibodies or soluble ligand receptor traps/decoys to target specific cancer cell surface proteins in clinical diagnosis and therapy. The epidermal growth factor receptor (EGFR) has been implicated in the development of a wide range of human cancers including breast, glioma and lung. The observation that its inhibition can interfere with the growth of such tumors has led to the design of new drugs including monoclonal antibodies and tyrosine kinase inhibitors currently used in clinic. However, some of these molecules can result in toxicity and acquired resistance, hence the need to develop novel kinds of EGFR-targeting drugs with high specificity and low toxicity.
Here we generated, by a cell-Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach, a nuclease resistant RNA-aptamer that specifically binds to EGFR with a binding constant of 10 nM. When applied to EGFR-expressing cancer cells the aptamer inhibits EGFR-mediated signal pathways causing selective cell death. Furthermore, at low doses it induces apoptosis even of cells that are resistant to the most frequently used EGFR-inhibitors, such as gefitinib and cetuximab, and inhibits tumor growth in a mouse xenograft model of human non-small-cell lung cancer (NSCLC). Interestingly, combined treatment with cetuximab and the aptamer shows clear synergy in inducing apoptosis in vitro and in vivo. In conclusion, we demonstrate that this neutralizing RNA aptamer is a promising bio-molecule that can be developed as a more effective alternative to the repertoire of already existing EGFR-inhibitors.
In-Silico Molecular Docking Analysis of Cancer Biomarkers
Currently, in the research scenario for cancer, the identification of anti-cancer drugs using immuno-modulatory proteins and other molecular agents to initiate apoptosis in cancer cells and to inhibit the signaling pathways of cancer biomarkers as a drug targeted therapy, for cancer cell proliferation assays by the researchers. In-Silico analysis is used to recognize anticancer compounds as a future prospective for In-Vitro and In-Vivo analysis. A large number of herbal remedies (e.g. garlic, mistletoe) are used by cancer patients for treating the cancer and/or reducing the toxicities of chemotherapeutic drugs. Some herbal medicines have shown potentially beneficial effects on cancer progression and may ameliorate chemotherapy-induced toxicities. (K. Gowri Shankar et al., In-Silico Molecular Docking Analysis of Cancer Biomarkers with Bioactive Compounds of Tribulus terrestris. Intl J NOVEL TRENDS PHARMAL SCI. 2013; 3(4).
Tribulus terrestris is mentioned in ancient Indian Ayurvedic medical texts dating back thousands of years. Tribulus terrestris has been widely used in the Ayurvedic system of medicine for the treatment of sexualdysfunction and various urinary disorders. The aim of the present study is to evaluate the interactions of some bioactive compounds of Tribulus terrestris for In-Silico anticancer analysis with cancer biomarkers as targets. The targeted biomarkers for analysis include NSE-Lung cancer, Follistatin-Prostrate cancer, GGT Hepatocellular carcinoma, Human Prostasin-Ovarian cancer.
GC-MS analysis of Tribulus terrestris whole plant methanol extract revealed the existence of the major compound like 3,7,11,15-tetramethylhexadec-2-en-1-ol, 1,2-Benzenedicarboxylic acid, disooctyl ester, 9,12,15-Octadecatrienoic acid, (z,z,z)-, 9,12-Octadecadienoic acid (z,z)-, Hexadecadienoic acid, ethyl ester, n-Hexadecadienoic acid, Octadecanoic acid, Phytol, α-Amyrin are chosen as ligands. Hence, by analyzing the minimum binding energy of the ligand binding complex with the receptors by dockinganalysis using AutoDock tools will show effective nature of inhibition of these receptors by the unique ligands. Based on the results low minimum binding energy ligands are identified and used as a future studies can be done for specific receptors docking.
Anti-Cancerous Effect of4,4′-Dihydroxychalcone ((2E,2′E)-3,3′-(1,4-Phenylene) Bis (1-(4-hydroxyphenyl) Prop-2-en-1-one)) on T47D Breast Cancer Cell Line
Narges Mahmoodi, T Besharati-Seidani, N Motamed, and NO Mahmoodi* Annual Research & Review in Biology 2014; 4(12): 2045-2052 SCIENCEDOMAIN international www.sciencedomain.org
Aims: The majority of human breast tumors are estrogen receptor α (ERα) positive. However, not all of the ERα+ breast cancers respond to anti-estrogens drugs for those women who do respond, initial positive responses can be of short duration. Thus, more effective drugs are needed to enhance the efficacy of anti-estrogens drugs or to be used separately in a period of time. In view of potential cytotoxicity associated with silybin as polyhydroxy compounds a synthetic 4-hydroxychalcones (bis-phenol) was considered to explore its anti-carcinogenic effects in comparison to silybin on ERα+ breast cancer cell line.
Methodology: We have studied the inhibitory effect of 4,4′-dihydroxychalcone on the T47D breast cancer cell line by MTT test and the IC50s were estimated using Pharm PCS.
Results: The 4,4′-dihydroxychalcone showed significant dose- and time-dependent cell growth inhibitory effects on T47D breast cancer cells. The IC50 of 4,4′-dihydroxychalcone on T47D cells after 24 and 48 hours was 160.88+/–1 μM, 62.20+/–1 μM and for silybin was 373.42+/-1 μM,176.98+/–1 μM respectively.
Conclusion: Our results strongly suggests that this premade synthetic 4,4′-dihydroxychalcone can promote anti carcinogenic actions on T47D cell line. All 4,4′-dihydroxychalcone doses had a much larger inhibitory effect on cell viability than silybin doses in T47D cells. The ratio of the IC50 of 4,4′-dihydroxychalcone to silybin after 24 and 48 hours was 1: 2.3 and 1: 2.8 respectively.
Anticancer and multidrug resistance-reversal effects of solanidine analogs synthetized from pregnadienolone acetate.
István Zupkó, Judit Molnár, Borbála Réthy, Renáta Minorics, Eva Frank, et al.
Molecules (Impact Factor: 2.43). 01/2014; 19(2):2061-76. http://dx.doi.org/10.3390/molecules19022061
Source: PubMed
ABSTRACT A set of solanidine analogs with antiproliferative properties were recently synthetized from pregnadienolone acetate, which occurs in Nature. The aim of the present study was an in vitro characterization of their antiproliferative action and an investigation of their multidrug resistance-reversal activity on cancer cells. Six of the compounds elicited the accumulation of a hypodiploid population of HeLa cells, indicating their apoptosis-inducing character, and another one caused cell cycle arrest at the G2/M phase. The most effective agents inhibited the activity of topoisomerase I, as evidenced by plasmid supercoil relaxation assays. One of the most potent analogs down-regulated the expression of cell-cycle related genes at the mRNA level, including tumor necrosis factor alpha and S-phase kinase-associated protein 2, and induced growth arrest and DNA damage protein 45 alpha. Some of the investigated compounds inhibited the ABCB1 transporter and caused rhodamine-123 accumulation in murine lymphoma cells transfected by human MDR1 gene, expressing the efflux pump (L5178). One of the most active agents in this aspect potentiated the antiproliferative action of doxorubicin without substantial intrinsic cytostatic capacity. The current results indicate that the modified solanidine skeleton is a suitable substrate for the rational design and synthesis of further innovative drug candidates with anticancer activities.
Nutrition and Cancer
Ascorbic Acid and Selenium Interaction: Its Relevance in Carcinogenesis
Michael J. Gonzalez
Journal of Orthomolecular Medicine 1990; 5(2)
Ascorbic acid and selenium are two nutrients that seem to have a preventive potential in the process of carcinogenesis; because of a possible synergistic action that may produce an enhanced anticarcinogenic effect. Interaction between these nutrients have been reported. Results indicate that the protective effect of the inorganic form of selenium (Na Selenite) was nullified by ascorbic acid, whereas the chemopreventive action of the organic form (seleno-DL-methionine) was not affected.
A possibility exists that Selenite is reduced by ascorbic acid to elemental selenium and is therefore not available for tissue uptake. In experiments using Selenite; plasma and erythrocyte glutathione peroxidase enzyme activity was directly related to the level of ascorbic acid fed.
Complementary RNA and Protein Profiling Identifies Iron as a Key Regulator of Mitochondrial Biogenesis
Mitochondria are centers of metabolism and signaling whose content and function must adapt to
changing cellular environments. The biological signals that initiate mitochondrial restructuring
and the cellular processes that drive this adaptive response are largely obscure. To better define
these systems, we performed matched quantitative genomic and proteomic analyses of mouse
muscle cells as they performed mitochondrial biogenesis. We find that proteins involved in
cellular iron homeostasis are highly coordinated with this process and that depletion of cellular iron results in a rapid, dose-dependent decrease of select mitochondrial protein levels and oxidative capacity. We further show that this process is universal across a broad range of cell
types and fully reversed when iron is reintroduced. Collectively, our work reveals that cellular iron
is a key regulator of mitochondrial biogenesis, and provides quantitative data sets that can be
leveraged to explore posttranscriptional and posttranslational processes that are essential for mitochondrial adaptation.
Avemar outshines new cancer ‘breakthrough’ drug
by Michael Traub
Townsend Letter / Oct, 2010
Many of us in the cancer research community were happy to hear about progress against metastatic melanoma reported this June at the annual meeting of the American Society of Clinical
Oncology (ASCO). since there has not been an improvement in overall survival from chemotherapy in over three decades.
Data from a phase III clinical trial of the experimental monoclonal antibody ipilimumab (pronounced “ep-eh-lim-uemab”) showed that patients with melanoma survived longer if they were taking ipilimumab than if they were not, regardless of whether they also were taking the other drug in the study, an experimental cancer vaccine. (1)
A Closer Look: How Big an Improvement, at What Cost to Patients?
Overall Survival: the ‘Gold Standard’ for Judging Cancer Therapies
Overall survival (OS) is the length of time that a patient actuallysurvives a cancer after treatment. It can also be measured as the percentage of patients surviving a specific time. It is the gold
standard by which the usefulness of a cancer treatment should be determined. Many things can help a patient, but the most important goal of doctors and patients is for the cancer patient to live longer, with a decent quality of life (QOL).
Among patients taking ipilimumab with or without the experimental vaccine, median overall survival was about 10 months. That is compared with 6.4 months’ overall survival among patients receiving the vaccine by itself. About 45.6% of patients taking ipilimumab survived one year, an improvement of some 7% over the 38% seen in some earlier studies. This very modest improvement in survival comes at quite a price.
Severe Side Effects in More Than One in Four Ipilimumab Patients Ipilimumab has some side effects that can be “both severe and long-lasting,” according to the study report. Among patients taking ipilimumab by itself (without the vaccine), 19.1% had side effects requiring hospitalization or invasive intervention, 3.8% died from the effects of the drug, and another 33.8% had life-threatening or disabling side effects. All totaled, 26.7% of the patients taking ipilimumab by itself– more than 1 in 4-had side effects that were severe, very severe, or fatal. Severe side effects included diarrhea, nausea, constipation, vomiting, abdominal pain, fatigue, cough, and headache. Vernon Sondak, MD, of the H. Lee Moffitt Cancer and Research Institute, said that “using the drug requires the medical team to be on guard to manage toxicity at all times.” But even with its severe side effects, the researchers said that the drug should be welcomed because it can increase median survival from 6.4 months to 10.1 months. That is because any lengthening of lives is welcome in a disease that hasn’t seen a new drug that can do that in many years.
Fermented Wheat Germ (Avemar) Improves Melanoma Survival Without Harsh Side Effects
But what if there already were such a treatment available-not a drug, but a safe, natural substance shown in clinical trials to have a remarkably similar ability to lengthen the lives of melanoma patients, without the severe side effects of the new drug?
What if the other substance had no significant side effects at all?
What if, instead of causing severe and sometimes fatal side effects, that other substance actually helped prevent and reduce serious side effects caused by chemotherapy and radiotherapy?
In fact, there is just such a treatment available. It is known as fermented wheat germ extract (FWGE) and by its trade name Avemar. It has been approved as a medical nutriment for cancer
patients in Europe for years and is available in the US as a dietary supplement. It has been compared to dacarbazine (DTIC), standard melanoma therapy, in a clinical trial with longer
follow-up than the ipilimumab trial. And with better results.
