Nathan Patrick Ward
Approved: 7 November 2016
The robust glycolytic metabolism of glioblastoma multiforme (GBM) has proven them susceptible to increases in oxidative metabolism induced by the pyruvate mimetic dichloroacetate (DCA). Recent reports demonstrate that the anti-diabetic drug metformin enhances the damaging oxidative stress associated with DCA treatment in cancer cells. We sought to elucidate the role of metformin’s reported activity as a mitochondrial complex I inhibitor in the enhancement of DCA cytotoxicity in the VM-M3 model of GBM. We demonstrated that metformin potentiated DCA-induced superoxide production and that this was required for enhanced cytotoxicity towards VM-M3 cells with the combination. Similarly, rotenone enhanced oxidative stress resultant from DCA treatment and this too was required for the noted augmentation of cytotoxicity. Adenosine monophosphate kinase (AMPK) activation was not observed with the concentration of metformin required to enhance DCA activity. Moreover, addition of an activator of AMPK did not enhance DCA cytotoxicity, whereas an inhibitor of AMPK heightened the cytotoxicity of the combination. We also show that DCA and metformin reduce tumor burden and prolong survival in VM-M3 tumor-burdened mice as individual therapies. In contrast to our in vitro work, we did not observe synergy between DCA and metformin in vivo. Our data indicate that metformin enhancement of DCA cytotoxicity is dependent on complex I inhibition. Particularly, that complex I inhibition cooperates with DCA-induction of glucose oxidation to enhance cytotoxic oxidative stress in VM-M3 GBM cells. This work supports further investigation and optimization of a DCA/metformin combination as a potential pro-oxidant combinatorial therapy for GBM.
Keywords: Cancer metabolism, mitochondrial glucose oxidation, complex I inhibition, oxidative stress, DCA, metformin
Copyright © 2017, Nathan P. Ward
Herein we provide a review of the metabolic programs employed by tumors to meet the biosynthetic requirements of tumorigenesis. The metabolism of tumors is intricately linked to the hallmarks of the disease and provides cancer cells with a survival advantage in response to the stresses imposed by the tumor microenvironment. An understanding of the metabolic characteristics of tumors provides a basis for rational targeting of these metabolic dependencies as a therapeutic strategy. Current approaches in targeting cancer metabolism are also discussed in this chapter.
Altered Energy Metabolism
Cancer is traditionally considered a genetic disease, characterized by genomic instability and frequent mutation that cooperate to promote a distinct cellular environment that permits unbridled proliferation (1). Genomic sequencing of tumors has identified a multitude of drug targetable mutations that have driven research and pharmaceutical development. Unfortunately, the promise of encouraging pre-clinical findings has not often translated to clinical efficacy. This has driven the field to consider additional hallmarks of tumor development and disease progression and devise alternative strategies for cancer management (1).
Resultant from this initiative was a renewed appreciation for the distinct metabolic activity of tumors (2). Beyond the dysregulation of the cell cycle and loss of deoxyribonucleic acid (DNA) quality control that accompany cancer cell proliferation is a fundamental demand for biomass. An intricate network of metabolic pathways converges to generate the molecular building blocks required for biosynthesis (3). Cancer cell metabolism is wired in such a manner that allows for the continuous production of the nucleotides, proteins and lipid membranes necessary for proliferation whilst also generating the energy and reduction potential required for cell survival (4). The past decade of research on cancer metabolism has encompassed a methodological renaissance for characterizing the metabolic dependencies of cancer cells and the intersection between metabolism and tumor biology (5-8). Most importantly, this work has demonstrated that targeting cancer metabolism may be a sustainable therapeutic alternative for the management of the devastating disease.
The notion of peculiar metabolism in cancer is not a recent phenomenon. Otto Warburg first observed a distinct difference in the metabolism of tumors compared to normal tissue in the early 20th century (9). Warburg reported that tumors took up significantly more circulating glucose than normal tissue, and whereas very little lactate was generated by the normal tissue, Warburg calculated that 66% of the consumed glucose was converted to lactate by the tumor. This suggests that the tumors were predominantly fermenting glucose rather than respiring on the sugar.
