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Oncogene (2011) 30, 4687–4696 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc REVIEW The autophagy conundrum in cancer: influence of tumorigenic metabolic reprogramming CH Eng and RT Abraham Pfizer Oncology Research Unit, Pearl River, NY, USA Tumorigenesis is often accompanied by metabolic changes that favor rapid energy production and increased biosynthetic capabilities. These metabolic adaptations promote the survival and proliferation of tumor cells, and in conjunction with the hypoxic and metabolically challenged tumor microenvironment, influence autophagic activity. Autophagy is a catabolic process that allows cellular macromolecules to be broken down and re-utilized as metabolic precursors. Stimulation of autophagy promotes the survival of tumor cells under stressful metabolic and environmental conditions, and counters the potentially deleterious effects of mitochondrial dysfunction and the ROS that these organelles generate. However, inhibition of autophagy has also been reported to fuel tumorigenesis. In spite of the advances in our understanding of the relationship between autophagy and tumorigenesis, it remains unclear whether the therapeutic approaches targeting autophagy should aim to increase or decrease autophagic flux in tumor tissues in human patients. Here, we review how metabolic reprogramming influences autophagic activity in tumors, and discuss how inhibition of autophagy might be exploited to target tumor cells that show altered metabolism. Oncogene (2011) 30, 4687–4696; doi:10.1038/onc.2011.220; published online 13 June 2011 Keywords: autophagy; cancer metabolism; glutaminolysis; glycolysis Introduction Autophagy, a term based on the Greek words for ‘self’ (auto) and ‘eating’ (phagy), describes a lysosomemediated degradative process where a cell recycles intracellular macromolecules and organelles to regenerate metabolic building blocks. In unicellular organisms such as yeast, autophagy is a mechanism that promotes survival when extracellular nutrients are limiting. Autophagy also supports cellular homeostasis in multicellular organisms during fasting. However, in metazoans, autophagy contributes to a diversity of other Correspondence: Dr CH Eng, Pfizer Oncology Research Unit, 401 N. Middletown Road, Building 200, Room 4603, Pearl River, NY 10965, USA. E-mail: christina.eng@pfizer.com Received 26 February 2011; revised and accepted 2 May 2011; published online 13 June 2011 processes, such as developmental cell death (Berry and Baehrecke, 2007; Denton et al., 2009), clearance of harmful protein aggregates (Renna et al., 2010) and mitochondrial clearance during erythrocyte maturation (Kundu et al., 2008; Sandoval et al., 2008; Novak et al., 2010). By targeting damaged macromolecules and organelles for degradation, autophagy also protects cells from both internal and external insults, including metabolic, oxidative and chemotherapeutic stress. These functions of autophagy are protective and have a prominent role in numerous pathological conditions, including neurodegeneration (Wong and Cuervo, 2010), Crohn’s disease (Brest et al., 2010) and pathogenic infections (Deretic, 2010). Its role in cancer, however, is less defined. The first genetic link between autophagy and cancer occurred with the discovery that Beclin-1, a tumor suppressor, was an autophagy-related protein. Beclin1 resides in a chromosomal region frequently deleted in sporadic human breast, ovarian and prostate cancers (Aita et al., 1999). In breast cancer cell lines with reduced Beclin-1 levels, re-expression of the protein restored autophagy and suppressed tumorigenesis (Liang et al., 1999), whereas mice heterozygous for beclin1 were more prone to tumorigenesis (Qu et al., 2003). A core complex consisting of Beclin-1, Vps34 (a class-III phosphatidylinositol-3-kinase (PI3K)) and Vps15 promotes autophagy by mediating autophagosome nucleation (Funderburk et al., 2010). Since the discovery of Beclin-1, other studies have also shown that autophagy-deficient cells are more tumorigenic than their autophagy-competent counterparts (Liang et al., 1999; Marino et al., 2007; Mathew et al., 2009), in part owing to an increase in the DNA damage response and chromosomal instability (Karantza-Wadsworth et al., 2007; Mathew et al., 2007). In addition, mice harboring deletions in autophagy-related (Atg) genes are more prone to tumorigenesis (Marino et al., 2007; Takamura et al., 2011). These data lead to the hypothesis that autophagy has tumor-suppressive functions. However, accumulating evidence also suggests that autophagy promotes rather than suppresses malignant progression. Inhibition of autophagy in pancreatic tumor cells grown as xenografts in nude mice prevents tumor growth (Yang et al., 2011). In more advanced tumors, autophagy protects cancer cells against chemotherapeutic or environmentally imposed metabolic stress, which is consistent with observations that cancer cells residing in poorly perfused tumor tissues often show markers of increased autophagic activity (Degenhardt Influence of metabolic reprogramming on tumor autophagic