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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 21^st century.  This has been partly rectified with the enlightening elucidation of genome modifications, cellular metabolic regulation, and signaling pathways.

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

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

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

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

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

John C. Schell, Kristofor A. Olson, Lei Jiang, Amy J. Hawkins, et al.
Molecular Cell Nov 6, 2014; 56: 400–413.
http://dx.doi.org/10.1016/j.molcel.2014.09.026

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

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

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

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

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

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

Yi Liu, Yanyan Cao, Weihe Zhang, Stephen Bergmeier, et al.
Mol Cancer Ther Aug 2012; 11(8): 1672–82
http://dx.doi.org://10.1158/1535-7163.MCT-12-0131

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

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

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

Valeria R. Fantin Julie St-Pierre and Philip Leder
Cancer Cell Jun 2006; 9: 425–434.
http://dx.doi.org:/10.1016/j.ccr.2006.04.02

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

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

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

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

S Langbein, M Zerilli, A zur Hausen, W Staiger, et al.
British Journal of Cancer (2006) 94, 578–585.
http://dx.doi.org:/10.1038/sj.bjc.6602962

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

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

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

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

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

Cancer Cell Jan 2007; 11: 37–51.
http://dx.doi.org:/10.1016/j.ccr.2006.10.020

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

A Metabolic Hypothesis of Cell Growth and Death in Pancreatic Cancer

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

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

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

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

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

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

Marco Gerlinger, Claudio R Santos, Bradley Spencer-Dene, et al.
J Pathol 2012; 227: 146–156
http://dx.doi.org:/10.1002/path.4006

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

http://dx.doi.org:/10.1016/j.gde.2009.10.009

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

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

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

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

Evaluation of HIF-1 inhibitors as anticancer agents

Gregg L. Semenza
Drug Discovery Today Oct 2007; 12(19/20).
http://dx.doi.org:/10.1016/j.drudis.2007.08.006

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

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

First, they must act by a described mechanism.

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

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

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

New anticancer strategies targeting HIF-1

Eun-Jin Yeo, Yang-Sook Chun, Jong-Wan Park
Biochemical Pharmacology 68 (2004) 1061–1069
http://dx.doi.org:/10.1016/j.bcp.2004.02.040

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

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

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

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

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

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

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

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

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

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

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

Cancer Cell Metabolism: Warburg & Beyond

Hsu PP & Sabatini DM
Cell  Sep 5, 2008; 134, 703-705
http://dx.doi.org:/10.1016/j.cell.2008.08.021

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

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

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

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

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

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

Daniel K. Bricker, Eric B. Taylor, John C. Schell, Thomas Orsak, et al.
Science Express 24 May 2012
http://dx.doi.org:/10.1126/science.1218099

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

Young S. Kim and John A. Milner
Journal of Biomedicine and Biotechnology 2011, Art ID 721213, 9 pages
http://dx.doi.org:/10.1155/2011/721213

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

Comment (Aurelian Udristioiu):

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

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

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

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

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

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

Shortened version of Comment –

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

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