Cancer is now recognized to be a disease arising from both genetic and metabolic abnormalities. In the mid-1900s, Otto Warburg described the phenomenon of elevated glucose consumption and aerobic glycolysis, and the dependence of cancer cells on this phenomenon for proliferation and growth. The Warburg effect has formed the basis of such diagnostic and prognostic imaging modalities as positron emission tomography (PET); however, we have not yet capitalized on this phenomenon for therapy. Several mechanisms have now been shown to contribute to the Warburg effect.
Ongoing studies are attempting to understand the reasons that tumor cells engage in aerobic glycolysis in lieu of oxidative phosphorylation, and the advantages that accrue to them as a result. In this review, we discuss known benefits to tumor cells from this metabolic switch, and we highlight key enzymes that play a role in aerobic glycolysis. We also describe novel therapeutic options targeting glucose metabolism and the importance of continuing to understand the metabolic plasticity of cancer.
Treatment of cancer has undergone several evolutionary changes in the last decade as discoveries of various pathways leading to oncogenesis have continued to unfold. Despite these advances, cancer deaths have decreased by only a little more than 1% over the last 10 years. A total of 1,638,910 new cancer cases and 577,190 deaths from cancer were projected to occur in the United States in 2012. One of the consistently encountered hurdles in the treatment of all cancers has been development of resistance to currently available treatment modalities. Thus, there is a need to develop newer treatment modalities to combat ensuing resistance and to improve survival in affected patients.
The Warburg Effect
Over the last century, cancer therapy has focused on understanding and targeting the genetic basis of tumor development and progression. While this approach has led to significant advances in personalized cancer therapy, the problem of how to target tumor cells that revert to alternative pathways to sustain survival and proliferation remains a stumbling block. The genetic heterogeneity of cancer in itself suggests the need for broader approaches that can target the wide range of epigenetic and genetic phenomena fueling cancer growth.
Cancer is also a disease of altered metabolism. This phenomenon, recognized close to 100 years ago, is now accepted as one of the fundamental hallmarks of cancer. Targeting altered cancer metabolism may, in fact, provide us with greater opportunities to target genetic heterogeneity within any particular cancer.
Normal cells generate adenosine triphosphate (ATP) primarily by oxidation of glucose in the presence of oxygen via the highly efficient mitochondrial oxidative phosphorylation (OXPHOS) pathway. In contrast, tumor cells rely on the inefficient glycolytic pathway for generation of ATP, even in the presence of oxygen, necessitating increased rates of glucose consumption to maintain energy production. This phenomenon, termed aerobic glycolysis, was first described by Otto Warburg in the mid-1900s and was originally thought to be a result of defects in oxidative phosphorylation. More recent studies have shown that tumor cells do contain functional mitochondria, yet they still produce excessive amounts of lactate, suggesting that the enhanced glycolytic flux may confer a growth advantage. In support of this notion, tumor cells forced to revert to OXPHOS by RNAi-mediated suppression of lactate dehydrogenase A (LDH-A; the enzyme responsible for conversion of pyruvate to lactate) or by chemical inhibition of LDH-A demonstrate reduced proliferation. Additionally, tumor cells treated with dichloroacetate (DCA), which inhibits pyruvate dehydrogenase kinase (PDK) and increases mitochondrial metabolism of pyruvate, demonstrate reduced rates of proliferation. These and other studies have now stimulated a renewed interest in therapies targeting glycolytic metabolism.
The ATP required for growth is mainly obtained through inefficient glycolysis; this occurs at an increased rate, which is necessary to sustain the high rate of cell proliferation seen in growing tumors.[10,11] It has been suggested that with an unlimited glucose supply, glycolysis is capable of producing energy at a much faster rate than OXPHOS. There is emerging literature to suggest that this switch to aerobic glycolysis from mitochondrial respiration also occurs to support anabolic growth. Glucose, in addition to being a source of ATP, can also serve as a precursor in a variety of biosynthetic pathways necessary for duplication of cell mass prior to cell division. Glucose-derived carbon is used to generate intermediates for nonessential amino acid synthesis, nucleotide synthesis, and fatty acid synthesis. Besides, many intermediate metabolites of glycolysis—such as glucose-6-phosphate, 3-phosphoglycerate, phosphoenolpyruvate, and pyruvate—are key precursors in the biosynthesis of several amino acids required for the growth of cancer cells. Figure 1 shows the impact of glucose on the synthesis of nucleotides, amino acids, fatty acids, and ATP—and on redox homeostasis in normal cells.