Glucose is the predominant energy metabolite in the body, and is preferentially metabolized by most tissues. Upon entering the cell, glucose is metabolized to pyruvate through the Embden-Meyerhof, or glycolytic pathway. Typically, pyruvate is then imported into the mitochondria where it is fully oxidized to carbon dioxide (CO2) as long as oxygen, the final electron acceptor of the electron transport chain is not limiting. Tissues are adequately perfused under normal physiological conditions, which facilitates the delivery of oxygen and permits mitochondrial respiration of glucose. In the context of limiting oxygen, such as in muscle during vigorous exercise, pyruvate is fermented to lactate by lactate dehydrogenase (LDH).
What is remarkable about Warburg’s findings is that the tumors were reported to be well perfused and thus oxygen was not limiting (9). Hence, the tumors were preferentially fermenting pyruvate to lactate in an aerobic environment. This aerobic fermentation of glucose is now widely recognized as a hallmark phenotype of most cancers and is now termed the Warburg effect (10). In fact, the robust uptake of glucose by tumors is the basis for diagnostic fluorodeoxyglucose positron emission tomography (FDG-PET) scanning (11).
A reliance on glycolytic metabolism seems counterintuitive for robust proliferation from a bioenergetics perspective. Generating the biomass required for cell division depends in part on the potential energy stored in adenosine triphosphate (ATP), a byproduct of certain catabolic reactions. Glycolysis is rather energy inefficient, generating only 2 moles (mol) of ATP per mol of glucose, whereas the complete oxidation of glucose yields ~36 mol ATP/mol glucose. Yet, cancer cells that exhibit this Warburg metabolism do not suffer from an ATP deficit (12). The conversion of pyruvate to lactate by LDH is coupled to the oxidation of reduced nicotinamide adenine dinucleotide (NADH) to its oxidized form, NAD+ . The regeneration of NAD+ maintains a high cytosolic NAD+ /NADH ratio that permits rapid glycolytic flux, as the glycolytic enzyme glyceraldehyde phosphate dehydrogenase (GAPDH) requires NAD+ as a cofactor.
Based on his original observation, Warburg hypothesized that aerobic fermentation in tumors was a result of an irreversible insult to oxidative capacity that prevented cancer cells from deriving sufficient energy from oxidative metabolism (13). Evidence suggests that ATP production is not a necessary function of cancer mitochondria, however mitochondrial metabolism is critical for cancer cell proliferation (14). In principle, the abundant generation of lactate as a result of the Warburg effect could restrict the flux of pyruvate into the mitochondria, where it is readily metabolized to acetyl coenzyme A (acetyl-CoA) and CO2 via the pyruvate dehydrogenase (PDH) complex.
Acetyl-CoA is a critical carbon carrier that is utilized extensively in central carbon metabolism. Acetyl-CoA is required for continuous flux of the Citric Acid (TCA) cycle, which generates the reducing equivalents NADH and reduced Flavin adenine dinucleotide (FADH2). These reducing equivalents are oxidized by protein complexes in or associated with the inner mitochondrial membrane (IMM) in reactions that couple the release of electrons with the movement of protons (H+ ) from the mitochondrial matrix into the intermembrane space. The movement of these electrons through subsequent protein complexes, that collectively make up the electron transport chain (ETC), is also coupled to the movement of H+ across the IMM. The translocation of these H+ generates a proton motive force and membrane potential across the IMM. ATP synthase harnesses this proton motive force to couple the movement of H+ back into the matrix with the generation of ATP from adenine diphosphate (ADP) and inorganic phosphate (Pi).
In addition to providing the reducing equivalents for oxidative phosphorylation, the TCA cycle intermediates are important for the biosynthesis of critical macromolecules. Reduced flux of glucose carbon through the PDH complex would thus restrict TCA cycling and decrease the levels of TCA cycle intermediates. Cancer cells, especially in culture, have upregulated glutamine metabolism to compensate for deficits in glucose carbon flux through the TCA cycle (15). Glutamine is an anaplerotic amino acid that is converted to glutamate in the mitochondria by glutaminase (GS). Glutamate can then be deaminated to ⍺-ketoglutarate (⍺-KG), a TCA cycle intermediate. ⍺-KG can then contribute to the replenishment of subsequent intermediates through traditional flux through the cycle or be converted to the upstream metabolite, isocitrate, through isocitrate dehydrogenase 2 (IDH2)-mediated reductive carboxylation.