activity CH Eng and RT Abraham 4688 et al., 2006). Thus, autophagy might be suppressed during the initial stages of transformation, and subsequently upregulated after the tumor is fully established (Chen and Debnath, 2010; Galluzzi et al., 2010; Mathew and White, 2011) (Amaravadi et al., 2011). Induction of autophagy by chemotherapeutics may also facilitate cellular survival, and this hypothesis is now being examined in human clinical trials. Hydroxychloroquine, a well-established inhibitor of lysosomal function and therefore autophagy, is being tested in patients with established tumors in combination with conventional cytotoxic chemotherapy (reviewed by Livesey et al., 2009). Upregulation of autophagy prolongs the survival of tumor cells treated with cytotoxic drugs; thus, concurrent inhibition of autophagy in patients receiving these drugs may augment tumor cell death (Livesey et al., 2009; Solomon and Lee, 2009; Amaravadi et al., 2011). However, certain cytotoxic drugs have been identified that induce cell death as a result of enhanced autophagic activity (Turcotte et al., 2008; Salazar et al., 2009). Thus, other groups continue to promote the idea that overstimulation of autophagy leads to cell death resulting from excessive self-cannibalization. Although autophagy as a cell death mechanism can occur in vitro with experimental manipulation, there is limited available evidence that physiological autophagic cell death occurs in mammalian cells in vivo (Kroemer and Levine, 2008). Confirmation of autophagic cell death is usually shown by increased survival upon knockdown of Atg proteins, but as these proteins may have roles in other cell death mechanisms (Yousefi et al., 2006), the notion of cell death resulting from autophagy remains controversial (Kroemer and Levine, 2008; Marino et al., 2011). Efforts to understand the roles of autophagy in tumorigenesis will require understanding how the events that drive tumorigenesis affect autophagy. One such notable tumorigenic event is the reprogramming of cellular metabolism. In order to sustain continuous cell division, tumor cells persistently engage in protein, nucleic acid and lipid biosynthesis. Although mutations in specific metabolic enzymes are rare, tumor cells alter their metabolic pathways to upregulate glycolytic, glutaminolytic and lipogenic programs. Physiological control of autophagy: mTOR Before examining the effect of tumorigenic metabolic perturbations on autophagy, we must first understand how autophagy is regulated in normal cells. Autophagy is initiated when a double-membrane structure termed the autophagosome engulfs cytoplasmic constituents and fuses with the lysosome. The resulting autolysosome represents the end of the line for the autophagosomal cargo; in the acidic microenvironment, lysosomal enzymes digest the macromolecules and the resulting degradation products are exported from the lysosomal lumen into the cytoplasm, where they are recycled back into metabolic pathways. Initially thought to be a nonselective process, recent studies show that recognition Oncogene and autophagic clearance of certain cargo, such as organelles, and ubiquitinated or aggregated proteins, are mediated by specific targeting proteins (Narendra et al., 2008; Kirkin et al., 2009; Filimonenko et al., 2010). Autophagy is activated by limitations in glucose, amino acids and oxygen. Early studies in budding yeast linked amino-acid starvation-induced autophagy to inhibition of the yeast orthologs of the mammalian target of rapamycin (mTOR), a central component of nutrient responsiveness in all eukaryotic cells. The mTOR complex-1 (mTORC1), a multi-protein complex defined by the signatory subunit Raptor, integrates signals relating to the availability of growth factors, amino acids, glucose, ATP and oxygen in the host cell. Inhibition of mTORC1 by lack of these factors, or by pharmacological agents such as rapamycin, suppresses mRNA translation (a major consumer of metabolic precursors and energy) and upregulates catabolism through stimulation of autophagic flux. The activity of mTORC1 is stimulated by the small GTPase Rheb, which itself is inhibited by the heterodimeric tuberous sclerosis complex (TSC) composed of TSC1 and TSC2. Although mTOR also resides in the mTOR complex-2 (mTORC2), defined by the Rictor subunit, the relationship between mTORC2 activity and autophagy appears to be more cell type- and context-dependent (Mammucari et al., 2007; Thoreen et al., 2009; Gurusamy et al., 2010). The recognition of nutrient availability by mTOR is modulated through multiple upstream regulators (Abraham and Eng, 2008). Glucose deprivation results in an elevated AMP/ATP ratio, leading to the activation of AMP-activated protein kinase (AMPK), which phosphorylates and activates TSC2, causing mTOR inactivation. Similarly, hypoxia induces the expression of REDD1 (for regulated in development and DNA damage responses), which stimulates TSC activity and, in turn, suppression of mTORC1. Finally, the presence of amino acids allows the activation of mTOR by Rheb, in part through the Rag GTPase-mediated translocation of mTOR to the