It was also proposed recently that persistent activation of glycolysis creates a state of metabolic acidosis that is toxic to normal cells through the p53-dependent apoptosis pathway triggered by increased caspase activity—but that is not toxic to cancer cells, presumably because of mutations in p53 or other components of the apoptotic pathway.[13,14] It has now been consistently shown that tumor cells exhibit maximal growth rates in a relatively acidic medium (~pH 6.8). This acidic environment has also been shown to support acquisition of angiogenesis and the ability to invade, both of which are hallmarks of cancer cells. The concept that cancer cells have an increased dependence on glycolysis has been further strengthened by the observation that most glycolysis inhibitors, such as oxamate, DCA, and PDK1 siRNA, induce apoptosis in cancers such as multiple myeloma. Some of these inhibitors, eg, DCA when combined with bortezomib (Velcade), have shown additive cytotoxic effects.
Several mechanisms have now been shown to contribute to the Warburg effect. Oncogenes and transcription factors, such as AKT, C-MYC, RAS, p53, and HIF1, upregulate various components of the glycolytic pathway, thus facilitating the glycolytic shift. The hypoxic microenvironment associated with the preangiogenic phase of tumor development promotes stabilization of HIF1 that can induce expression of glucose transporter 1 (GLUT1), LDH-A, C-MYC and PDK3, facilitating aerobic glycolysis. A switch to the embryonic isoform of pyruvate kinase (PK)—ie, PKM2—is also thought to enhance anabolic processes dependent on glycolytic intermediates, thereby facilitating tumor cell proliferation, while overexpression of hexokinase (HK) II in various cancers also facilitates glycolysis.
The genetic and epigenetic changes in a cancer cell that contribute to the glycolytic phenotype also contribute to the progressive development of resistance to chemotherapeutics that in part may be a consequence of increased glucose catabolism. This is evident from studies demonstrating that stromal cell co-culture of leukemia cells induces the expression of uncoupling proteins that promote the switch to aerobic glycolysis and an accompanying induction of chemoresistance. In addition, the switch to glycolysis can promote chemoresistance by suppressing activation of BAX and BAD, pro-apoptotic signaling effectors that are regulated by glucose.[24,25]
In sum, altered metabolism, including increased glycolysis with enhanced glucose consumption, is consistently observed in various cancers. The Warburg effect has formed the basis of positron emission tomography (PET) scanning with fluorine-18 fluorodeoxyglucose (18F-FDG), which is now increasingly being used in the diagnosis and prognosis of various malignancies. PET exploits the increased glucose uptake by malignant cells compared with most normal cells; FDG uptake is, therefore, directly proportional to tumor burden. Figure 2 shows a PET scan of a patient with large B-cell lymphoma of the skin; the scan demonstrates numerous active metabolic lesions with increased uptake of glucose. Two large independent clinical myeloma studies demonstrated the early appearance of PET-positive lesions (in 76% of 192 patients tested), indicating an early reliance on elevated glucose metabolism[27,28]; more importantly, both studies demonstrated that the persistence and extent of PET positivity correlated with lower event-free survival,[27,28] underscoring the usefulness of targeting aberrant glucose utilization in myeloma.
Previous studies have demonstrated the utility of caloric restriction in inducing chemosensitization to a wide range of chemotherapeutics. It was shown that starvation induced sensitization to radio- or chemotherapy and led to extended survival in an in vivo glioma model of astrocytoma and glioblastoma multiforme. The same phenomenon was also demonstrated in a mouse neuroblastoma model in which fasting cycles and chemotherapy together resulted in long-term cancer-free survival. The same group went on to explore 4T1 breast cancer cells, in which it was seen that short-term starvation resulted in increased phosphorylation of the stress-sensitizing Akt and S6 kinases, and increased oxidative stress, caspase-3 cleavage, DNA damage, and apoptosis.
Given the diagnostic and prognostic usefulness of PET, there is a great potential for cancer therapeutics that target glucose utilization. The obstacle to targeting altered glucose metabolism is the identification of tumor-specific rate-limiting steps—and importantly, the metabolic plasticity that cells may engage in to bypass inhibition of these rate-limiting steps.
Targeting Glucose Metabolism
Recent research has focused on ways to target the increased dependence of cancer cells on glycolysis, with the objective of developing new chemotherapeutic and chemosensitizing treatments. Several glycolytic targets are currently being explored. This list includes targets such as HK, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-2; PFKFB3), and PDK4, with newer investigations exploring such avenues as enzymes like PKM2, an isoenzyme of pyruvate kinase. PKM2 exhibits lower enzymatic activity compared to PKM1 and is normally expressed in embryonic and proliferating cells. However, tumor cells switch to expression of the PKM2 isoform that promotes the Warburg effect and glycolysis, in part by constraining oxidative metabolism, as well as by translocating to the nucleus to promote the expression of genes associated with tumor cell proliferation. Several compounds that target HK have been developed and tested, including 3-bromopyruvate, lonidamine, and 2-deoxyglucose.[37-39] However, dose-limiting toxicities and administration issues have prevented these compounds from moving ahead in clinical trials.
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