The abnormal conditions present in the tumor microenvironment play a major role in determining the metabolic phenotype of tumor cell (Lunt et al, 2009). The relationship between the tumor microenvironment and cellular metabolism is not one of simple cause-and-effect in which biochemical conditions in the tumor influence cellular metabolism. Because metabolite concentrations are governed by both supply via the vasculature and demand by the tissue (or vice versa), changes in metabolism of both the tumor and normal stromal cell have a profound effect on local microenvironmental conditions. Because of the dynamic nature of the tumor microenvironment, it is likely that the metabolic phenotype of tumor cells can change to adapt to the prevailing local conditions.
Critical phenotypes characteristic of tumor cells are caused by a series of mutational events that combine to alter multiple cellular signaling pathways affecting proliferation and survival. Thousands of point mutations, translocations, amplifications, and deletions have been detected in cancer cells and the mutational spectrum can differ even among seemingly identical tumors. Bioinformatic analyses of large data sets have suggested that cancer-related driver mutations affect a dozen or more core signaling pathways and processes responsible for tumorigenesis, many of which are described in other chapters. Multiple molecular mechanisms, both intrinsic and extrinsic, converge to alter cellular metabolism to support growth and survival of the cancer cells. Furthermore, some of these metabolic alterations appear to be absolutely required for malignant transformation.
Genetic alterations to oncogenes and tumor suppressors (see Chap. 7) and the cellular response to extracellular microenvironmental conditions both contribute to the development of an abnormal metabolic phenotype. These metabolic alterations provide support for 3 of the most basic needs of dividing cells (Fig. 12–11):
Determinants of the tumor metabolic phenotype. The metabolic phenotype of tumor cells is controlled by intrinsic genetic mutations and external responses to the tumor microenvironment. Oncogenic signaling pathways controlling growth and survival are often activated by loss of tumor suppressors (such as p53) or activation of oncogenes (such as PI3K). The resulting altered signaling modifies cellular metabolism to match the requirements of cell division. Abnormal microenvironmental conditions such as hypoxia, low pH, and/or nutrient deprivation elicit responses from tumor cells that further affect metabolic activity. These adaptations serve to optimize tumor cell metabolism for proliferation by providing appropriate levels of energy in the form of adenosine triphosphate (ATP), biosynthetic capacity, and maintenance of balanced reduction-oxidation (redox) status.
Rapid generation of energy in the form of adenosine triphosphate (ATP);
Biosynthesis of the macromolecular building blocks required to generate daughter cells; and
Maintenance of appropriate cellular reduction-oxidation (redox) status required to prevent excessive oxidative damage and to provide reducing power for anabolic enzymatic reactions.
To meet these needs, cancer cells acquire alterations to the metabolism of all 4 major classes of macromolecules: carbohydrates, proteins, lipids, and nucleic acids. Many similar metabolic alterations are also observed in rapidly proliferating normal cells, where they represent appropriate responses to physiological growth signals (Newsholme et al, 1985; Vander Heiden et al, 2009). However, for cancer cells, these adaptations represent responses to the inappropriate growth and survival signals generated by acquired genetic mutations. Furthermore, these metabolic alterations must be implemented in a stressful and dynamic tumor microenvironment, where concentrations of critical nutrients and waste products, such as glucose, glutamine, oxygen, and lactate, are spatially and temporally heterogeneous (see Sec. 12.2).
12.3.1 Aerobic Glycolysis (The Warburg Effect)
In addition to the ATP required to maintain normal cellular homeostasis, proliferating tumor cells must also generate the energy required to support cell division. Furthermore, tumor cells must evade the checkpoint controls that would normally block proliferation under the stressful metabolic conditions characteristic of the abnormal tumor microenvironment. During the course of transformation and progression, these selective pressures result in a reprogramming of core metabolic pathways. The best characterized, and most universal metabolic phenotype observed in tumor cells is an increase in glycolysis. In this process, glucose is converted to lactate and secreted from the cell, rather than being completely oxidized to CO2 via oxidative phosphorylation in the mitochondria (see Fig. 12–10). This is referred to as the Warburg effect, after Otto Warburg, who first described the phenomena (Warburg, 1956). This mode of glucose utilization and ATP production is similar to the Pasteur effect, which is the normal shift toward glycolytic ATP production that occurs during periods of oxygen limitation. However, the Warburg effect is observed in tumor cells even under normal oxygen concentrations (Warburg, 1956). As a result, unlike most normal cells, many transformed cells derive a substantial amount of their energy from aerobic glycolysis, converting the majority of incoming glucose into lactate rather than metabolizing it in the mitochondria via oxidative phosphorylation. Although ATP production by glycolysis can be more rapid than by oxidative phosphorylation, it is far less efficient in terms of ATP generated per molecule of glucose consumed. This shift therefore demands that tumor cells increase their rate of glucose uptake so as to meet their energy requirements.