In certain tumor species, the branched-chain amino acids (BCAAs) leucine and valine can be used as anaplerotic substrates (5). Moreover, glucose carbon can enter the TCA cycle in a PDH-independent manner through pyruvate carboxylase (PC), which converts cytosolic pyruvate to oxaloacetate. This oxaloacetate is then converted to malate via malate dehydrogenase (MDH). Malate can be taken up into the mitochondria through the malate-aspartate shuttle and incorporate into the TCA cycle. Together, these pathways provide alternative means for maintaining TCA function.
The advent of isotope-labeled metabolite tracing has demonstrated that aerobic fermentation does not fully restrict glucose oxidation, rather the tracing of 13C-glucose metabolic flux shows concurrent fermentation and oxidation of glucose carbon in certain cancers (16). Cellular energy metabolism is dependent on the regulated movement of electrons between metabolic intermediates and enzymatic cofactors through a series of oxidative-reduction (redox) reactions. Recent evidence suggests that mitochondrial oxidation is critical for the cell proliferation independent of the generation of ATP.
Stimulation of ETC activity through oxidation of reducing equivalents promotes redox balance through regeneration of NAD+ and oxidized Flavin adenine dinucleotide (FAD+ ), which are critical electron acceptors. Electron acceptors are necessary for continuous metabolic flux, especially in the context of meeting the biosynthetic demands of rapid proliferation (17). Oxygen serves as the terminal electron acceptor in oxidative metabolism and this reduction of oxygen is considered the most vital aspect of mitochondrial oxidative metabolism for proliferating cells (18). The amino acid aspartate is also shown to serve as an essential electron acceptor for proliferation (17, 18).
Maintenance of the mitochondrial membrane potential (ΔΨm) is generally dependent on the continual regulated flux of electrons through the ETC resultant from oxidative metabolism. The preservation of ΔΨm is critical to the proliferative capacity of cells independent of its coupling to ATP production (19). In fact, cancer mitochondria are often hyperpolarized, suggesting inefficient flux of H+ back into the matrix for the purposes of ATP generation (20).
In addition to glucose, fatty acids can serve as a substrate for mitochondrial oxidative metabolism. The beta-oxidation of fatty acids (FAO) yields acetyl-CoA, which is incorporated into the TCA cycle, and NADH and FADH2 for electron transport and the potential generation of ATP. FAO is shown to be essential for survival and growth under conditions of metabolic stress (21). Certain haematopoietic malignancies exhibit increased FAO (22, 23). Diffuse large B cell lymphoma (DLBCL) appear to rely on FAO largely to maintain cellular ATP levels. Whereas leukemia cells often display enhanced FAO that is associated with preventing the toxic buildup of fatty acids (21). Additionally, some leukemia cells require FAO for maintenance of cytosolic redox balance in the form of citrate-dependent reduced nicotinamide adenine dinucleotide phosphate (NADPH) generation.
Maintenance of Redox Balance
As mentioned above, cellular metabolism is dependent on the coordinated movement of electrons through intermediate metabolites and the oxidation state of important electron carriers. Cells harness the reducing power of NADH and NAPDH for the catabolic and biosynthetic reactions necessary for growth and viability. The ratios of NAD+ /NADH and oxidized nicotinamide adenine dinucleotide phosphate (NADP+ )/NADPH are indicators of the redox state of the cell. Metabolic flux and the activity of bidirectional metabolic enzymes are dependent on the status of these ratios. The redox state of the cell is compartmentalized within organelles, as there are distinct metabolic mechanisms for regulating NAD+ /NADH and NADP+/NADPH in the cytosol and mitochondrial matrix for example. Yet, these are not completely independent of each other as there are mechanisms for the exchange of metabolites between compartments that facilitate alterations to these ratios.
Maintenance of the NAD+/NADH ratio is predominantly mediated in the cytosol through glycolysis and through the TCA cycle in the mitochondrial matrix. As previously mentioned, cancer cells exhibit enhanced LDH activity, which recycles the NADH generated through glycolysis to NAD+, facilitating the rapid glycolytic flux associated with Warburg metabolism (4). The shuttling of pyruvate between the cytosol and matrix links the NAD+/NADH pools of the two compartments and is tightly regulated in cancer (24).