In 2008, data were published in the research journal Cancer Biotherapy and Radiopharmaceuticals from seven years’ follow-up on a trial at the N. N. Blokhin Cancer Center in Moscow,
Russia, involving 52 patients who had taken or not taken Avemar while taking dacarbazine for the year following surgical removal of their stage III melanoma tumors. (2) Patients who got only dacarbazine survived 44.7 months. Those who got Avemar along with their dacarbazine survived 66.2 months. This is an improvement in overall survival time of over 48%. In the Russian study,
just as it has in other studies, Avemar reduced side effects of the chemotherapy. Among those taking only dacarbazine, 11 % experienced severe (grade 3 or grade 4) side effects that required hospitalization or invasive intervention. None of the Avemar patients had grade 3 or 4 side effects. Since it is difficult to compare length of survival between the recent ipilimumab study and the Avemar melanoma study, because the ipilimumab study tested mostly stage 4 melanoma patients and the Avemar study tested mostly stage 3 melanoma patients, it is most instructive to look at
the percentage improvement in overall survival from adding either treatment to the regimen. Ipilimumab and Avemar both produced very similar improvements in OS (56% vs. 48%, respectively),
Avemar Ameliorates Conventional Treatment Side Effects
The improvement of survival and the amelioration of chemotherapy side effects by Avemar seen in the Russian melanoma study is typical of Avemar’s effects when used in treating other cancers, including in combination with chemotherapy or radiotherapy. Among 170 colorectal cancer patients in a 2003 study published in the British journal of Cancer, Avemar improved overall survival
and reduced metastasis and recurrences after surgery, chemotherapy, and radiotherapy. (3) Taking Avemar for six months during and after those conventional treatments resulted in a 61.8% reduction in the death rate among those patients, compared with those who received only the conventional treatment. Those taking Avemar experienced lower rates of recurrences and metastases
as well, even though most patients in the Avemar group came into the study with more advanced disease, had more radiation earlier, and had been diagnosed longer. Side effects of Avemar, as in
other Avemar trials., were rare, mild, and transient, with no serious adverse events occurring.
In a 2004 study published in the journal of Pediatric Hematology and Oncology, childhood cancer patients taking Avemar during and after conventional therapies had a 42.8% reduction in the
low white blood cell counts and high fever known as febrile neutropenia, which can be a life-threatening consequence of chemotherapy and radiation. (4) This and similar results with
Avemar in other cancers are consistent with animal studies showing that Avemar helps the immune system recover a full white blood cell count after chemotherapy and radiation faster
than would otherwise happen. This study also demonstrated the safety of Avemar for children.
Why Avemar Works in Many Different Kinds of Cancer
Extensive studies in cells and animals have shown how Avemar works. Perhaps its most important action is to restrict cancer cells’ use of glucose. (5) Cancer cells use up to 50 times more glucose
than normal cells, a phenomenon known as the Warburg effect. (6) They use those enormous amounts of glucose to make ribose, the backbone sugar of DNA, much faster than normal cells can. To
do this, they must use a different series of biochemical reactions (“pathway”) than normal cells. Avemar makes this very difficult for cancer cells to do, because it inhibits the activity of the key enzyme in that pathway, transketolase (TK). (7) With the TK pathway blocked, cancer cells cannot use large amounts of glucose to make DNA fast enough to support the proliferation that makes them so dangerous.(8-10)
In experiments in the US and abroad, scientists have learned that Avemar has these additional effects. It:
* lowers the levels of a DNA repair enzyme known as poly (ADPribose) polymerase (PARP).” With this effect, cancer cells are forced to self-destruct, preventing them from proliferating and
producing a synergistic cancer-cell killing effect when given with chemotherapy, which also works to damage cancer cells’ DNA;
* reduces the number of molecules on cancer cells that identify them as originating within the body (MHC-1 molecules). (12) With cancer cells stripped of that protection, the immune system,
which recognizes the cancer cells as abnormal, no longer gives them the pass given to cells originating in the body. The cancer cells are attacked by the immune system’s natural killer (NK)
cells and destroyed;
* increases levels of molecules called intercellular adhesion molecule-1 (ICAM-1) on the blood vessels of cancer tumors. (13). The increase helps immune system cells pass through the walls of the blood vessels supplying the tumor blood flow, moving directly into the tumor to attack its cancer cells; increases the activity of the primary anticancer cytokine, tumor necrosis factor alpha (TNF-a), and produces a synergistic effect in interaction with other anticancer cytokines. (14) Cytokines are substances produced by cells to act directly on other cells. TNF-a helps force cancer cells into the programmed death known as apoptosis and inhibits tumorigenesis, the process through which new tumors are formed;
* inhibits the activity of ribonucleotide reductase (RR), a key enzyme that cells must have to make new DNA so that each cancer cell can divide to make two more like it. (15) With DNA
production slowed, increases in cancer cell growth and replication are inhibited.
Antimetastatic and Immune-Boosting Effects Are Key to Survival
Because the biochemical changes listed above have consistently been shown in both animal and human studies to be directly linked to reducing cancer’s ability to metastasize and to
improving the immune system’s ability to fight cancer, scientists count them as among the most likely main causes of improved survival seen in cancer patients when Avemar is used alone or,
more often, as an adjuvant in addition to standard-of-care therapies such as chemotherapy, radiotherapy, or the combination of the two. (16-23)
Extending Life: How Long, Exactly, and At What Cost in Quality of Life?
Any improvement in advanced melanoma survival, no matter how small, is certainly an achievement. But ipilimumab had severe side effects requiring hospitalization or invasive intervention in
over one-quarter of patients treated with it. And it increased median survival only by 3-plus months. On the other hand, Avemar added to dacarbazine improved survival very markedly, with no severe side effects. If actually improving overall survival substantially without significant side effects means that a drug should be considered as the new standard of care for first-line therapy, then there is no need to wait for further results. Avemar has already demonstrated very significant improvement in survival over chemotherapy alone and has a safety profile unmatched by
conventional therapies.
Michael Traub, ND, FABNO, is in private practice and serves as a member of Oncology Association of Naturopathic Physicians board of examiners.
Notes
(1.) Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010 Jun 14.
(2.) Demidov LV. Manziuk LV, Kharkevitch GY, Pirogova NA, Artamonova EV. Adjuvant fermented wheat germ extract (Avemar) nutraceutical improves survival of high-risk skin
melanoma patients; a randomized, pilot, phase ll clinical study with a 7-year follow-up. Cancer Biother Radiopharm. 2008 Aug. 23(4):477-482. Erratum in: Cancer Biother Radiopharm. 2008
Oct;2315):669.
(3.) Jakab F, Shoenfeld Y, Balogh A. et al. A medical nutriment has supportive value in the treatment of colorectal cancer. Br J Cancer. 2001 Aug 4;89(3):465-9.
(4.) Garami M, Schuler D, Babosa M, et al. Fermented wheat germ extract reduces chemotherapy-induced febrile neutropenia in pediatric cancer patients, J Pediatr Hematol Oncol. 2004
Oct;26(10):631-635.
(5.) Boros I.G, Lapis K, Szende B, et al. Wheat germ extract decreases glucose uptake and RNA ribose formation but increases fatty acid synthesis in MIA pancreatic adenocarcinoma
cells. Pancreas. 2001 Aug:23(2):141-147.
(6.) Warburg, O. On the origin of cancer cells. Science. 1956 Feb 24; 123(31 91):309-314.
(7.) Boros LG, Lee VVN, Go VL., A metabolic hypothesis of cell growth and death in pancreatic cancer, Pancreas. 2002 Jan;
24:(1):26 33.
(8.) Boros LG, Lapis K, Szende B, et al. Op cit.
(9.) Comin-Anduix B, Boros LG, Marin S, et al. Fermented wheat germ extract inhibits glycolysis/pentose cycle enzymes and induces apoptosis through poly(ADP-ribose) polymerase
activation in Jurkat T-cell leukemia tumor cells. J Biol Chem. 2002 Nov 29;277 (48):46408-46414. Epub 2002 Sep 25.
(23.) Garami M, Schuler D, Babosa M, et al. Fermented wheat germ extract reduces chemotherapy-induced febrile neutropenia in pediatric cancer patients. J Pediatr Hematol Oncol. 2004 Oct;
26(10):631-635.
by Michael Traub, ND, FABNO
COPYRIGHT 2010 The Townsend Letter Group
COPYRIGHT 2010 Gale, Cengage Learning
Nanotechnology in Cancer Drug Delivery and Selective Targeting
Nanoparticles are rapidly being developed and trialed to overcome several limitations of traditional drug delivery systems and are coming up as a distinct therapeutics for cancer treatment. Conventional chemotherapeutics possess some serious side effects including damage of the immune system and other organs with rapidly proliferating cells due to nonspecific targeting, lack of solubility, and inability to enter the core of the tumors resulting in impaired treatment with reduced dose and with low survival rate.
Nanotechnology has provided the opportunity to get direct access of the cancerous cells selectively with increased drug localization and cellular uptake. Nanoparticles can be programmed for recognizing the cancerous cells and giving selective and accurate drug delivery avoiding interaction with the healthy cells. This review focuses on cell recognizing ability of nanoparticles by various strategies having unique identifying properties that distinguish them from previous anticancer therapies. It also discusses specific drug delivery by nanoparticles inside the cells illustrating many successful researches and how nanoparticles remove the side effects of conventional therapies with tailored cancer treatment.
(Kumar Bishwajit Sutradhar and Md. Lutful Amin. Hindawi Publ. Corp. 2014, Article ID 939378, 12 pages
Cancer, the uncontrolled proliferation of cells where apoptosis is greatly disappeared, requires very complex process of treatment. Because of complexity in genetic and phenotypic levels, it shows clinical diversity and therapeutic resistance. A variety of approaches are being practiced for the treatment of cancer each of which has some significant limitations and side effects. Cancer treatment includes surgical removal, chemotherapy, radiation, and hormone therapy. Chemotherapy, a very common treatment, delivers anticancer drugs systemically to patients for quenching the uncontrolled proliferation of cancerous cells. Unfortunately, due to nonspecific targeting by anticancer agents, many side effects occur and poor drug delivery of those agents cannot bring out the desired outcome in most of the cases. Cancer drug development involves a very complex procedure which is associated with advanced polymer chemistry and electronic engineering.
The main challenge of cancer therapeutics is to differentiate the cancerous cells and the normal body cells. That is why the main objective becomes engineering the drug in such a way as it can identify the cancer cells to diminish their growth and proliferation. Conventional chemotherapy fails to target the cancerous cells selectively without interacting with the normal body cells. Thus they cause serious side effects including organ damage resulting in impaired treatment with lower dose and ultimately low survival rates.
Nanotechnology is the science that usually deals with the size range from a few nanometers (nm) to several hundrednm, depending on their intended use. It has been the area of interest over the last decade for developing precise drug delivery systems as it offers numerous benefits to overcome the limitations of conventional formulations . It is very promising both in cancer diagnosis and treatment since it can enter the tissues at molecular level.
Cisplatin-incorporated nanoparticles of poly(acrylic acid-co-methyl methacrylate) copolymer
K Dong Lee, Young-Il Jeong, DH Kim, Gyun-Taek Lim, Ki-Choon Choi. Intl J Nanomedicine 2013:8 2835–2845.
Although cisplatin is extensively used in the clinical field, its intrinsic toxicity limits its clinical use. We investigated nanoparticle formations of poly(acrylic acid-co-methyl methacrylate) (PAA-MMA) incorporating cisplatin and their antitumor activity in vitro and in vivo.
Methods: Cisplatin-incorporated nanoparticles were prepared through the ion-complex formation between acrylic acid and cisplatin. The anticancer activity of cisplatin-incorporated nanoparticles was assessed with CT26 colorectal carcinoma cells.
Results: Cisplatin-incorporated nanoparticles have small particle sizes of less than 200 nm with spherical shapes. Drug content was increased according to the increase of the feeding amount of cisplatin and acrylic acid content in the copolymer. The higher acrylic acid content in the copolymer induced increase of particle size and decrease of zeta potential. Cisplatin-incorporated nanoparticles showed a similar growth-inhibitory effect against CT26 tumor cells in vitro. However, cisplatin-incorporated nanoparticles showed improved antitumor activity against an animal tumor xenograft model.
Conclusion: We suggest that PAA-MMA nanoparticles incorporating cisplatin are promising carriers for an antitumor drug-delivery system.
Researchers Say Molecule May Help Overcome Cancer Drug Resistance By Estel Grace Masangkay
A group of researchers from the University of Delaware has discovered that a deubiquitinase (DUB) complex, USP1-UAF1, may present a key target in helping fight resistance to platinum-based anticancer drugs. The research team’s findings were published online in Nature Chemical Biology.