Aerobic glycolysis is a common feature of many tumors, but there remains debate about the selective advantage that glycolytic metabolism provides to proliferating tumor cells. Initial work by Warburg and others focused on the concept that tumor cells develop defects in mitochondrial function, and that aerobic glycolysis was a necessary adaptation to cope with a decrease in ATP generation by oxidative phosphorylation. However, functional mitochondrial defects in tumors are relatively rare (Frezza and Gottlieb, 2009), and most tumor cells retain the capacity for oxidative phosphorylation and continue to consume oxygen at rates similar to those observed in normal tissues (Weinhouse, 1976). In fact, mitochondrial function is critical for transformation in some model tumor systems (Funes et al, 2007). Other explanations for the shift toward aerobic glycolysis include the concept that glycolysis has the capacity to generate ATP at a higher rate compared to oxidative phosphorylation, so it could be advantageous as long as glucose supplies are not limited. Alternatively, it has been proposed that glycolytic metabolism arises as an adaptation to the hypoxic conditions that develop during the early avascular phase of tumor growth, as it allows for ATP production and maintenance of bioenergetic homeostasis in the absence of oxygen. Adaptation to the resulting acidic microenvironment caused by excess lactate and carbonic acid production may further drive the evolution of the glycolytic phenotype (Gillies et al, 2008).
Another explanation for increased aerobic glycolysis in tumors is that this pathway serves to balance the need of proliferating cells for energy with the equally important need for macromolecular building blocks and maintenance of redox homeostasis (see Sec. 12.3.4). Several important subsidiary biosynthetic metabolic pathways rely on glycolytic intermediates, including the hexosamine pathway, uridine diphosphate (UDP)-glucose synthesis, glycerol synthesis, and the pentose phosphate pathway. The pentose phosphate pathway is also a key means of producing nicotinamide adenine dinucleotide phosphate (NADPH), which provides reducing power for many biosynthetic reactions and maintenance of antioxidant systems. In addition, by removing the burden of energy production from the mitochondria, high rates of aerobic glycolysis allow the tricarboxylic acid (TCA) cycle to act as a hub for biosynthesis of fatty acids and amino acids rather than as a site of energy generation.
The reliance of cancer cells on increased glucose uptake has proven useful for tumor detection and monitoring. The glycolytic phenotype of tumor cells serves as the basis for clinical [18F] fluorodeoxyglucose positron emission tomography (FDG-PET) imaging (see Chap. 14, Sec. 14.3.3). FDG-PET employs a radioactive glucose analog that is taken up by cells along with glucose, but cannot be further metabolized and is trapped intracellularly. Imaging of fluorodeoxyglucose (FDG) detects regions of high glucose uptake, and allows identification and monitoring of many tumor types, as well as generating data regarding the importance of glucose as a fuel for malignancies (Jadvar et al, 2009). Attempts to exploit the glycolytic phenotype of tumor cells for therapy have been less successful: There have been attempts to block aerobic glycolysis, using compounds such as 2-deoxyglucose, but effective therapeutic strategies have yet to be devised. Several novel therapeutic approaches targeting the glycolytic process are undergoing evaluation, including the inhibition of lactate dehydrogenase, and the inactivation of the monocarboxylate transporters (MCT) responsible for conveying lactate across the plasma membrane (see Fig. 12–10) (Le et al, 2010).
Although aerobic glycolysis is the most well documented metabolic phenotype of tumor cells, it is not a universal feature of human cancers (Moreno-Sanchez et al, 2007). Moreover, even in relatively glycolytic tumors, oxidative phosphorylation is not completely shut down. Clinical FDG-PET data, as well as in vitro and in vivo experimental studies show that tumor cells are capable of using alternative fuel sources. It is estimated that up to 30% of human tumors are considered FDG-PET–negative depending on the tumor type (Jadvar et al, 2009). Amino acids, fatty acids, and even lactate have been shown to act as fuels for tumor cells in certain genetic and microenvironmental contexts.
Much of the work on cancer metabolism has focused on rapidly proliferating tumor models and cells grown in vitro. Because the rate of cell division can alter the metabolism of normal cells and tissues, some of the metabolic properties associated with malignancy may simply relate to rapid cell proliferation. It is unclear to what extent the metabolic phenotypes of tumor cells, including the Warburg effect, will prove to be important in low-grade slow-growing tumors, where metabolic demands may not be as extreme. Future clinical data describing the metabolic profiles of human tumors will be required to determine which metabolic alterations are most prevalent in specific tumor types and how they relate to changes in common oncogenic signaling pathways.