NADPH provides the reducing power for biosynthesis and is a critical component of cellular antioxidant capacity, both of which will be thoroughly discussed later in this review. Cytosolic NADPH is generated through two enzymatic reactions in the pentose phosphate pathway (PPP), via the conversion of malate to pyruvate by malic enzyme (ME) and oxidation of isocitrate to ⍺-KG by IDH1. The exchange of citrate between the matrix and cytosol links the NADPH pools of the two compartments. Reductive carboxylation of glutamine is shown to contribute to the cytosolic pools of NADPH through citrate, which can be metabolized to oxaloacetate by citrate lyase and subsequently to malate via MDH. Ultimately, this citrate-derived malate is converted to pyruvate by ME, generating NADPH (25, 26). 13C-glutamine tracing demonstrated that a significant fraction of mitochondrial NADPH is derived from folate metabolism (27, 28). Additional contributing factors to the matrix NADPH pool are IDH2 and the IMMassociated enzyme nicotinamide nucleotide transhydrogenase (NNT), which harnesses the proton motive force across the IMM and the reducing power of NADH to generate NADPH.
Cellular redox state is also affected by oxidative stress, a natural byproduct of metabolism. Oxidative stress is caused by the generation of highly reactive free radical oxygen- or nitrogen-containing species (ROS, RNS) that exhibit an array of biological functions, both cell-sustaining and cytotoxic. For instance, electron transport is not a totally efficient process. Electrons can be prematurely released from the ETC to reduce molecular oxygen to superoxide anion (·O2 – ). This occurs on the matrix side of the IMM at complex I (NADH dehydrogenase) and complex II (succinate dehydrogenase) of the ETC. Additionally, ·O2 – can be generated on both the matrix- and intermembrane space (IMS)-facing sides of the IMM at complex III (coenzyme Q: cytochrome c oxidoreductase). ·O2 – can also be generated in the cytosol and matrix through an NADPH-dependent process catalyzed by NADPH oxidases (NOXs).
In the presence of nitric oxide (NO), a byproduct of arginine metabolism, superoxide contributes to the formation of the very reactive peroxynitrite ion (ONOO- ). Additionally, this ·O2 – can be dismutated to hydrogen peroxide (H2O2) by superoxide dismutases (SODs). H2O2 can subsequently be detoxified to water through a number of enzymatic systems. Peroxiredoxins (PRXs) undergo H2O2-mediated oxidation that initiates a catalytic cycle in which thioredoxin (TRX), thioredoxin reductase (TrxR) and NADPH cooperate to regenerate reduced PRXs. Glutathione peroxidases (GPXs) utilize reduced glutathione (GSH) to detoxify H2O2. Glutathione reductase then utilizes NADPH to convert the oxidized glutathione (GSSG) to GSH. Finally, catalase can also convert to H2O2 to water. Alternatively, in the presence of ferrous (Fe2+) or cupric (Cu+ ) ions, H2O2 can generate hydroxyl radical (·OH) through Fenton reactions.
Collectively, these detoxifying enzymes contribute to the antioxidant capacity of the cell, which prevents the accumulation of the free radicals that potentiate oxidative stress. Transcriptional regulation of these enzymes is controlled by the master regulator of cellular antioxidant machinery, nuclear factor-like 2 (Nrf2). Nrf2 activity is stimulated by oxidative stress, resulting in the upregulation of a host of detoxifying enzymes and a metabolic program that boosts antioxidant capacity. The balance between ROS and RNS generation and antioxidant detoxification greatly influences cell function and viability and is a critical component of tumorigenesis (29).
Consequences of Cancer Metabolism
Tumors exist as a heterogeneous population of cells that are under severe selection pressures that drive an evolutionary response. The mutations acquired during tumorigenesis must either confer a survival advantage or passively permit unbridled proliferation (1, 2). Tumors are subject to the constraints of natural selection and those mutations that reduce cancer cell fitness are ultimately selected against (30). Given that altered cellular metabolism is a consistent hallmark of cancer, there must be a survival benefit associated with the metabolism of neoplastic cells. Herein, I describe the consequences of cancer metabolism that provide a survival benefit to cancer cells and contribute to disease progression.