Zhihao Zhuang, associate professor in the Department of Chemistry and Biochemistry at UD, and his team studied a DNA damage tolerance mechanism called translesion synthesis (TLS). Enzymes known as TLS polymerases synthesize DNA over damaged nucleotide bases, followed by replication after lesion. The enzymes have been linked with building cancer cell resistance to certain cancer drugs including cisplatin. Cisplatin is used in treatment of ovarian, bladder, and testicular cancers which have spread.
“Cancer drugs like cisplatin work by damaging DNA and thereby preventing cancer cells from replicating the genomic DNA and dividing. However, cancer cells quickly develop resistance to cisplatin, and we and other researchers suspect that a polymerase known as Pol η is involved in overcoming cisplatin-induced lesions,” Professor Zhuang said.
The team found that USP1-UAF1 may play a crucial role in regulating DNA damage response. A new molecule ML323 can be used to inhibit processes such as translesion synthesis. Zhuang said, “Using ML323, we studied the cellular response to DNA damage and revealed new insights into the role of deubiquitination in both the TLS pathway and another one called the Fanconi anemia, or FA, pathway. We’re very encouraged by the fact that a single molecule is effective at inhibiting the USP1-UAF1 DUB complex and disrupting two essential DNA damage tolerance pathways.”
A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro
Cui-yan Han, Li-ling Yue, Ling-yu Tai, Li Zhou et al. Intl J Nanomedicine 2013:8 1541–1549
The discovery of suitable ligands that bind to cancer cells is important for drug delivery specifically targeted to tumors. Monoclonal antibodies and fragments that serve as ligands have specific targets. Natural ligands have strong mitogenic and neoangiogenic activities. Currently, small peptides are pursued as targeting moieties because of their small size, low immunogenicity, and their ability to be incorporated into certain delivery vectors.
The epidermal growth factor receptor (EGFR) serves an important function in the proliferation of tumors in humans and is an effective target for the treatment of cancer. The epidermal growth factor receptor (EGFR) is a transmembrane protein on the cell surface that is overexpressed in a wide variety of human cancers. EGFR is an effective tumor-specific target because of its significant functions in tumor cell growth, differentiation, and migration. EGFR-targeted small molecule peptides such as YHWYGYTPQNVI have been successfully identified using phage display library screening; by contrast, the peptide LARLLT has been generated using computer-assisted design (CAD).
These peptides can be conjugated to the surfaces of liposomes that are then delivered selectively to tumors by the specific and efficient binding of these peptides to cancer cells that express high levels of EGFR.
In this paper, we studied the targeting characteristics of small peptides (AEYLR, EYINQ, and PDYQQD) These small peptides were labeled with fluorescein isothiocyanate (FITC) and used the peptide LARLLT as a positive control, which bound to putative EGFR selected from a virtual peptide library by computer-aided design, and the independent peptide RALEL as a negative control.
Analyses with flow cytometry and an internalization assay using NCI-H1299 and K562 with high EGFR and no EGFR expression, respectively, indicated that FITC-AEYLR had high EGFR targeting activity. Biotin-AEYLR that was specifically bound to human EGFR proteins demonstrated a high affinity for human non-small-cell lung tumors.
We found that AEYLR peptide-conjugated, nanostructured lipid carriers enhanced specific cellular uptake in vitro during a process that was apparently mediated by tumor cells with high-expression EGFR. Analysis of the MTT assay indicated that the AEYLR peptide did not significantly stimulate or inhibit the growth activity of the cells. These findings suggest that, when mediated by EGFR, AEYLR may be a potentially safe and efficient delivery ligand for targeted chemotherapy, radiotherapy, and gene therapy.
Arginine-based cationic liposomes for efficient in vitro plasmid DNA delivery with low cytotoxicity
SR Sarker Y Aoshima, R Hokama T Inoue et al. Intl J Nanomedicine 2013:8 1361–1375.
Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group.
Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000.
We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular.
Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity.
The gene delivery efficiency of amino acid-based cationic assemblies is influenced by the amino acids (ie, arginine or lysine) present as the hydrophilic head group and their associated counterions.
Molecularly targeted approaches herald a new era of non-small-cell lung cancer treatment
H Kaneda, T Yoshida, I Okamoto. Cancer Management and Research 2013:5 91–101.
The discovery of activating mutations in the epidermal growth-factor receptor (EGFR) gene in 2004 opened a new era of personalized treatment for non-small-cell lung cancer (NSCLC). EGFR mutations are associated with a high sensitivity to EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib. Treatment with these agents in EGFR-mutant NSCLC patients results in dramatically high response rates and prolonged progression-free survival compared with conventional standard chemotherapy. Subsequently, echinoderm microtubule-associated protein-like 4 (EML4)–anaplastic lymphoma kinase (ALK), a novel driver oncogene, has been found in 2007. Crizotinib, the first clinically available ALK tyrosine kinase inhibitor, appeared more effective compared with standard chemotherapy in NSCLC patients harboring EML4-ALK. The identification of EGFR mutations and ALK rearrangement in NSCLC has further accelerated the shift to personalized treatmentbased on the appropriate patient selection according to detailed molecular genetic characterization. This review summarizes these genetic biomarker-based approaches to NSCLC, which allow the instigation of individualized therapy to provide the desired clinical outcome.
Non-small-cell lung cancer (NSCLC) has a poor prognosis and remains the leading cause of death related to cancer worldwide. For most individuals with advanced, metastatic NSCLC, cytotoxic chemotherapy is the mainstay of treatment on the basis of the associated moderate improvement in survival and quality of life. However, the outcome of chemotherapy in such patients has reached a plateau in terms of overall response rate (25%–35%) and overall survival (OS; 8–10 months). This poor outcome, even for patients with advanced NSCLC who respond to such chemotherapy, has motivated a search for new therapeutic approaches.
Recent years have seen rapid progress in the development of new treatment strategies for advanced NSCLC, in particular the introduction of molecularly targeted therapiesand appropriate patient selection. First, the most important change has been customization of treatment according to patient selection based on the genetic profile of the tumor. Small-molecule tyrosine kinase inhibitors (TKIs) that target the epidermal growth-factor receptor (EGFR), such as gefitinib and erlotinib, are especially effective in the treatment of NSCLC patients who harbor activating EGFR mutations.
Ido Bachelet’s moonshot to use nanorobotics for surgery has the potential to change lives globally. But who is the man behind the moonshot?
Ido graduated from the Hebrew University of Jerusalem with a PhD in pharmacology and experimental therapeutics. Afterwards he did two postdocs; one in engineering at MIT and one in synthetic biology in the lab of George Church at the Wyss Institute at Harvard.
Now, his group at Bar-Ilan University designs and studies diverse technologies inspired by nature.
They will deliver enzymes that break down cells via programmable nanoparticles.
Delivering insulin to tell cells to grow and regenerate tissue at the desired location.
Surgery would be performed by putting the programmable nanoparticles into saline and injecting them into the body to seek out remove bad cells and grow new cells and perform other medical work.
Effects of Two Disiloxanes ALIS-409 and ALIS-421 on Chemoprevention in Model Experiments
H TOKUDA,…. L AMARAL and J MOLNAR.. ANTICANCER RESEARCH 33: 2021-2028 (2013).
Figure 1. Chemical structures of ALIS-409 and ALIS-421.
Morpholino-disiloxane (ALIS-409) and piperazinodisiloxane (ALIS-421) compounds were developed as inhibitors of multidrug resistance of various types of cancer cells. In the present study, the effects of ALIS-409 and ALIS-421 compounds were investigated on cancer promotion and on co-existence of
tumor and normal cells. The two compounds were evaluated for their inhibitory effects on Epstein-Barr virus immediate early antigen (EBV-EA) expression induced by tetradecanoylphorbolacetate (TPA) in Raji cell cultures. The method is known as a primary screening test for antitumor effect, below the (IC50) concentration. ALIS-409 was more effective in inhibiting EBV-EA (100 μg/ml) and tumor promotion, than
ALIS-421, in the concentration range up to 1000 μg/ml. However, neither of the compounds were able to reduce tumor promotion significantly, expressed as inhibition of TPA-induced tumor antigen activation. Based on the in vitro results, the two disiloxanes were investigated in vivo for their effects on mouse skin tumors in a two-stage mouse skin carcinogenesis study.
Curation, HealthCare System in the US, and Calcium Signaling Effects on Cardiac Contraction, Heart Failure, and Atrial Fibrillation, and the Relationship of Calcium Release at the Myoneural Junction to Beta Adrenergic Release
Curator and e-book Contributor: Larry H. Bernstein, MD, FCAP
Curator and BioMedicine e-Series Editor-in-Chief: Aviva Lev Ari, PhD, RN
This portion summarises what we have covered and is now familiar to the reader. There are three related topics, and an extension of this embraces other volumes and chapters before and after this reading. This approach to the document has advantages over the multiple authored textbooks that are and have been pervasive as a result of the traditional publication technology. It has been stated by the founder of ScoopIt, that amount of time involved is considerably less than required for the original publications used, but the organization and construction is a separate creative process. In these curations we amassed on average five articles in one curation, to which, two or three curators contributed their views. There were surprises, and there were unfulfilled answers along the way. The greatest problem that is being envisioned is the building a vision that bridges and unmasks the hidden “dark matter” between the now declared “OMICS”, to get a more real perspective on what is conjecture and what is actionable. This is in some respects unavoidable because the genome is an alphabet that is matched to the mino acid sequences of proteins, which themselves are three dimensional drivers of sequences of metabolic reactions that can be altered by the accumulation of substrates in critical placements, and in addition, the proteome has functional proteins whose activity is a regulatory function and not easily identified. In the end, we have to have a practical conception, recognizing the breadth of evolutionary change, and make sense of what we have, while searching for more.
We introduced the content as follows:
1. We introduce the concept of curation in the digital context, and it’s application to medicine and related scientific discovery.
Topics were chosen were used to illustrate this process in the form of a pattern, which is mostly curation, but is significantly creative, as it emerges in the context of this e-book.
Alternative solutions in Treatment of Heart Failure (HF), medical devices, biomarkers and agent efficacy is handled all in one chapter.
PCI for valves vs Open heart Valve replacement
PDA and Complications of Surgery — only curation could create the picture of this unique combination of debate, as exemplified of Endarterectomy (CEA) vs Stenting the Carotid Artery (CAS), ischemic leg, renal artery stenosis.
2. The etiology, or causes, of cardiovascular diseases consist of mechanistic explanations for dysfunction relating to the heart or vascular system. Every one of a long list of abnormalities has a path that explains the deviation from normal. With the completion of the analysis of the human genome, in principle all of the genetic basis for function and dysfunction are delineated. While all genes are identified, and the genes code for all the gene products that constitute body functions, there remains more unknown than known.
3. Human genome, and in combination with improved imaging methods, genomics offers great promise in changing the course of disease and aging.
4. If we tie together Part 1 and Part 2, there is ample room for considering clinical outcomes based on individual and organizational factors for best performance. This can really only be realized with considerable improvement in information infrastructure, which has miles to go.
Curation
Curation is an active filtering of the web’s and peer reviewed literature found by such means – immense amount of relevant and irrelevant content. As a result content may be disruptive. However, in doing good curation, one does more than simply assign value by presentation of creative work in any category. Great curators comment and share experience across content, authors and themes.
Great curators may see patterns others don’t, or may challenge or debate complex and apparently conflicting points of view. Answers to specifically focused questions comes from the hard work of many in laboratory settings creatively establishing answers to definitive questions, each a part of the larger knowledge-base of reference. There are those rare “Einstein’s” who imagine a whole universe, unlike the three blindmen of the Sufi tale. One held the tail, the other the trunk, the other the ear, and they all said this is an elephant!
In my reading, I learn that the optimal ratio of curation to creation may be as high as 90% curation to 10% creation. Creating content is expensive. Curation, by comparison, is much less expensive. The same source says “Scoop.it is my content marketing testing “sandbox”. In sharing, he says that comments provide the framework for what and how content is shared.
Healthcare and Affordable Care Act
We enter year 2014 with the Affordable Care Act off to a slow start because of the implementation of the internet signup requiring a major repair, which is, unfortunately, as expected for such as complex job across the US, and with many states unwilling to participate. But several states – California, Connecticut, and Kentucky – had very effective state designed signups, separate from the federal system. There has been a very large rush and an extension to sign up. There are many features that we can take note of:
1. The healthcare system needed changes because we have the most costly system, are endowed with advanced technology, and we have inexcusable outcomes in several domains of care, including, infant mortality, and prenatal care – but not in cardiology.
2. These changes that are notable are:
The disparities in outcome are magnified by a large disparity in highest to lowest income bracket.