12.3.2 Regulation of Aerobic Glycolysis
18.104.22.168 The PI3K pathway
The PI3K (phophatidylinositol-3 kinase) pathway is one of the most commonly altered signaling pathways in human cancers (see Chap. 7, Sec. 7.5.4 and Chap. 8, Sec. 8.2.5). This pathway is activated by mutations in tumor-suppressor genes such as PTEN, mutations in components of the PI3K complex itself, or by aberrant signaling from receptor tyrosine kinases (Wong et al, 2010). Once activated, the PI3K pathway not only provides strong growth and survival signals to tumor cells but also has profound effects on their metabolism.
The best-studied effector molecule downstream of PI3K is AKT1, also known as protein kinase B (PKB). Upon activation, AKT1 exerts its effects by phosphorylating key signaling substrates involved in proliferation, survival and metabolism. AKT1 is an important driver of the tumor glycolytic phenotype and stimulates ATP generation via multiple mechanisms, ensuring that cells have the bioenergetic capacity required to respond to parallel growth signals. AKT1 stimulates glycolysis by increasing the expression and membrane translocation of glucose transporters, and by phosphorylating key glycolytic enzymes such as hexokinase and phosphofructokinase-2 (Robey and Hay, 2009). The elevated and prolonged AKT1 signaling that is associated with transformation inhibits the forkhead box subfamily O (FOXO) transcription factors, resulting in a host of complex transcriptional changes that also increase glycolytic capacity (Khatri et al, 2010). Finally, AKT1 strongly activates mTOR kinase by phosphorylating and inhibiting its negative regulator, tuberous sclerosis 2 (TSC2) (Robey and Hay, 2009). The mTOR protein functions as a key metabolic integration point, coupling growth signals to nutrient availability (see Sec. 12.2.3). Normally, activated mTOR stimulates protein and lipid biosynthesis and cell growth in response to sufficient nutrient and energy conditions and growth signals, but it is often constitutively activated in tumors (Guertin and Sabatini, 2007). At the molecular level, mTOR directly stimulates messenger RNA (mRNA) translation and ribosome biogenesis, and indirectly causes other metabolic changes by activating transcription factors such as HIF-1 (see Sec. 12.2.3). The subsequent HIF-1–dependent metabolic changes are a major determinant of the glycolytic phenotype downstream of PI3K, AKT1, and mTOR (see Fig. 12–10).
The HIF-1 and HIF-2 complexes are the major transcription factors responsible for changes in gene expression during the cellular response to hypoxia (see Sec. 12.2.3). Although these 2 transcription factors transactivate an overlapping set of genes, the effects on central metabolism have been better characterized for HIF-1. In addition to its stabilization under hypoxic conditions, HIF-1 can be activated under normoxic conditions by oncogenic signaling pathways, including PI3K, and by mutations in tumor-suppressor proteins such as VHL (Kaelin, 2008), succinate dehydrogenase (SDH), and fumarate hydratase (FH) (King et al, 2006), which cause defects in its normal degradation. HIF-1 amplifies the transcription of genes encoding glucose transporters and most of the glycolytic enzymes, increasing the capacity of the cell to perform glycolysis (Semenza, 2010). In addition, HIF-1 activates the pyruvate dehydrogenase kinases (PDKs), which phosphorylate and inactivate the mitochondrial pyruvate dehydrogenase complex, thereby reducing the flow of glucose-derived pyruvate into the TCA cycle (Papandreau et al, 2006; see Fig. 12–10). This reduction in pyruvate flux into the TCA cycle decreases the rate of oxidative phosphorylation and oxygen consumption, reinforcing the glycolytic phenotype and sparing oxygen under hypoxic conditions. Inhibitors of HIF-1 or the PDKs might reverse some of the metabolic effects of tumorigenic HIF-1 signaling, and several such candidates, including the PDK inhibitor dichloroacetic acid (DCA), are under evaluation for their therapeutic utility.
The oncogenic transcription factor MYC also has a number of important effects on cell metabolism (see Chap. 7, Sec. 7.5.2). MYC can collaborate with HIF in the activation of a number of glucose transporters, glycolytic enzymes, lactate dehydrogenase A, and PDK1 (Dang et al, 2008). However, unlike HIF, MYC also activates the transcription of targets that increase mitochondrial function, especially the metabolism of glutamine, which is discussed below.