This is also reflected in educational status, and which plays out in childhood school lunches, and is also affected by larger class size and cutbacks in school programs.
This is not helped by a large paralysis in the two party political system and the three legs of government unable to deal with work and distraction.
Unemployment is high, and the banking and home construction, home buying, and rental are in realignment, but interest rates are problematic.
3. The medical care system is affected by the issues above, but the complexity is not to be discounted.
The medical schools are unable at this time to provide the influx of new physicians needed, so we depend on a major influx of physicians from other countries
The technology for laboratories, proteomic and genomic as well as applied medical research is rejuvenating the practice in cardiology more rapidly than any other field.
In fields that are imaging related the life cycle of instruments is shorter than the actual lifetime use of the instruments, which introduces a shortening of ROI.
Hospitals are consolidating into large consortia in order to maintain a more viable system for referral of specialty cases, and also is centralizing all terms of business related to billing.
There is reduction in independent physician practices that are being incorporated into the hospital enterprise with Part B billing under the Physician Organization – as in Partners in Greater Boston, with the exception of “concierge” medical practices.
There is consolidation of specialty laboratory services within state, with only the most specialized testing going out of state (Quest, LabCorp, etc.)
Medicaid is expanded substantially under the new ACA.
The federal government as provider of services is reducing the number of contractors for – medical devices, diabetes self-testing, etc.
The current rearrangements seeks to provide a balance between capital expenses and fixed labor costs that it can control, reduce variable costs (reagents, pharmaceutical), and to take in more patients with less delay and better performance – defined by outside agencies.
Cardiology, Genomics, and calcium ion signaling and ion-channels in cardiomyocyte function in health and disease – including heart failure, rhythm abnormalities, and the myoneural release of neurotransmitter at the vesicle junction.
This portion is outlined as follows:
2.1 Human Genome: Congenital Etiological Sources of Cardiovascular Disease
2.2 The Role of Calcium in Health and Disease
2.3 Vasculature and Myocardium: Diagnosing the Conditions of Disease
Genomics & Genetics of Cardiovascular Disease Diagnoses
disruption of Ca2+ homeostasis cardiac & vascular smooth muscle
synaptotagmin as Ca2+ sensor & vesicles
atherosclerosis & ion channels
It is increasingly clear that there are mutations that underlie many human diseases, and this is true of the cardiovascular system. The mutations are mistakes in the insertion of a purine nucleotide, which may or may not have any consequence. This is why the associations that are being discovered in research require careful validation, and even require demonstration in “models” before pursuing the design of pharmacological “target therapy”. The genomics in cardiovascular disease involves very serious congenital disorders that are asserted early in life, but the effects of and development of atherosclerosis involving large and medium size arteries has a slow progression and is not dominated by genomic expression. This is characterized by loss of arterial elasticity. In addition there is the development of heart failure, which involves the cardiomyocyte specifically. The emergence of regenerative medical interventions, based on pleuripotent inducible stem cell therapy is developing rapidly as an intervention in this sector.
Finally, it is incumbent on me to call attention to the huge contribution that research on calcium (Ca2+) signaling has made toward the understanding of cardiac contraction and to the maintenance of the heart rhythm. The heart is a syncytium, different than skeletal and smooth muscle, and the innervation is by the vagus nerve, which has terminal endings at vesicles which discharge at the myocyte junction. The heart specifically has calmodulin kinase CaMK II, and it has been established that calmodulin is involved in the calcium spark that triggers contraction. That is only part of the story. Ion transport occurs into or out of the cell, the latter termed exostosis. Exostosis involves CaMK II and pyruvate kinase (PKC), and they have independent roles. This also involves K+-Na+-ATPase. The cytoskeleton is also discussed, but the role of aquaporin in water transport appears elsewhere, as the transport of water between cells. When we consider the Gibbs-Donnan equilibrium, which precedes the current work by a century, we recall that there is an essential balance between extracellular Na+ + Ca2+ and the intracellular K+ + Mg2+, and this has been superceded by an incompletely defined relationship between ions that are cytoplasmic and those that are mitochondrial. The glass is half full!
We have previously covered the Warburg Effect, and there has been a number of comments about the chicken or the egg! There is an underlying factor that makes it difficult to comprehend that the initiation of cancer is mutation driven, although we are clear that smoking and a number of environmental factors are instigators of the change. The main problem that I have referred to is the chemical, thermodynamic, and evolutionary state of our existence. I strongly refer to the work of Ilya Prigogene. There is a progressive series of changes over time, and it is not possible to determine the initial state. Consequently, a progressive series of adaptations progresses, involving gene expression, non-genetic changes, and metabolic equilibrium that is maintained, but becomes non-adaptive.
Radoslav Bozov
Date: 3/26/2013
Subject: RE: comment
The process of genomic evolution cannot be revealed throughout comparative genomics as structural data representation does not illuminate either the integral path of particles-light interference, as Richard Feynman suggests, in stable forms of matter such as interference/entanglement of the nature of particles/strings/waves to first approximation as I have claimed. Towards the compressibility principle realization, I have claimed that DNA would be entropic- favorable stable state going towards absolute ZERO temp in the space defined itself. In other words themodynamics measurement in subnano discrete space would go negative towards negativity. DNA is sort of like a cold melting/growing crystal, quite stable as it appears not due to hydrogen bonding , but due to interference of C-N-O. That force is contradicted via proteins onto which we now know large amount of negative quantum redox state carbon attaches. Chemistry is just a language as it is math following certain rules based on observation. Most stable states are most observed ones. The more locally one attempts to observe, the more hidden variables would emerge as a consequence of discrete energy spaces opposing continuity of matter/time. Still, stability emerges out of non stability states. And if life was in absolute stability, there will be neither feelings nor freedom. What is feelings and freedom is a far reaching philosophical question with sets of implications, to one may be a driving car, to another riding a horse or a bicycle etc cetera or simply seeing the unobservable …No wonder genome size differs among organisms and even tissue types as an outcome of carbon capacity.
PIM2 phosphorylates PKM2 and promotes Glycolysis in Cancer Cells
Yu Z, Huang L, Zhang T, Yang F, Xie L, Liu J, Song S, Miao P, Zhao L, Zhao X, Huang G.
Shanghai Jiao Tong University, China;
J Biol Chem. 2013 Oct 18. [Epub ahead of print]
Pyruvate kinase M2 (PKM2) is a key player in the Warburg effect of cancer cells.
the mechanisms of regulating PKM2 are not fully elucidated.
we identified the serine/threonine protein kinase PIM2, a known oncogene,
as a novel binding partner of PKM2.
The interaction between PIM2 and PKM2 was confirmed by multiple biochemical approaches in vitro and in cultured cells. Importantly, we found that
PIM2 could directly phosphorylate PKM2 on the Thr454 residue, resulting in
an increase of PKM2 protein levels.
Compared to wild-type, PKM2 with the phosphorylation-defective mutation
displayed a reduced effect on glycolysis, co-activating HIF-1α and β-catenin, and cell proliferation,
while enhanced mitochondria respiration and chemotherapeutic sensitivity of cancer cells.
These findings demonstrate that PIM2-dependent phosphorylation of PKM2 is critical for regulating the Warburg effect in cancer,
highlighting PIM2 as a potential therapeutic target.
Different mtDNA mutations modify tumor progression in dependence of the degree of respiratory complex I impairment.
Iommarini L, Kurelac I, Capristo M, Calvaruso MA, Giorgio V, Bergamini C, Ghelli A, et al.
Dipartimento di Farmacia e Biotecnologie (FABIT).
Hum Mol Genet. 2013 Nov 11. [Epub ahead of print]
Mitochondrial DNA mutations are currently investigated as modifying factors impinging on tumor growth and aggressiveness,
having been found in virtually all cancer types and
most commonly affecting genes encoding mitochondrial complex I (CI) subunits.
It is still unclear whether they exert a pro- or anti-tumorigenic effect.
We here analyzed the impact of three homoplasmic mtDNA mutations (m.3460G>A/MT-ND1, m.3571insC/MT-ND1 and m.3243A>G/MT-TL1) on osteosarcoma progression,
chosen since they induce different degrees of oxidative phosphorylation impairment.
In fact, the m.3460G>A/MT-ND1 mutation caused only a reduction in CI activity, whereas
the m.3571insC/MT-ND1 and the m.3243A>G/MT-TL1 mutations induced a severe structural and functional CI alteration.
As a consequence, this severe CI dysfunction determined an energetic defect associated with a compensatory increase in glycolytic metabolism and AMP-activated protein kinase activation.
Osteosarcoma cells carrying such marked CI impairment
displayed a reduced tumorigenic potential both in vitro and in vivo, when compared with cells with mild CI dysfunction, suggesting that
mtDNA mutations may display diverse impact on tumorigenic potentialdepending on
the type and severity of the resulting oxidative phosphorylation dysfunction.
The modulation of tumor growth was independent from reactive oxygen species production but correlated with
hypoxia-inducible factor 1α stabilization, indicating that
structural and functional integrity of CI and oxidative phosphorylation are required for hypoxic adaptation and tumor progression.
PMID: 24163135 [PubMed – as supplied by publisher]
Systematic Identification of Molecular Subtype-Selective Vulnerabilities in Non-Small-Cell Lung Cancer
Hyun Seok Kim, Saurabh Mendiratta, Jiyeon Kim, Chad Victor Pecot, Jill E. Larsen, et al.
Cell, 24 Oct 2013; 155 (3): 552-566, doi:10.1016/j.cell.2013.09.041 Systematic isolation of context-dependent vulnerabilities in NSCLC
Highlights
NLRP3 mutations drive addiction to FLIP expression
Lysosome maturation is a metabolic bottleneck for KRAS/LKB1 tumors
Selective sensitivity to an indolotriazine discriminates a NSCLC expression subtype
Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
Curator, Reporter, EAW: Larry H Bernstein, MD, FCAP
Article 1.5 Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
This discussion is a continuation of an earlier piece on the technologic framework for , proteomics, nutrigenomics, and translational medicine. The last decade has seen the emergence of a genomic science that is changing the trajectory of biological sciences and medicine. It has not resolved all of our problems by any means, but it has begun to redraw the map, which began with the elucidation of major metabolic pathways in the first half of the 20th century, was then captured by the transformation of genetics with the discovery of the “Watson-Crick Model”, and then later was recharged with the discovery of the Toll-like receptor and the drawing of “signaling pathways”. What we have seen in an unraveling of protein-genome interactions, small peptide regulators, and dynamic changes in pathway dominance, bloackage, and reentry, depending on genetic, dietary, and environmental conditions, mostly expressed in what we refer to as “oxidative stress”.
Unraveling the multitude of nutrigenomic, proteomic, and metabolomic patterns that arise from the ingestion of foods or their bioactive food components will not be simple but is likely to provide insights into a tailored approach to diet and health. The use of new and innovative technologies, such as microarrays, RNA interference, and nanotechnologies, will provide needed insights into molecular targets for specific bioactive food components and how they harmonize to influence individual phenotypes. A challenging aspect of omic technologies is the refined analysis of quantitative dynamics in biological systems.
In recent years, nutrition research has moved from classical epidemiology and physiology to molecular biology and genetics. The new era of nutrition research translates empirical knowledge to evidence-based molecular science. Following this trend, Nutrigenomics has emerged as a novel and multidisciplinary research field in nutritional science that aims to elucidate how diet can influence human health. It is already well known that bioactive food compounds can interact with genes affecting transcription factors, protein expression and metabolite production. The study of these complex interactions requires the development of advanced analytical approaches combined with bioinformatics. The Institute of Medicine recently convened a workshop to review the state of the various domains of nutritional genomics research and policy and to provide guidance for further development and translation of this knowledge into nutrition practice and policy. Nutritional genomics holds the promise to revolutionize both clinical and public health nutrition practice and facilitate the establishment of
genome-informed nutrient and food-based dietary guidelines for disease prevention and healthful aging,
individualized medical nutrition therapy for disease management, and
better targeted public health nutrition interventions (including micronutrient fortification and supplementation) that maximize benefit and minimize adverse outcomes within genetically diverse human populations.
For metabolomics, gas and liquid chromatography coupled to mass spectrometry are well suited for coping with high sample numbers in reliable measurement times with respect to both technical accuracy and the identification and quantitation of small-molecular-weight metabolites. This potential is a prerequisite for the analysis of dynamic systems. Thus, metabolomics is a key technology for systems biology. The bioavailability of bioactive food constituents as well as dose-effect correlations are key information to understand the impact of food on defined health outcomes. Both strongly depend on appropriate analytical tools to identify and quantify minute amounts of individual compounds in highly complex matrices–food or biological fluids–and to monitor molecular changes in the body in a highly specific and sensitive manner. Based on these requirements, mass spectrometry has become the analytical method of choice with broad applications throughout all areas of nutrition research.