22.214.171.124 Adenosine Monophosphate-Activated Protein Kinase
Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a critical sensor of energy status and plays an important role in cellular responses to metabolic stress, coupling energy status to growth signals. AMPK opposes the effects of AKT1 by acting as a potent inhibitor of mTOR (see Fig. 12–10). The AMPK complex thus functions as a metabolic checkpoint, regulating the cellular response to available metabolic energy. AMPK becomes activated in response to an increased AMP:ATP ratio, and is responsible for shifting cells to an oxidative metabolic phenotype and inhibiting cell proliferation (Shackelford and Shaw, 2009). Tumor cells must overcome this checkpoint in order to proliferate in response to activated growth signaling pathways, especially in a nutrient deficient microenvironment. A number of oncogenic mutations and signaling pathways can suppress AMPK signaling (Shackelford and Shaw, 2009), effectively uncoupling fuel signals from growth signals, and allowing tumor cells to continue to divide under abnormal nutrient conditions. This uncoupling permits tumor cells to respond to inappropriate growth signaling pathways activated by oncogenes and the loss of tumor suppressors. Accordingly, many cancer cells exhibit a loss of appropriate AMPK signaling, which may also contribute to their glycolytic phenotype.
Given the role of AMPK, it is not surprising that the gene which encodes liver kinase B1 (LKB1), the upstream kinase necessary for AMPK activation, has been identified as a tumor-suppressor gene (see Fig. 12–10). Inherited mutations in LKB1 are responsible for Peutz-Jeghers syndrome (Jenne et al, 1998). This syndrome is characterized by the development of benign gastrointestinal and oral lesions, and an increased risk of developing a broad spectrum of malignancies. LKB1 is also frequently mutated in sporadic cases of non–small cell lung cancer and cervical carcinoma. Recent evidence suggests that LKB1 mutations are tumorigenic as a consequence of the resulting decrease in AMPK signaling and loss of mTOR inhibition (Shackelford and Shaw, 2009). The loss of AMPK signaling permits the activation of mTOR and HIF-1, and therefore may also support the shift toward glycolytic metabolism. Clinically, there is interest in evaluating whether AMPK agonists can be used to recouple fuel and growth signals in tumor cells and shut down cell growth. Two such agonists are the commonly used antidiabetic drugs metformin and phenformin.
Although the transcription factor and tumor-suppressor p53 is best known for its functions in the DNA damage response (DDR) and apoptosis pathways (see Chap. 5, Sec. 5.4 and Chap. 7, Fig. 7–12), it is also an important regulator of metabolism (Vousden and Ryan, 2009). p53 activates the expression of hexokinase II, which converts glucose to glucose-6-phosphate (G6P). G6P then either enters the glycolytic pathway to produce ATP, or enters the pentose phosphate pathway (PPP), which supports macromolecular biosynthesis by producing reducing potential in the form of reduced NADPH and/or ribose building blocks for nucleotide synthesis. p53 inhibits the glycolytic pathway by upregulating the expression of p53-induced glycolysis and apoptosis regulator (TIGAR), an enzyme that decreases levels of the glycolytic activator fructose-2,6-bisphosphate (see Fig. 12–10) (Vousden and Ryan, 2009). Wild-type p53 also acts to support the expression of PTEN, which inhibits the PI3K pathway, thereby suppressing glycolysis (Stambolic et al, 2001). Furthermore, p53 promotes oxidative phosphorylation by activating the expression of SCO2, which is required for assembly of the cytochrome c oxidase complex of the electron transport chain (Matoba et al, 2006). Thus, the loss of p53 may also be a major force behind the acquisition of the glycolytic phenotype.
Pyruvate kinase (PK) catalyzes the final, and ATP-generating step of glycolysis, in which phosphoenolpyruvate (PEP) is converted to pyruvate (Mazurek et al, 2005). Multiple isoenzymes of PK exist in mammals: type L, found in the liver and kidneys; type R, expressed in erythrocytes; type M1, found in muscle and brain; and type M2, present in self-renewing cells such as embryonic and adult stem cells (Mazurek et al, 2005). PKM2 is also overexpressed by many tumor cells, and PKM2 expression by lung cancer cells was found to confer a tumorigenic advantage over cells expressing the PKM1 isoform (Christofk et al, 2008). While PKM1 is highly active, and would be expected to efficiently promote glycolysis and rapid energy generation, the tumor associated PKM2 is characteristically found in a less active state (Mazurek et al, 2005; Christofk et al, 2008). Initially, this observation seemed paradoxical: PKM2 represented a tumor-specific glycolytic enzyme that slowed ATP generation and antagonized the Warburg effect. However, the switch to PKM2 provides an advantage to tumor cells, because by slowing the last step of glycolysis, it allows glucose-derived carbohydrate metabolites to be diverted to other metabolic pathways. Interestingly, the oncoprotein MYC has been found to promote the expression of PKM2 over PKM1 by modulating exon splicing (David et al, 2010). MYC upregulates the expression of heterogeneous nuclear ribonucleoproteins (hnRNPs), which bind to PK mRNA, leading to preferential production of PKM2.
Although glutamine is not an essential amino acid, cell culture medium must be supplemented with high concentrations of glutamine in order to support robust cell proliferation, and some cultured tumor cell lines derive the majority of their ATP from glutamine catabolism (Reitzer et al, 1979). More recently, it was found that oncogenic transformation can stimulate glutamine uptake and catabolism (glutaminolysis) and that many tumor cells are critically dependent on this amino acid (Wise et al, 2008). After glutamine enters the cell via specific plasma membrane transporters, glutaminase enzymes remove an ammonium ion, converting it to glutamate, which has several fates (Fig. 12–12). Glutamate can be converted directly into glutathione (GSH) by the enzyme glutathione cysteine ligase (GCL). Reduced GSH is one of the most abundant antioxidants found in mammalian cells, and is vital to controlling the redox state of all subcellular compartments. Glutamate can also be converted to α-ketoglutarate (α-KG) and enter the TCA cycle. This process, termed anaplerosis, supplies the alternative carbon input required for the TCA cycle to act as a biosynthetic hub and permits the production of other amino acids and fatty acids. Some glutamine-derived carbon can exit the TCA cycle as malate and serve as a substrate for malic enzyme 1 (ME1) which produces NADPH (DeBerardinis et al, 2007). The mechanisms regulating the fate of glutamine in tumor cells are not completely understood, and it is likely that genetic background and microenvironmental factors play a role.
Glutamine metabolism. Glutamine enters cells via specific transporters (SLC5A1 and SLC5A7) and is converted to glutamate by the enzyme glutaminase (GLS). Glutamate can be converted to glutathione so as to combat oxidative stress or enter the TCA cycle as α-ketoglutarate so as to provide ATP and macromolecular building blocks for cell growth and division. Key products of glutamine metabolism are shown in bold. Oncogenic proteins, including MYC, can enhance this process at several levels and render tumor cells highly dependent on exogenous glutamine.
One factor that plays a major role in regulating glutaminolysis is the oncogene MYC (see Chap. 7, Sec. 7.5.2). Thus MYC promotes not only proliferation but also the production of the macromolecules and reducing power required for cell growth and division. MYC increases glutamine uptake by directly increasing the expression of the glutamine transporters SLC5A1 and SLC7A1 (Gao et al, 2009). Furthermore, MYC indirectly increases the level of glutaminase 1 (GLS1), the key first enzyme of glutaminolysis, by repressing the expression of microRNAs-23a/b, which function to inhibit GLS1 (see Chap. 2, Sec. 2.4.3) (Gao et al, 2009). It is now clear that MYC supports antioxidant capacity by driving the production of NADPH via the PPP, by promoting the PKM2 isoform as described above, and also by increasing the synthesis of GSH through glutaminolysis. New techniques for measuring glutamine and its metabolites should soon permit the detailed examination of glutamine metabolism and MYC expression in tumors in patients.
12.3.4 Metabolic Alterations Support Balanced Redox Status
Redox status refers to the balance of the reduced versus the oxidized state of a biochemical system. In biological systems, this balance is influenced by the level of reactive oxygen species (ROS) relative to the capacity of antioxidant systems to eliminate ROS, as well as the relative concentrations of key substrates involved in oxidation reduction reactions. ROS are a diverse class of molecules produced in all cells as a normal byproduct of metabolic processes. ROS are heterogeneous in their chemical properties and cause a number of effects, depending on their concentrations. At low levels, ROS can act as signaling molecules to increase cell proliferation and survival through post-translational modification of kinases and phosphatases (Lee et al, 2002). Production of these low levels of ROS is required for homeostatic signaling events. At moderate levels, ROS induce the expression of stress-responsive genes downstream of transcription factors such as HIF-1α and NRF2, which trigger the expression of proteins providing prosurvival signals and antioxidant defense mechanisms (Gao et al, 2007). However, at high levels, ROS can overwhelm antioxidant systems, and damage macromolecules, including DNA, proteins, and lipids. Cells counteract the detrimental effects of ROS by producing antioxidant molecules such as reduced GSH and thioredoxin (TRX), along with enzymes such as superoxide dismutase and catalase. These molecules act to reduce excessive ROS so as to prevent irreversible cellular damage. Several of these antioxidant systems, including GSH and TRX, rely on the reducing power of NADPH to maintain their activities. In highly proliferative tumor cells, ROS regulation is critical because of the presence of oncogenic mutations that promote aberrant metabolism and protein translation, resulting in elevated rates of ROS production. Transformed cells counteract the accumulation of ROS by further upregulating antioxidant systems, creating a dynamic equilibrium between high levels of ROS production and high levels of antioxidant molecules (Trachootham et al, 2009; Fig. 12–13).