Dynamic Construct of the –Omics
Metabolomics is a term that encompasses several types of analyses, including
metabolic fingerprinting, which measures a subset of the whole profile with little differentiation or quantitation of metabolites;
metabolic profiling, the quantitative study of a group of metabolites, known or unknown, within or associated with a particular metabolic pathway; and
target isotope-based analysis, which focuses on a particular segment of the metabolome by analyzing only a few selected metabolites that comprise a specific biochemical pathway.
Any unifying concept of the metabolome was incomplete or debatable in the first 30 years of the 20th century. It was only known that insulin is anabolic and that insulin deficiency (or resistance) would have consequences in the point of entry into the citric acid cycle, which generates 28-32 ATPs. In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In the case of this cycle there is a tie in with both catabolism and anabolism.
For bypass of the Pyruvate Kinase reaction of Glycolysis, cleavage of 2 ~P bonds is required. The free energy change associated with cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP), since PEP has a higher negative DG of phosphate hydrolysis than ATP. The two enzymes that catalyze the reactions for bypass of the Pyruvate Kinase reaction are the following:
Pyruvate Carboxylase (Gluconeogenesis) catalyzes pyruvate + HCO3- + ATP — oxaloacetate + ADP + Pi
PEP Carboxykinase (Gluconeogenesis) catalyzes: oxaloacetate + GTP —- phosphoenolpyruvate + GDP + CO2
Many high throughput methods have been employed to get some insight into the whole process and several examples of successful research. Proteomics and metabolomics need to encompass large numbers of experiments and linked data. Due to the nature of the proteins, as well as due to the properties of various metabolites, experimental approaches require the use of comprehensive high throughput methods and a sufficiency of analysed tissue or body fluids.
An important and revolutionary aspect of ‘The 2010 Project’ is that it implicitly endorses the allocation of resources to attempts to assign function to genes that have no known function. This represents a significant departure from the common practice of defining and justifying a scientific goal based on the biological phenomena. The rationale for endorsing this radical change is that for the first time it is feasible to envision a whole-systems approach to gene and protein function. I shall not discuss the emerging field of bioinformatics that makes this possible. In this review, the end-of-the line “detector will be considered having been covered. The entire focus proceeds to a discussion of separation methods. Separation methods have always been tricky, time consuming, and a multiple step process that depended on using anionic and cationic resins as intermediate steps in bulk separation, and then molecular size separation. Therapeutic Targets will be identified as they are seen.
Affinity Chromatography The rapid development of biotechnology and biomedicine requires more reliable and efficient separation technologies for the isolation and purification of biopolymers such as therapeutic proteins, antibodies, enzymes and nucleic acids. In particular, monoclonal antibodies are centrally important as therapeutics for the treatment of cancer and other diseases, leading to recombinant monoclonal antibodies that dominate today’s biopharmaceutical pipeline. The large-scale production of therapeutic biopolymers requires
a manufacturing process that delivers reliability and in high-yield, as well as
an effective purification process affording extremely pure products.
Because of its high selectivity, affinity chromatography has been used extensively to isolate a variety of biopolymers. The retention of solutes is based on specific, reversible interactions found in biological systems, such as the binding of an enzyme with an inhibitor or an antibody with an antigen. These interactions are exploited in affinity chromatography by immobilizing an affinity ligand onto a support, and using this as a stationary phase. Non-porous particles having an average diameter of 2.1 mm were prepared by co-polymerization of styrene, methyl methacrylate and glycidyl methacrylate, which was abbreviated as P(S–MMA–GMA). The particles were mechanically stable due to the presence of benzene rings in the backbone of polymer chains, and could withstand high pressures when a column packed with these particles was operated in the HPLC mode.
The polymer particles were advantaged by immobilization of ligands via the epoxy groups on the particle surface that were introduced by one of the monomers, glycidyl methacrylate. As a model system, Cibacron Blue 3G-A was covalently immobilized onto the non-porous copolymer beads. The dye-immobilized P(S–MMA–GMA) particles were slurry packed into a 1.0 cm30.46 cm I.D. column. This affinity column was effective for the separation of turkey egg white lysozyme from a protein mixture. The bound lysozyme could be eluted to yield a sharp peak by using a phosphate buffer containing 1 M NaCl. For a sample containing up to 8 mg of lysozyme, the retained portion of proteins could be completely eluted without any slit peak. Due to the use of a shorter column, the analysis time was shorter in comparison with other affinity systems reported in the literature. The retention time could be reduced significantly by increasing the flow-rate, while the capacity factor remained at the same level. CH Chen, WC Lee. Affinity chromatography of proteins on non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate. Journal of Chromatography A 2001; 921: 31–37.
Affinity separation membranes, consisting of electrospun nanofibers, have been developed recently. Affinity ligands are attached to the surface of the constituent fibers, offering a potential solution to some of the problems of traditional, column-based, affinity chromatography. Electrospun fibers are good candidates for use in affinity separation because of their
unique characteristics of high surface area to volume ratio, resulting in
high ligand loading, and
their large porosity, resulting in
high throughput operation.
A number of polymers have been used for electrospun fiber mesh-based affinity membrane separations including poly (ether-urethane-urea), cellulose, poly(ethylene terephthalate, polysulphone, and polyacrlonitrile. Typically, very thin electrospun fiber meshes are produced by electrostatically collecting negatively charged fibers on a collector electrode. These very thin 2D electrospun fiber mesh mats provide excellent solution permeability as compared to 3D column packed with affinity beads. M Miyauchi, J Miao, TJ Simmons, JS Dordick and RJ Linhardt. Flexible Electrospun Cellulose Fibers as an Affinity Packing Material for the Separation of Bovine Serum Albumin. J Chromatograph Separat Techniq 2011; 2:2 http://dx.doi.org/10.4172/2157-7064.1000110
Dye Affinity Chromatography Biomimetic Dyes Affinity adsorbents based on immobilized triazine dyes offer important advantages circumventing many of the problems associated with biological ligands. The main drawback of dyes is their moderate selectivity for proteins. Rational attempts to tackle this problem are realized through the biomimetic dye concept according to which new dyes, the biomimetic dyes, are designed to mimic natural ligands. Biomimetic dyes are expected to exhibit increased affinity and purifying ability for the targeted proteins.
Biocomputing offers a powerful approach to biomimetic ligand design. The successful exploitation of contemporary computational techniques in molecular design requires the knowledge of the three-dimensional structure of the target protein, or at least, the amino acid sequence of the target protein and the three-dimensional structure of a highly homologous protein. From such information one can then design, on a graphics workstation,
the model of the protein and also
a number of suitable synthetic ligands which mimic natural biological ligands of the protein.
There are several examples of enzyme purifications
Interactions between Cibacron Blue F3GA (CB F3GA), as a model of triazine dye, and 2-hydroxypropyl-b-cyclodextrin (HP-b-CD), as a model of cyclodextrin, were investigated by monitoring the spectral shift that accompanies the binding phenomena. Matrix analysis of the difference spectral titration of CB F3GA with HP-b-CD revealed only two absorbing species, indicating a host–guest ratio of 1:1. The dissociation constant for this HP-b-CD–CB F3GA complex, K , was found d to be 0.43 mM. The data for HP-b-CD forming inclusion complexes with CB F3GA were used to develop the concept of competitive elution by inclusion complexes in dye-affinity chromatography. When this concept was applied to the elution of L-lactate dehydrogenase from a CB F3GA affinity matrix, it was shown to be an effective elution strategy. It provided a 15-fold purification factor with 89% recovery and sharp elution profile (0.8 column volumes for 80% recovery), which is as good as that obtained by specific elution with NADH (16-fold, 78% recovery and 1.8 column volumes). In addition, the new elution strategy showed a better purification factor and sharper elution profile than traditional non-specific. JA Lopez-Mas, SA Streitenberger, F Garcıa-Carmona, AA Sanchez-Ferrer. Cyclodextrin biospecific-like displacement in dye-affinity chromatography. Journal of Chromatography A 2001; 911: 47–53.
Affinity chromatography uses biospecific binding usually between an antibody and an antigen, an enzyme and a substrate or other pairs of key-lock type of matching molecules. Due to its high selectivity, it is able to purify proteins and other macromolecules from very dilute solutions. In this work, a general rate model for affinity chromatography was used for scale-up studies. Parameters for the model were estimated from existing correlations, or from experimental results obtained on a small column with the same packing material. As anexample, Affi-Gel with 4.5mol cm−3 Cibacron Blue F-3GA as immobilized ligands covalently attached to cross-linked 6% agarose was used for column packing. Cibacron Blue F-3GA was also used as a soluble ligand in the elution stage. Satisfactory scale-up predictions were obtained for a 98.2 ml column and a 501 ml column based on a few experimental data obtained on a 7.85 ml small column. T. Gu, K.-H. Hsu and M.-J. Syu, “Scale-Up of Affinity Chromatography for Purification of Enzymes and Other Proteins.” Enzyme and Microbial Technology 2003; 33:433-437.
Affinity Column with AAAA as a Model Sense Ligand The degeneracy of antisense peptides was studied by high-performance affinity chromatography. A model sense peptide (AAAA) and its antisense peptides (CGGG, GGGG, RGGG, SGGG) were designed and synthesized according to the degeneracy of genetic codes. An affinity column with AAAA as the ligand was prepared. The affinity chromatographic behaviors of antisense peptides on the column were evaluated. The results indicated that model antisense peptides have clear retention on the immobilized AAAA affinity column. RGGG showed the strongest affinity interaction. R Zhao, X Yu, H Liu, L Zhai, S Xiong, et al. Study on the degeneracy of antisense peptides using affinity chromatography. Journal of Chromatography A 2001; 913: 421–428.
Frontal AC for Biomolecular Interactions Frontal affinity chromatography is a method for quantitative analysis of biomolecular interactions. We reinforced it by incorporating various merits of a contemporary liquid chromatography system. As a model study, the interaction between an immobilized Caenorhabditis elegans galectin (LEC-6) and fluorescently labeled oligosaccharides (pyridylaminated sugars) was analyzed. LEC-6 was coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (100 mm diameter), and packed into a miniature column (e.g., 1034.0 mm, 0.126 ml). The volume of the elution front (V) determined graphically for each sample was compared with that obtained in the presence of an excess amount of hapten saccharide, lactose (V ); and the dissociation constant, K , was calculated according to the literature. This system also proved to be useful for an inverse confirmation; that is, application of galectins to an immobilized glycan column (in the present case, asialofetuin was immobilized on Sepharose 4 Fast Flow), and the elution profiles were monitored by fluorescence based on tryptophan. The newly constructed system proved to be extremely versatile. It enabled rapid (analysis time 12 min/ cycle) and sensitive (20 nM for pyridylaminated derivatives, and 1 mg/ml for protein) analyses of lectin–carbohydrate interactions. J Hirabayashi, Y Arata, K Kasai. Reinforcement of frontal affinity chromatography for effective analysis of lectin–oligosaccharide interactions. Journal of Chromatography A 2000; 890:261–271.
Immobilized Metal Ion Affinity New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal–chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant b-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media.
IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins. For example, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole).
The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions. However, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface, but the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface. C Mateo , G Fernandez-Lorente , BCC Pessela , A Vian, et al. Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions. Journal of Chromatography A 2001; 915:97–106. The underlying principle of immobilized metal ion affinity chromatography (IMAC) of proteins is the coordination between the electron donor groupings on a protein surface (histidine, tryptophan, cysteine) and chelated (iminodiacetate; IDA) transition metal ions [IDA-M(II)]. This principle of immobilized metal ion affinity (IMA) has been presented by now in some detail. The practice of IMAC in the purification of proteins has had its empirical phase. There is now a need, from the body of data, to establish somewhat more detailed ground rules that would allow for the use of IMAC in a more predictive manner. Immobilized metal ion affinity chromatography (IMAC) has been explored as a probe into the topography of histidyl residues of a protein molecule. An evaluation of the chromatographic behavior of selected model proteins-
thioredoxin
ubiquitin
calmodulin
lysozyme
cytochrome c
myoglobin
on immobilized transition metal ions
Co2+
Ni2+
Cu2+
Zn2
-allows establishment of the following facets of the histidyl side chain distribution:
either interior or surface;
when localized on the surface, accessible or unaccessible for coordination;
single or multiple;
When multiple, either distant or vicinal.