Relationship between ROS level and cancer. The impact of ROS on cell fate depends on ROS levels. Low levels of ROS provide a beneficial effect, supporting cell proliferation and survival pathways. However, once ROS levels become excessively high, they cause detrimental oxidative stress that can lead to cell death. To counter such oxidative stress, cells employ antioxidant systems that prevent ROS from accumulating to high levels. In cancer cells, aberrant metabolism, protein translation, and microenvironmental conditions generate abnormally high ROS levels. Through additional mutations and adaptations, cancer cells exert tight regulation of ROS and antioxidants in such a way that the cells survive and ROS are reduced to moderate levels.
126.96.36.199 Rb, PTEN, and p53 Contribute to Redox Maintenance
Fully transformed tumor cells alter their metabolic pathways and regulatory mechanisms so that ROS and antioxidants are tightly controlled and maintained at elevated levels compared to normal cells. However, during the process of tumorigenesis, the initial loss of tumor suppressors may cause cells to become overloaded with the products of aberrant metabolism and lose control of redox balance. Several mechanisms downstream of commonly mutated tumor suppressors may contribute to increased oxidative stress (Fig. 12–14). For example, when the tumor-suppressor TSC2 is deleted, mTOR becomes hyperactive. This mTOR activity leads to an upregulation of protein translation and increased ROS production (Ozcan et al, 2008). Data from experimental systems indicates that cells lacking retinoblastoma (Rb) tumor-suppressor function, which normally participates in the antioxidant response, are more sensitive to apoptosis as a consequence of this cellular stress (Li et al, 2010). Similar results have been seen with loss of PTEN, where increased activation of AKT leads to FOXO transcription factor inactivation and increased oxidative stress because of a reduction in the antioxidant defense molecules normally maintained by FOXO (Nogueira et al, 2008).
Tumor suppressors influence oxidative stress. Loss of the tumor supressors tuberous sclerosis 1 (TSC), phosphatase and tensin homolog (PTEN), p53, and retinoblastoma 1 (Rb), shaded in gray, leads to increased oxidative stress by increasing ROS production or by preventing the induction and maintenance of appropriate antioxidant defense mechanisms. TSC inhibits the mammalian target of rapamycin (mTOR), reducing the oxidative stress resulting from metabolism associated with protein translation. PTEN inhibits the ability of AKT1 to inhibit the FOXO transcription factor, which also increases oxidative stress. p53 can bolster antioxidant defenses by increasing the activity of the NRF2 antioxidant trascription factor via the p21 protein, and by stimulating glutathione production by upregulating glutaminase 2 (GLS2). The oncogenic DJ1 protein can contribute to both of these pathways by inhibiting PTEN, and by helping to stabilize NRF2. The Rb tumor suppressor enhances antioxidant defenses via a number of downstream mechanisms.
The tumor-suppressor p53 may promote oxidative stress during the induction of apoptosis (see Chap. 9, Sec. 9.4.3 and Fig. 9–12), yet it also plays a significant role in reducing oxidative stress as a defense mechanism. Glutaminase 2 (GLS2) is upregulated by p53 and drives de novo GSH synthesis (Suzuki et al, 2010). Furthermore, via the p53 target gene CDK inhibitor 1A (CDKN1A), which encodes p21 (see Chap. 9, Sec. 9.2.2), p53 promotes the stabilization of the NRF2 transcription factor (see Fig. 12–14) (Chen et al, 2009). NRF2 is an important antioxidant transcription factor that upregulates the expression of several antioxidant and detoxifying molecules. Loss of p53 in a cancer cell inactivates this redox maintenance mechanism. Thus it might be possible to exploit clinically loss-of-function p53 mutations or other tumor-suppressor genes by applying additional oxidative stress, since malignant cells might be selectively killed.
Much of our understanding of ROS and oxidative stress has emerged from the field of neurodegenerative disease. Similar mechanisms maintain appropriate redox status in both neurons and cancer cells. One protein involved in preventing neurodegeneration that has been investigated in the context of cancer is DJ1 (also known as PARK7). Similar to p21, DJ1 acts to stabilize NRF2 and thereby promotes antioxidant responses (Clements et al, 2006). DJ1 is mutated and inactive in several neurodegenerative disorders, most notably Parkinson disease (Gasser et al, 1997). In these disorders, it is believed that loss of DJ1 function leads to elevated oxidative stress in the brain, and increased neuronal cell death (see Fig. 12–14). DJ1 has also been described as an oncogene, and in patients with lung, ovarian, and esophageal cancers, high DJ1 expression in the tumor predicts a poor outcome (Kim et al, 2005). DJ1 stimulates AKT activity both in vitro and in vivo by regulating the function of the tumor-suppressor PTEN (see Fig. 12–14) (Kim et al, 2005). Although this function appears to be a logical candidate for the mechanism underlying a tumorigenic role of DJ1, high DJ1 expression may also promote tumor progression by reducing the oxidative stress caused by aberrant cell proliferation and thereby preventing ROS-induced cell death.
Supporting the notion that loss of DJ1 prevents appropriate redox control in cancers, an inverse correlation has been reported between cancer risk and Parkinson disease. A recent metaanalysis of patients with Parkinson disease determined that they have an approximately 30% lower risk of developing cancers compared with controls (Bajaj et al, 2010). The lower risk was associated with several different cancer types, including lung, prostrate, and colorectal cancers. Additional investigation of the cancer risk of patients with other neurodegenerative disorders may provide key insights into potential therapeutic exploitation of the heightened need to maintain redox balance in a cancer cell.
12.3.5 Metabolic Oncogenes and Tumor Suppressors
Although many signaling pathways commonly altered in tumors have profound effects on cellular metabolism, few key metabolic enzymes themselves are consistently mutated. However, several metabolic enzymes have been shown to be important oncogenes and tumor suppressors.
188.8.131.52 Fumarate Hydratase and Succinate Dehydrogenase
The TCA cycle enzymes FH and SDH have been identified as classical tumor suppressors (see Chap. 7, Sec. 7.6). Loss-of-function mutations in these enzymes cause disruption of the TCA cycle and lead to hereditary cancer syndromes, predisposing patients to paraganglioma and pheochromocytoma in the case of SDH, and to leiomyoma and renal cell carcinoma in the case of FH (Selak et al, 2005). The biochemical mechanisms responsible for driving tumorigenesis in these situations are still being clarified, but they appear to involve the induction of a pseudohypoxic phenotype, caused by inhibition of the PHD enzymes responsible for causing the degradation of HIF (Selak et al, 2005). However, other mechanisms, including an increase in ROS production and alterations to other PHD substrates, may be involved. The restricted tumor spectrum observed in these syndromes indicates that the capacity of these mutations to cause cancer may be dependent on cellular context.
184.108.40.206 Isocitrate Dehydrogenases
Isocitrate dehydrogenase 1 and 2 (IDH1/2) have been identified as genes commonly mutated in glioma and acute myeloid leukemia (AML) (Mardis, 2009; Parsons et al, 2008). These genes normally function to regulate cellular redox status by producing NADPH during the conversion of isocitrate to α-KG in the cytoplasm and mitochondria, respectively. IDH1 and IDH2 are homologous, and structurally and functionally distinct from the nicotinamide adenine dinucleotide (NAD)-dependent enzyme IDH3, which functions in the TCA cycle to produce the reduced form of nicotinamide adenine dinucleotide (NADH) required for oxidative phosphorylation.
The IDH1 and IDH2 mutations associated with the development of glioma and AML are restricted to critical arginine residues required for isocitrate binding in the active site of the protein (R132 in IDH1 and R172 or R140 in IDH2) (Dang et al, 2009). Affected patients are heterozygous for these mutations, suggesting that these alterations cause an oncogenic gain-of-function. The spectrum of mutation differs in the 2 diseases, with the IDH1 R132H mutation predominating in gliomas (>90%), whereas a more diverse collection of mutations in both IDH1 and IDH2 are found in AML.
The specific mutations cause the IDH1 and IDH2 proteins to acquire a novel enzymatic activity that converts α-KG to 2-hydroxyglutarate (2-HG) in an NADPH-dependent manner (Dang et al, 2009; Gross et al, 2010). This change causes the mutated IDH1 and IDH2 enzymes to switch from NADPH production to NADPH consumption, with consequences for cellular redox balance. The product of the novel reaction, 2-HG, is a poorly understood metabolite normally present at low concentrations in cells and tissues. However, in patients bearing somatic IDH1 or IDH2 mutations, 2-HG builds up to high levels in glioma tissues, and in the leukemic cells and sera of AML patients and may be directly oncogenic (Dang et al, 2009; Gross et al, 2010).
Studies of IDH1 and IDH2 have established a new paradigm in oncogenesis: a driver mutation that confers a new metabolic enzymatic activity that produces a potential oncometabolite. The molecular mechanisms by which IDH1 and IDH2 mutations contribute to tumorigenesis are under investigation, as is the possibility that these mutant enzymes may be useful targets for therapy. Although IDH1 and IDH2 mutations are clearly powerful drivers of glioma and AML, they appear to be rare or absent in other tumor types, illustrating the importance of specific cellular context in understanding metabolic perturbations in cancer cells.