Moreover, proteins displaying single histidyl side chains on their surfaces may, in some instances, be resolved by IMAC; apparently, the microenvironments of histidyl residues are sufficiently diverse to result in different affinities for the immobilized metal ions. IMAC, previously introduced as an approach to the fractionation of proteins, has become also, upon closer examination, a facile probe into the topography of histidyl residues. This is possible because of the inherent versatility of IMAC; an appropriate metal ion (M2+) can be selected to suit the analytical purpose and a particular chromatographic protocol can be applied (isocratic pH, falling pH, and imidazole elution). We now report that IMAC may be exploited as an analytical tool in addition to its use as a protein purification technique. IMAC can be used to ascertain several facets of the status of a histidyl residue(s) in a protein molecule:
localization (interior vs. surface)
coordination potential as defined by the steric accessibility and the state of protonation
A novel, two-step preparative technique is described for the purification of authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coli cells. The first step is based on immobilized metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exclusion chromatography is used for further purification to .99.5% purity. The methodology is reproducible, leading to 77% recovery. Identity and purity of the rhPRL were demonstrated using sodium dodecylsulphate–polyacrylamide electrophoresis, isoelectric focusing, mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC and high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg.
Adenosine Affinity Ligand for Glutamine Synthase Glutamine synthetase has been purified from both procaryotic and eucaryotic sources using various types of affinity chromatography. For example, ADP-agarose has been used to purify glutamine synthetase from photosynthetic bacteria, while the related “Blue” chromatography media (e.g. Affigel Blue) have been used to purify glutamine synthetases from a variety of sources. In addition, 2’,5’-ADPSepharose 4B has been used to purify glutamine synthetase from procaryotes, plants and insects. However, this latter affinity ligand resembles NADP more than ADP, particularly with respect to the position of the phosphate moieties. This is reflected in the more general use of this affinity ligand in the purification of NADPH-dependent enzymes. In the present report, we characterize the ability of glutamine synthetase to be purified by three different adenosine-affinity ligands: 5’-ADP-agarose (an ADP analogue), 2’,5’-ADP-Sepharose 4B (an NADP analogue) and 3’,5’-ADP-agarose (a cyclic AMP analogue). We report conditions for the successful purification of insect flight muscle glutamine synthetase using each of these three different affinity ligands. The enzyme bound most strongly to the
ADP analogue (S-ADP-agarose),
followed by the NADPH analogue (2’,5’-ADP-Sepharose 4B), and least strongly to
the cyclic AMP analogue (3’J’-ADP-agarose).
In all cases, binding was strongest in the presence of Mn2+ when compared to Mg”. These results suggest that the binding of glutamine synthetase to adenosine-affinity media is related to the participation of Mn. ADP in the y-glutamyl transferase reaction that is catalyzed by glutamine synthetase. M Dowton, IR Kennedy. Purification of glutamine synthetase by adenosine-affinity chromatography. Journal of Chromatography A 1994; 664: 280-283
Aptamer Based Stationary Phase An anti-adenosine aptamer was evaluated as a stationary phase in packed capillary liquid chromatography. Using an 21 aqueous mobile phase containing 20 mM Mg , adenosine was strongly retained on the column. A gradient of increasing 21 Ni (to 18 mM), which is presumed to complex with nitrogen atoms in adenosine involved in binding to the aptamer, eluted adenosine in a narrow zone. The adenosine assay, which required no sample preparation, was used on microdialysis samples. Total analysis times were short so samples could be injected every 5 min. Q Deng, CJ Watson, RT Kennedy. Aptamer affinity chromatography for rapid assay of adenosine in microdialysis samples collected in vivo. Journal of Chromatography A 2003; 1005:123–130.
We will realize the full power of proteomics only when we can measure and compare the proteomes of many individuals to identify biomarkers of human health and disease and track the blood-based proteome of an individual over time. Because the human proteome contains an estimated 20,000 proteins – plus splicing and post-translational variants – that span a concentration range of ,12 logs, identifying and quantifying valid biomarkers is a great technical challenge. Proteomic measurements demand
extreme sensitivity
specificity
dynamic range
accurate quantification.
We describe a new class of DNA-based aptamers enabled by a versatile chemistry technology that endows nucleotides with protein-like functional groups. These modifications greatly expand the repertoire of targets accessible to aptamers. The resulting technology provides efficient, large-scale selection of exquisite protein-binding reagents selected specifically for use in highly multiplexed proteomics arrays. Aptamers are a class of nucleic acid-based molecules discovered twenty years ago, and have since been employed in diverse applications including
therapeutics
catalysis
proteomics
Aptamers are short single-stranded oligonucleotides, which fold into diverse and intricate molecular structures that bind with high affinity and specificity to
proteins
peptides
small molecules.
Aptamers are selected in vitro from enormously large libraries of randomized sequences by the process of Systematic Evolution of Ligands by EXponential enrichment (SELEX). A SELEX library with 40 random sequence positions has 440 (,1024) possible combinations and a typical selection screens 1014–1015 unique molecules. This is on the order of 105 times larger than standard peptide or protein combinatorial molecular libraries.
The interrogation of proteomes (‘‘proteomics’’) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 mL of serum or plasma).
Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (,100 fM–1 mM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray.
Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes.
This is a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine. L Gold, D Ayers, J Bertino, Christopher Bock, et al. Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery. PlosONE 2010; 5 (12): e15004
Biomarker Discovery, Diagnostics, and Therapeutics Progression from health to disease is accompanied by complex changes in protein expression in both the circulation and affected tissues. Large-scale comparative interrogation of the human proteome can offer insights into disease biology as well as lead to
the discovery of new biomarkers for diagnostics
new targets for therapeutics
can identify patients most likely to benefit from treatment.
Although genomic studies provide an increasingly sharper understanding of basic biological and pathobiological processes, they ultimately only offer a prediction of relative disease risk, whereas proteins offer an immediate assessment of “real-time” health and disease status. We have recently developed a new proteomic technology, based on modified aptamers, for biomarker discovery that is capable of simultaneously measuring more than a thousand proteins from small volumes of biological samples such as plasma, tissues, or cells. Our technology is enabled by SOMAmers (Slow Off-rate Modified Aptamers), a new class of protein binding reagents that contain chemically modified nucleotides that greatly expand the physicochemical diversity of nucleic acid-based ligands. Such modifications introduce functional groups that are absent in natural nucleic acids but are often found in protein-protein, small molecule-protein, and antibody-antigen interactions. The use of these modifications expands the range of possible targets for SELEX (Systematic Evolution of Ligands by EXponential Enrichment), results in improved binding properties, and facilitates selection of SOMAmers with slow dissociation rates. Our assay works by transforming protein concentrations in a mixture into a corresponding DNA signature, which is then quantified on current commercial DNA microarray platforms. In essence, we take advantage of the dual nature of SOMAmers as
both folded binding entities with defined shapes and
unique nucleic acid sequences recognizable by specific hybridization probes.
Aptamers and Smart Drug delivery Targeting In this review, the strategies for using functional nucleic acids in creating smart drug delivery devices will be explained, as their has been very recent progress in controlled drug release based on molecular gating achieved with aptamers. Aptamers are functional nucleic acid sequences which can bind specific targets. An artificial combinatorial methodology can identify aptamer sequences for any target molecule, from ions to whole cells. Drug delivery systems seek to increase efficacy and reduce side-effects by concentrating the therapeutic agents at specific disease sites in the body. This is generally achieved by specific targeting of inactivated drug molecules. Aptamers which can bind to various cancer cell types selectively and with high affinity have been exploited in a variety of drug delivery systems for therapeutic purposes. Recent progress in selection of cell-specific aptamers has provided new opportunities in targeted drug delivery. Especially functionalization of nanoparticles with such aptamers has drawn major attention in the biosensor and biomedical areas.
Nucleic acids are recognized as attractive building materials in nanomachines because of their unique molecular recognition properties and structural features. An active controlled delivery of drugs once targeted to a disease site is a major research challenge. Stimuli-responsive gating is one way of achieving controlled release of nanoparticle cargoes. Recent reports incorporate the structural properties of aptamers in controlled release systems of drug delivering nanoparticles.
Nanoparticle-encapsulated drug delivery aims to deliver the active therapeutic ingredients to the disease site in stable compartments in order to reduce premature release. This ensures that the effects of drug are maximized and the side effects are reduced. An encapsulated nanoparticle system requires a specific targeting mechanism and at the same time the retention of drugs inside the container should be high. The balance between specificity of targeting and the extent of premature leakage determines the success of a given delivery system.
Nanotechnology research approaches in drug delivery include a wide variety of nanomaterials ranging from soft hydrogels to solid polymeric particles. Large surface area, high drug loading efficiency and potential combination with other organic/inorganic materials are the main properties of hollow nanostructures that are attractive for biomedical applications.
Packaging of small-molecule drugs
improves their availability
compatibility
reduces toxicity
Controlling the drug release profile is the main challenge in drug delivery development when the drug is to be successfully targeted to a specific site. Stimuli-responsive materials have been created by using biological, physical and chemical properties of materials for heat-activated, light-activated or pH-activated delivery. Nucleic acids are utilized to construct rationally designed nanostructures at molecular levels for nanotechnology applications. Integration of the properties of nucleic acids can offer many opportunities for drug delivery systems, including stimuli-responsive nanogates for nanocarriers and molecular sensors. Favorable drug release kinetics can be achieved at the target sites by aptamer-based capping systems.
Activity Based Profiling Powerful strategies for the gel-free analysis of proteomes have emerged, including isotope-coded affinity tagging (ICAT) for quantitative proteomics and multidimensional protein identification technology (MudPIT) for comprehensive proteomics, both of which utilize liquid chromatography (LC) and MS for protein separation and detection, respectively. Nonetheless, these methods, like 2DE-MS, still focus on measuring changes in protein abundance and, therefore, provide only an indirect estimate of dynamics in protein function. Indeed, several important forms of post-translational regulation, including protein–protein and protein–small-molecule interactions, may elude detection by abundance-based proteomic methods. To facilitate the analysis of protein function, several proteomic methods have been introduced to characterize the activity of proteins on a global scale. These include large-scale yeast two-hybrid screens and epitope tagging immunoprecipitation experiments, which aim to construct comprehensive maps of protein–protein interactions, and protein microarrays, which aim to provide an assay platform for the rapid assessment of protein activities. A chemical proteomic strategy referred to as activity-based protein profiling (ABPP) has emerged that utilizes active site-directed probes to profile the functional state of enzyme families directly in complex proteomes.
Recent advances in genomic and proteomic technologies have begun to address the challenge of assigning molecular and cellular functions to the numerous protein products encoded by prokaryotic and eukaryotic genomes. In particular, chemical strategies for proteome analysis have emerged that enable profiling of protein activity on a global scale. Herein, we highlight these chemical proteomic methods and their application to the discovery and characterization of disease-related enzyme activities.
Cells with fundamental metabolic alterations commonly arise during tumorigenesis, and it is these types of changes that help to establish a biochemical foundation for disease progression and malignancy. A seminal example of this was discovered in the 1920s when Otto Warburg found that cancer cells consume higher levels of glucose and secrete most of the glucose carbon as lactate rather than oxidizing it completely. Since then, studies by multiple groups have uncovered a diverse array of metabolic changes in cancer, including alterations in
glycolytic pathways
the citric acid cycle
glutaminolysis
lipogenesis
lipolysis
proteolysis
These in turn modulate the levels of cellular building blocks
lipids, nucleic acids and amino acids,
cellular energetics,
oncogenic signaling molecules
the extracellular environment to confer protumorigenic and malignant properties.
Despite these advances, our current understanding of cancer metabolism is far from complete and would probably benefit from experimental strategies that are capable of profiling enzymatic pathways on a global scale. To this end, conventional genomic and proteomic methods, which comparatively quantify the expression levels of transcripts and proteins, respectively, have yielded many useful insights. These platforms are, however, limited in their capacity to identify changes in protein activity that are caused by posttranslational mechanisms.
Annotating biochemical pathways in cancer is further complicated by the potential for enzymes to carry out distinct metabolic activities in tumor cells that might not be mirrored in normal physiology. In addition, a substantial proportion of the human proteome remains functionally uncharacterized, and it is likely that at least some of these poorly understood proteins also have roles in tumorigenesis. These challenges require new proteomic technologies that can accelerate the assignment of protein function in complex biological systems, such as cancer cells and tumors.
Metabolomics has emerged as a powerful approach for investigating enzyme function in living systems. Metabolomic experiments in the context of enzyme studies typically start with
the extraction of metabolites from control and enzyme-disrupted biological systems,
followed by metabolite detection and comparative data analysis.
For example, lipophilic metabolites can be enriched from cells or tissues by organic extraction. Mass spectrometry (MS) has become a primary analytical method for surveying metabolites in complex biological samples, with upfront separation accomplished by liquid chromatography (LC–MS) or gas chromatography (GC–MS). MS experiments can be carried out using
targeted or untargeted approaches,
depending on whether the objective is
to profile and quantitate known metabolites or
to broadly scan for metabolites across a large mass range, respectively.
As metabolomic experiments generate a large amount of data, powerful software tools are needed for identification and quantitation of ions in LC–MS data sets (see the figure; the mass to charge ratio (m/z) is indicated). One such program is XCMS95, which
aligns,
quantifies and
statistically ranks ions that are altered between two sets of metabolomic data.
This program can be used to rapidly identify metabolomic signatures of various disease states or to assess metabolic networks that are regulated by an enzyme using pharmacological or genetic tools that modulate enzyme function. Additional databases assist in metabolite structural characterization, such as HMDB96,97, METLIN98,99 and LIPID MAPS100. In this Review, we discuss one such proteomic platform, termed activity based protein profiling (ABPP), and its implementation in the discovery and functional characterization of deregulated enzymatic pathways in cancer. We discuss the evidence that, when coupled with other large scale profiling methods, such as metabolomics and proteomics, ABPP can provide a compelling, systems level understanding of biochemical networks that are important for the development and progression of cancer.
Large-scale profiling methods have uncovered numerous gene and protein expression changes that correlate with tumorigenesis. However, determining the relevance of these expression changes and which biochemical pathways they affect has been hindered by our incomplete understanding of the proteome and its myriad functions and modes of regulation. Activity-based profiling platforms enable both the discovery of cancer-relevant enzymes and selective pharmacological probes to perturb and characterize these proteins in tumour cells. When integrated with other large-scale profiling methods, activity-based proteomics can provide insight into the metabolic and signaling pathways that support cancer pathogenesis and illuminate new strategies for disease diagnosis and treatment.
Representative activity-based probes and their application to cancer research
enzyme class applications in cancer
Serine hydrolases increased KIAA1363 and MAGL
aggressive human cancer lines
uPA and tPA serine protease aggressive cancers
RBBP9 activity in pancreatic carcinoma
Metalloproteinases neprilysin activity in melanoma cell lines
Cysteine proteases cathepsin cysteine protease in pancreatic islet tumours
Kinases Inhibitor selectivity profiling of kinase inhibitors
Caspases visualization of apoptosis in colon tumour-bearing mice treated with Apomab
Deubiquitylases Identified increased carboxy-terminal hydrolase UCHL3 and UCH37 activity in HPV cervical carcinomas
Cytochrome P450s Identified the aromatase inhibitor anastrazole as an inducer of CYP1A2
Serine hydrolases KIaa1363 and MaGL regulate lipid metabolic pathways that support cancer pathogenesis. Activity-based protein profiling (ABPP) identified
KIAA1363 and
monoacylglycerol (MAG) lipase (MAGL)
as being increased in aggressive human cancer cells from multiple tumour types. Pharmacological and/or RNA interference ablation of KIAA1363 and MAGL coupled with metabolomic analysis revealed specific roles for KIAA1363 and MAGL in cancer metabolism. Disruption of KIAA1363 by the small-molecule inhibitor AS115 lowered monoalkylglycerol ether (MAGE), alkyl lysophosphatidic acid (alkyl LPA) and alkyl lysophosphatidyl choline (alkyl LPC) levels in cancer cells. Disruption of MAGL by the small-molecule inhibitor JZL184 raised MAG levels and reduced free fatty acid, lysophosphatidic acid (LPA) and prostaglandin E2 (PGE2) levels in cancer cells. Disruption of KIAA1363 and MAGL leads to impairments in cancer cell aggressiveness and tumour growth, PAF, platelet-activating factor.
At a glance
• Activity-based protein profiling (ABPP) facilitates the discovery of deregulated enzymes in cancer. • Competitive ABPP yields selective inhibitors for functional characterization of cancer enzymes. • ABPP can be integrated with metabolomics to map deregulated enzymatic pathways in cancer. • ABPP can be integrated with other proteomic methods to map proteolytic pathways in cancer. • ABPP probes can be used to image tumour development in living animals.
Activity-based protein profiling (ABPP), the use of active site-directed chemical probes to monitor enzyme function in complex biological systems, is emerging as a powerful post-genomic technology. ABPP probes have been developed for several enzyme classes and have been used to inventory enzyme activities en masse for a range of (patho)physiological processes.
ABPP uses active site–directed, small molecule–based covalent probes to report on the functional state of enzyme activities directly in native biological systems. ABPP probes are designed or selected to target a subset of the proteome based on shared principles of binding and/or reactivity and have been successfully developed for many enzyme classes, including
serine
cysteine,
aspartyl
metallo hydrolases
kinases
glycosidases
histone deacetylases and
oxidoreductases.
These probes have been shown to selectively label active enzymes but not their inactive precursor (zymogen) or inhibitor-bound forms, thus allowing researchers to capture functional information that is beyond the scope of standard proteomic methods. By presenting specific examples, we show here that ABPP provides researchers with a distinctive set of chemical tools to embark on the assignment of functions to many of the uncharacterized enzymes that populate eukaryotic and prokaryotic proteomes.
Vinyl-methylester UL from HSV-1 Deubiquitinating enzyme (DUB)
Aryl 2-deoxy-2-fluoro glycoside Cfx from C. fimi Glycosidase (β-1-4-glycanase) Fluorophosphonate SAE Serine hydrolase
Examples of enzymes assigned to specific mechanistic classes by ABPP
ABPP can also be implemented as a direct assay for inhibitor discovery, allowing researchers to develop potent and selective pharmacological probes for uncharacterized enzymes.
Examples of enzymes assigned to specific mechanistic classes by ABPP.
Probe Leu-Asp-αCA probe selectively labeled Upβ
Substrate the endogenous Upβ substrate, N-carbamoyl-β-alanine
Substrate mimicry of an ABPP probe.
Multidimensional profiling strategy for the annotation of the cancer-related enzyme KIAA1363. ABPP using fluorophosphonate probes identified KIAA1363 as a highly elevated enzyme activity in aggressive cancer cells. Competitive ABPP was then used to develop a selective KIAA1363 inhibitor (AS115). Metabolomic analysis of cancer cells treated with AS115 determined a role for this enzyme in the regulation of MAGE lipids in cancer cells. Biochemical studies confirmed that KIAA1363 acts as 2-acetyl MAGE hydrolase in a metabolic network that bridges the platelet activating factor and lysophosphatidic acid classes of signaling lipids. Assignment of enzyme mechanism by ABPP
There are multiple levels of annotation for enzymes. The most basic level is assignment to a specific mechanistic class based on the general chemical reaction catalyzed by the enzyme (for example, hydrolase, kinase, oxidoreductase and others). Additional annotation involves determining the endogenous substrates and products for the enzyme. Finally, complete annotation requires an understanding of how the specific chemical transformation(s) catalyzed by an enzyme integrate into larger metabolic and signaling pathways to influence cell physiology and behavior.
Many of the predicted enzymes uncovered by genome sequencing projects can be assigned to a mechanistic class or ascribed a putative biochemical function based on sequence homology to well-characterized enzymes. But some enzymes have insufficient sequence relatedness for class assignment or have a function different from that predicted by sequence comparisons. ABPP has facilitated class annotation for several such uncharacterized enzymes.
A principal goal of modern biomedical research is to discover, assemble, and experimentally manipulate molecular pathways in cells and organisms to reveal new disease mechanisms.
Toward this end, complete genome sequences for numerous bacteria and higher organisms, including humans, have laid the fundamental groundwork for understanding the molecular basis of life in its many forms. However, the information content of DNA sequences is limited and, on its own, cannot describe most physiological and pathological processes.
Unlike oligonucleotides, proteins are a very diverse group of biomolecules that display a wide range of chemical and biophysical features, including
membrane-binding,
hetero/homo-oligomerization, and
posttranslational modification.
The biochemical complexity intrinsic to protein science intimates that several complementary analytical strategies will be needed to achieve the ultimate goal of proteomics – a comprehensive characterization of the expression, modification state, interaction map, and activity of all proteins in cells and tissues.
A powerful LC-MS strategy for proteomics involves the use of isotope-coded affinity tags (ICAT). This approach enables the comparison of protein expression in proteomes by treating samples with isotopically distinct forms of a chemical labeling reagent. ICAT methods provide superior resolving power compared to gel-based methods and improve access to membrane-associated proteins. More recently, isotope-free MS methods for quantitative proteomics have emerged.
Reverse protein microarrays have also been described in which proteomes themselves are arrayed and the antibodies used for detection in a format analogous to Western blotting. In addition to increasing the throughput of proteomic experiments by integrating the protein separation and detection steps, microarrays consume much less material than conventional proteomic methods. Still, the general application of microarrays for proteomics is currently limited by the availability of high-quality capture reagents (e.g., antibodies, aptamers, etc).
These approaches, by measuring protein abundance provide, like genomics, only an indirect assessment of protein activity and may fail to detect important posttranslational events that regulate protein function, such as protein–protein or protein–small-molecule interactions. To address these limitations, complementary strategies for the functional analysis of proteins have been introduced. Prominent among these functional proteomic efforts is the use of chemistry for the design of active site-directed probes that measure enzyme activity in samples of high biological complexity.
Many post-translational modes of enzyme regulation share a common mechanistic foundation – they perturb the active site such that catalytic power and/or substrate recognition is impaired. Accordingly, it was hypothesized that chemical probes capable of reporting on the integrity of enzyme active sites directly in cells and tissues might serve as effective functional proteomic tools. These activity based protein profiling (ABPP) probes consist of at least two general elements:
a reactive group for binding and covalently modifying the active sites of many members of a given enzyme class or classes
a reporter tag for the detection, enrichment, and identification of probe-labeled proteins
ABPP probes have been successfully developed for more than a dozen enzyme classes, including
all major classes of proteases
kinases
phosphatases
glycosidases
GSTs
oxidoreductases.
Post-translational regulation of enzyme activity. Many enzymes are produced as inactive precursors, or zymogens, which require proteolytic processing for activation. Enzyme activity can be further regulated by interactions with endogenous protein inhibitors. The field of proteomics aims to develop and apply technologies for the characterization of protein function on a global scale. Toward this end, synthetic chemistry has played a major role by providing new reagents to profile segments of the proteome based on activity rather than abundance. Small molecule probes for activity-based protein profiling have been created for more than a dozen enzyme classes and used to discover several enzyme activities elevated in disease states. These innovations have inspired complementary advancements in analytical chemistry, where new platforms have been introduced to augment the information content achievable in chemical proteomics experiments. Here, we will review these analytical platforms and discuss how they have exploited the versatility of chemical probes to gain unprecedented insights into the function of proteins in biological samples of high complexity.
Advanced analytical platforms utilize a range of separation and detection strategies, including LC-MS, CELIF, and antibody microarrays, to achieve an unprecedented breadth and depth of proteome coverage in ABPP investigations. The complementary strengths and weaknesses of each of these methods suggest that the selection of an appropriate analytical platform should be guided by the specific experimental question being addressed. SA Sieber and BF Cravatt. Analytical platforms for activity-based protein profiling – exploiting the versatility of chemistry for functional proteomics. Chem. Commun. 2006, 2311–2319. http://www.rsc.org/chemcomm
Diagnostic Therapeutics in Activity Based Probes Activity-based chemical proteomics-an emerging field involving a combination of organic synthesis, biochemistry, cell biology, biophysics and bioinformatics-allows the detection, visualisation and activity quantification of whole families or selected sub-sets of proteases based upon their substrate specificity. This approach can be applied for drug target/lead identification and validation, the fundamentals of drug discovery. The activity-based probes discussed in this review contain three key features;
a ‘warhead’ (binds irreversibly but selectively to the active site),
a ‘tag’ (allowing enzyme ‘handling’, with a combination of fluorescent, affinity and/or radio labels),
a linker region between warhead and tag.