Autophagy has emerged as an important link between cellular metabolism and hypoxia in the microenvironment of solid tumors. Autophagy literally means "self-eating" and is a process that is responsible for the basal turnover (degradation) of long-lived cellular proteins. It occurs through generation of a double-membrane vesicle referred to as an autophagosome that captures cytoplasmic contents destined for degradation. The outer membrane of the autophagosome fuses with a lysosome to form an autolysosome, resulting in the degradation of the inner membrane and its contents by enzymes present within the lysosome. The degradation products (amino acids, lipids) are delivered back to the cytoplasm and can be used in essential metabolic processes. Autophagy occurs at a low basal rate in all cells, but can be strongly induced by particular forms of cell stress, including those often present in the microenvironment of tumors.
Autophagy serves at least 2 critical cellular functions that are relevant in cancer. First, it is important for the removal and degradation of damaged organelles and misfolded protein aggregates. This includes removal of damaged mitochondria that would otherwise "leak" electrons and produce ROS through a selective form of autophagy sometimes referred to as mitophagy. The ability to remove and degrade these otherwise toxic cellular components is thought to provide a tumor-suppressor function in some instances during early tumorigenesis. Indeed, mutations in genes, such as Beclin1, that regulate the initiation of autophagy have been observed in some types of cancer. Second, and perhaps more important, autophagy plays an essential metabolic function and is required to maintain cell survival during conditions of extreme metabolic stress. Autophagy is activated as part of a "starvation" response when cells are unable to import nutrients to sustain essential metabolic pathways. In this role, autophagy functions to degrade cytoplasmic components to provide essential products for sustaining cellular metabolism and energy homeostasis. This function is particularly important within hypoxic regions of solid tumors where maintaining energy homeostasis becomes critical for cell survival, and each of the HIF, UPR, and mTOR pathways described above are capable of stimulating rates of autophagy during hypoxia (Fig. 12–15). In some tumors, activation of growth-promoting oncogenes including K-ras is sufficient to render cells constitutively dependent on autophagy for their continued survival. Thus, in most advanced cancers, autophagy rates are markedly induced and function to promote cell survival. Consequently, autophagy and the genes/pathways that regulate it, have emerged as new therapeutic targets. Current clinical strategies for inhibiting autophagy include the use of agents such as hydroxychloroquine that disrupt lysosomal pH regulation and thus prevent autolysosome formation and degradation of captured cytoplasmic content.
Hypoxia and autophagy. Immunostaining of a head and neck cancer xenograft reveals colocalization of hypoxia (green; pimonidazole immunohistochemistry) and autophagy (red; LC3 immunohistochemistry). Blood vessels are shown in blue; N = necrosis. Activation of autophagy can protect hypoxic and other tumor cells experiencing metabolic stress by promoting energy homeostasis. (From Rouschop et al, 2010.)
The growth of tumors is dependent on the rate of proliferation and of death of the cells within them. In many human tumors, the rate of cell production is only slightly higher than the rate of cell death and many cells may not be actively cycling, so that the median doubling time of tumors (typically about 2 months for common human solid tumors) is much longer than the cell cycle time of the proliferating tumor cells (typically about 2 to 3 days).
Factors that influence the rates of proliferation and cell death in tumors include nutrient molecules in the microenvironment, which, in turn, depend on angiogenesis and the expansion of the vascular network of the tumor, and the molecular signals that are influenced by endogenous and exogenous factors.
Tumor cells grow within a unique microenvironment characterized by deficiencies in vasculature and vascular function. This results in the generation of highly heterogeneous regions where tumor cells are exposed to elevated levels of waste products (lactate), low pH, high IFP, and hypoxia. Each of these features can influence tumor biology and tumor response to treatment in adverse ways.
Mutations in oncogenes and tumor-suppressor genes affect the core metabolism of tumor cells, reengineering it to support cell growth and division. Such metabolic alterations help to balance 3 critical requirements of proliferating cells: supply of energy in the form of ATP; supply of macromolecular building blocks for cell growth and division; and maintenance of redox homeostasis. The unique and stressful microenvironmental conditions in solid tumors further distort the metabolic phenotype, affecting progression, treatment response, and patient outcome.
Tumor hypoxia, in particular, leads to a series of biological changes that can promote enhanced malignancy primarily through the action of HIF transcription factors driving several oxygen-sensitive signaling pathways. In addition, very low levels of oxygen induce the unfolded protein response to reduce metabolic demand under conditions of severe energy "starvation."
Autophagy is a further important mechanism of cell survival in the adverse microenvironment of solid tumors. It is activated as part of the "starvation" response when cells are unable to obtain nutrients to sustain essential metabolic pathways. Autophagy functions to degrade cytoplasmic components to provide essential products for sustaining cellular metabolism and energy homeostasis. This function is particularly important within hypoxic regions of solid tumors where maintaining energy homeostasis becomes critical for cell survival.
A more complete understanding of the unique aspects of tumor metabolism may reveal opportunities for novel diagnostic and therapeutic strategies.