From the design and synthesis of the linker arise some of the latest developments discussed here; not only can the physical properties (e.g., solubility, localisation) of the probe be tuned, but the inclusion of a cleavable moiety allows selective removal of tagged enzyme from affinity beads etc. Heal WP, Wickramasinghe SR, Tate EW. Activity based chemical proteomics: profiling proteases as drug targets. Curr Drug Discov Technol 2008; 5(3):200-12. PMID: 18690889
The genomic revolution has created a wealth of information regarding the fundamental genetic code that defines the inner workings of a cell. However, it has become clear that analyzing genome sequences alone will not lead to new therapies to fight human disease. Rather, an understanding of protein function within the context of complex cellular networks will be required to facilitate the discovery of novel drug targets and, subsequently, new therapies directed against them. The past ten years has seen a dramatic increase in technologies that allow large-scale, systems-based methods for analysis of global biological processes and disease states.
In the field of proteomics, several well-established methods persist as a means to resolve and analyze complex mixtures of proteins derived from cells and tissues. However, the resolving power of these methods is often challenged by the diverse and dynamic nature of the proteome. The field of activity-based proteomics, or chemical proteomics, has been established in an attempt to focus proteomic efforts on subsets of physiologically important protein targets. This new approach to proteomics is centered around the use of small molecules termed activity-based probes (ABPs) as a means to tag, enrich, and isolate, distinct sets of proteins based on their enzymatic activity. Berger AB, Vitorino PM, Bogyo M. Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery. Am J Pharmacogenomics. 2004;4(6):371-81.
Recent advances in global genomic and proteomic methods have led to a greater understanding of how genes and proteins function in complex networks within a cell. One of the major limitations in these methodologies is their inability to provide information on the dynamic, post-translational regulation of enzymatic proteins. In particular proteases are often synthesized as inactive zymogens that need to be activated in order to carry out specific biological processes. Thus, methods that allow direct monitoring of protease activity in the context of a living cell or whole animal will be required to begin to understand the systems-wide functional roles of proteases. In this review, we discuss the development and applications of activity based probes (ABPs) to study proteases and their role in pathological processes. Specifically we focus on application of this technique for biomarker discovery, in vivo imaging and drug screening.
Proteases, in particular, are known for their multilayered post-translational activity regulation that can lead to a significant difference between protease abundance levels and their enzyme activity. To address these issues, the field of activity-based proteomics has been established in order to characterize protein activity and monitor the functional regulation of enzymes in complex proteomes.
As a result of the recent enormous technological progress, experimental structure determination has become an integral part of the development of drugs against disease-related target proteins. The post-translational modification of proteins is an important regulatory process in living organisms; one such example is lytic processing by peptidases. Many different peptidases represent disease targets and are being used in structure-based drug design approaches. The development of drugs such as aliskiren and tipranavir, which inhibit renin and HIV protease, respectively, testifies to the success of this approach.
Presenilin is the catalytic component of γ-secretase, a complex aspartyl protease and a founding member of intramembrane-cleaving proteases. γ-Secretase is involved in the pathogenesis of Alzheimer’s disease and a top target for therapeutic intervention. However, the protease complex processes a variety of transmembrane substrates, including the Notch receptor, raising concerns about toxicity. Nevertheless, γ-secretase inhibitors and modulators have been identified that allow Notch processing and signaling to continue, and promising compounds are entering clinical trials.
Molecular and biochemical studies offer a model for how this protease hydrolyzes transmembrane domains in the confines of the lipid bilayer. Progress has also been made toward structure elucidation of presenilin and the γ-secretase complex by electron microscopy as well as by studying cysteine-mutant presenilins. The signal peptide peptidase (SPP) family of proteases are distantly related to presenilins. However, the SPPs work as single polypeptides without the need for cofactors and otherwise appear to be simple model systems for presenilin in the γ-secretase complex.
Critical clues to the identity of γ-secretase included: (1) Genes encoding the multi-pass membrane proteins presenilin-1 and presenilin-2 are, like APP, associated with familial, early-onset Alzheimer’s disease. The disease-causing missense mutations were found to alter how γ-secretase cuts APP, leading to increased proportions of longer, more aggregation-prone forms of Aβ. (2) Knockout of presenilin genes eliminates γ-secretase cleavage of APP. (3) Peptidomimetics that inhibit γ-secretase contain moieties typically found in aspartyl protease inhibitors. These findings led to the identification of two conserved transmembrane aspartates in the multi-pass presenilins that are critical for γ-secretase cleavage of APP, evidence that presenilins are aspartyl proteases. Presenilin is endoproteolytically cleaved into two polypeptides, an N-terminal fragment (NTF) and a C-terminal fragment (CTF), the formation of which is
regulated
metabolically stable
part of a high-molecular weight complex
suggesting that the NTF-CTF heterodimer is the biologically active form. NTF and CTF each contribute one of the essential and conserved aspartates, and transition-state analogue inhibitors of γ-secretase, compounds designed to interact with the active site of the protease, bind directly to presenilin NTF and CTF. Presenilins are also required for Notch signaling (Levitan and Greenwald, 1995), a pathway essential for cell differentiation during development and beyond.
The highly conserved role of γ-secretase in Notch signalling and its importance in development led to genetic screens in Caenorhabditis elegans that identified three other integral membrane proteins besides presenilin that modify Notch signaling. Designed inhibitors have proven to be useful tools in understanding the mechanism of γ-secretase and substrate recognition – affinity labelling with transition-state analogue inhibitors showed binding at the interface between the presenilin NTF and CTF subunits, consistent with the active site residing at this interface, with each presenilin subunit contributing one of the essential aspartates. The concept of presenilin as the catalytic component for γ-secretase was considerably strengthened when
signal peptide peptidase (SPP) was found to be a similar intramembrane aspartyl protease
SPP is exploited by the hepatitis C virus for the maturation of its core protein, suggesting that this protease may be a suitable target for antiviral therapy
SPP was identified by affinity labeling with a peptidomimetic inhibitor, and the protein sequence displayed similarities with presenilin.
SPP contains two conserved aspartates, each predicted to lie in the middle of a transmembrane domain, and the aspartate-containing sequences resemble those found in presenilins.
SPP appears to be less complicated than γ-secretase.
Expression of human SPP in yeast reconstituted the protease activity, suggesting that the protein has activity on its own and does not require other mammalian protein cofactors.
Aspartyl I-CLiPs are found in all forms of life and play essential roles in biology and disease. How these enzymes carry out hydrolysis in the membrane is a fascinating question that is not entirely resolved, but evidence suggests an initial substrate docking site and a lateral gate into a pore where water and the active site aspartates reside. Designed inhibitors have been critical in elucidating these mechanisms, but inhibitors targeting γ-secretase for the treatment of Alzheimer’s disease must avoid interfering with Notch signaling.
MS Wolfe. Structure, Mechanism and Inhibition of γ-Secretase and Presenilin-Like Proteases. Biol Chem. 2010 August; 391(8): 839–847. doi: 10.1515/BC.2010.086. PMCID: PMC2997569. NIHMSID: NIHMS254540 Study Suggests Expanding the Genetic Alphabet May Be Easier than Previously Thought Genomics Monday, June 4, 2012 A new study led by scientists at The Scripps Research Institute suggests that the replication process for DNA—the genetic instructions for living organisms that is composed of four bases (C, G, A and T)—is more open to unnatural letters than had previously been thought.
An expanded “DNA alphabet” could carry more information than natural DNA, potentially coding for a much wider range of molecules and enabling a variety of powerful applications, from precise molecular probes and nanomachines to useful new life forms. The new study, which appears in the June 3, 2012 issue of Nature Chemical Biology, solves the mystery of how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases. “We now know that the efficient replication of our unnatural base pair isn’t a fluke, and also that the replication process is more flexible than had been assumed,” said Floyd E. Romesberg, principal developer of the new DNA bases.
Adding to the DNA Alphabet Romesberg and his lab have been trying to find a way to extend the DNA alphabet since the late 1990s. In 2008, they developed the efficiently replicating bases NaM and 5SICS, which come together as a complementary base pair within the DNA helix, much as, in normal DNA, the base adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).
The following year, Romesberg and colleagues showed that NaM and 5SICS could be efficiently transcribed into RNA. But these bases’ lack the ability to form the hydrogen bonds that join natural base pairs in DNA. Such bonds had been thought to be an absolute requirement for successful DNA replication‑—a process in which a large enzyme, DNA polymerase, moves along a single, unwrapped DNA strand and stitches together the opposing strand, one complementary base at a time.
An early structural study of a very similar base pair in double-helix DNA added to Romesberg’s concerns. The data strongly suggested that NaM and 5SICS do not even approximate the edge-to-edge geometry of natural base pairs—termed the Watson-Crick geometry, after the co-discoverers of the DNA double-helix. Instead, they join in a looser, overlapping, “intercalated” fashion. “Their pairing resembles a ‘mispair,’ such as two identical bases together, which normally wouldn’t be recognized as a valid base pair by the DNA polymerase.” Yet in test after test, the NaM-5SICS pair was efficiently replicable.
Edge to Edge The NaM-5SICS pair maintain an abnormal, intercalated structure within double-helix DNA—but remarkably adopt the normal, edge-to-edge, “Watson-Crick” positioning when gripped by the polymerase during the crucial moments of DNA replication. “The DNA polymerase apparently induces this unnatural base pair to form a structure that’s virtually indistinguishable from that of a natural base pair.” NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by “hydrophobic” forces, which cause certain molecular structures to be repelled by water molecules, and thus to cling together in a watery medium. “It’s very possible that these hydrophobic forces have characteristics that enable the flexibility and thus the replicability of the NaM-5SICS base pair.”
An Arbitrary Choice? The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could some day be used to extend the DNA alphabet. It also hints that Evolution’s choice of the existing four-letter DNA alphabet—on this planet—may have been somewhat arbitrary. “It seems that life could have been based on many other genetic systems.” Source: The Scripps Research Institute
DNA damage response (DDR) network
Eukaryotic cells have evolved an intricate system to resolve DNA damage to prevent its transmission to daughter cells. This system, collectively known as the DNA damage response (DDR) network, includes many proteins that detect DNA damage, promote repair, and coordinate progression through the cell cycle. Because defects in this network can lead to cancer, this network constitutes a barrier against tumorigenesis. The modular BRCA1 carboxyl-terminal (BRCT) domain is frequently present in proteins involved in the DDR, can exist either as an individual domain or as tandem domains (tBRCT), and can bind phosphorylated peptides. We performed a systematic analysis of protein-protein interactions involving tBRCT in the DDR.
We identified 23 proteins containing conserved BRCT domains and generated a human protein-protein interaction network for seven proteins with tBRCT. This study also revealed previously unknown components in DNA damage signaling, such as COMMD1 and the target of rapamycin complex mTORC2. Additionally, integration of tBRCT domain interactions with DDR phosphoprotein studies and analysis of kinase-substrate interactions revealed signaling subnetworks that may aid in understanding the involvement of tBRCT in disease and DNA repair.
Mitochondria have various essential functions in metabolism and in determining cell fate during apoptosis. In addition, mitochondria are also important nodes in a number of signaling pathways. For example, mitochondria can modulate signals transmitted by second messengers such as calcium. Because mitochondria are also major sources of reactive oxygen species (ROS), they can contribute to redox signaling—for example, by the production of ROS such as hydrogen peroxide that can reversibly modify cysteine residues and thus the activity of target proteins. Mitochondrial ROS production is thought to play a role in hypoxia signaling by stabilizing the oxygen-sensitive transcription factor hypoxia-inducible factor–1α. New evidence has extended the mechanism of mitochondrial redox signaling in cellular responses to hypoxia in interesting and unexpected ways. Hypoxia altered the microtubule-dependent transport of mitochondria so that the organelles accumulated in the perinuclear region, where they increased the intranuclear concentration of ROS. The increased ROS in turn enhanced the expression of hypoxia-sensitive genes such as VEGF (vascular endothelial growth factor) not by reversibly oxidizing a protein, but by oxidizing DNA sequences in the hypoxia response element of the VEGF promoter. This paper and other recent work suggest a new twist on mitochondrial signaling: that the redistribution of mitochondria within the cell can be a component of regulatory pathways.
A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ.
2012pharmaceutical
Amandeep Kaur
Aashir Awan, Phd
Abhisar Anand
Adina Hazan
Alan F. Kaul, PharmD., MS, MBA, FCCP
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anamikasarkar
apreconasia
aviralvatsa
David Orchard-Webb, PhD
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Demet Sag, Ph.D., CRA, GCP
Dror Nir
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Ethan Coomber
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Gail S Thornton
Irina Robu
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Ed Kislauskis
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Madison Davis
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Dr. Pati
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Rick Mandahl
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Srinivas Sriram
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Dr. Sudipta Saha
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.png){kind=link}
.png){kind=link}
.png){kind=link}
.png){kind=link}