lysosomal surface (Sancak et al., 2010). Amino-acid starvation, which inhibits mTOR, also facilitates autophagosome–lysosome fusion through lysosome clustering (Korolchuk et al., 2011). Inhibition of mTOR stimulates autophagy through an evolutionarily conserved pathway involving the protein kinase ULK1, a homolog of the yeast Atg1 kinase. ULK1 is found in complex with the Atg proteins FIP200 (which is thought to be the functional homolog of yeast Atg17) and Atg13. In response to nutrients, mTOR binds to the ULK1–Atg13–FIP200 complex and phosphorylates both ULK1 and Atg13. The binding of mTOR to the complex and phosphorylation of ULK1 by mTOR inhibits the kinase activity of ULK1 (Hosokawa et al., 2009; Jung et al., 2009). Nutrient limitation or treatment with rapamycin results in the inhibition of mTOR, dissociation of mTOR from the complex, and a reduction in ULK1 and Atg13 phosphorylation. Recent studies identified AMPK as a positive regulator of the ULK1 complex activity, which is necessary for autophagy (Lee et al., 2010; Egan et al., 2011; Kim et al., 2011a). Influence of metabolic reprogramming on tumor autophagic activity CH Eng and RT Abraham 4689 Autophagy is also stimulated through mTOR-independent pathways, although the exact mechanisms are less well-understood. Numerous pharmacological agents have been reported to stimulate autophagy in an mTOR-independent manner (Sarkar et al., 2007; Zhang et al., 2007), with some involvement of Ca2 þ and inositol 1,4,5-trisphosphate signaling (Williams et al., 2008). Amino-acid regulation of autophagy also occurs in an mTOR-independent manner in muscle cells (Mordier et al., 2000) through FoxO3-mediated transcription of the autophagy genes MAP1LC3B (microtubule-associated protein 1 light chain 3 b) and BNIP3 (BCL2/ adenovirus E1B 19 kDa protein-interacting protein 3) (Mammucari et al., 2007), and in liver cells (Kanazawa et al., 2004). A small interfering RNA screen for autophagy modulators also identified a number of growth factors and cytokines that suppress autophagy independently of mTOR through inhibition of class-III PI3K (Lipinski et al., 2010). Effects of metabolic reprogramming on autophagy: glycolysis The first connection between tumorigenesis and metabolic reprogramming was made in the 1920s, when the legendary German biochemist Otto Warburg first noted that tumor slices showed an unusually high level of glycolytic activity, even under normoxic conditions, in contrast to normal differentiated cells, which primarily metabolize glucose through oxidative phosphorylation. This metabolic aberration is the basis of a clinically validated tumor-imaging modality termed 18F-deoxyglucose positron emission tomography (FDG–PET). Tumors are visualized by FDG–PET based on their elevated rates of glucose uptake compared with most normal tissues within the body (Vander Heiden et al., 2009). Warburg hypothesized that this alteration in glucose metabolism resulted from mitochondrial dysfunction. It is now appreciated that cancer cell mitochondria are not globally dysfunctional, but rather reprogrammed to support their increased biosynthetic needs. As mitochondria in tumor cells are re-purposed to produce the precursors necessary for protein, lipid and nucleic acid biosynthesis, the activity of the ATP-generating electron transport chain is reduced and glycolytic metabolism is increased to meet the bioenergetic and biosynthetic needs of cancer cells. Although less efficient, the increased pace of energy production by aerobic glycolysis compared with oxidative phosphorylation is also beneficial to rapidly proliferating cells (DeBerardinis et al., 2008). The switch from oxidative phosphorylation to glycolysis is induced by the activation of several oncogenes. Transformation induced by Ras or Src increases glucose uptake (Flier et al., 1987). Overexpression of myc, which occurs in over 40% of human cancers, activates the transcription of numerous enzymes involved in glucose metabolism, including lactate dehydrogenase, hexokinase, glucose transporter-1 (GLUT-1) and pyruvate kinase M2 (Dang et al., 2009). Finally, activation of the PI3K pathway, which frequently occurs in malignant cells through deregulated growth factor receptor signaling, mutations in the p110a catalytic subunit, loss of phosphatase and tensin homolog (PTEN) or amplification of Akt, also results in increased glucose uptake and glycolysis (Elstrom et al., 2004). In order to maximize cell mass accumulation during tumorigenesis, one might expect catabolic processes such as autophagy to be suppressed. This hypothesis is supported by data showing that transformation driven by oncogenes such as Ras (Furuta et al., 2004), PI3K (Petiot et al., 2000) and myc (Balakumaran et al., 2009), or loss of the tumor suppressor PTEN (Arico et al., 2001), suppresses autophagy. Activation of mTOR by these transforming events (Shaw and Cantley, 2006; Ravitz et al., 2007) is one mechanism by which autophagy might be downregulated in response to oncogenic signaling. Cytokine receptor engagement also triggers signaling events that suppress autophagy (Lipinski et al., 2010). These studies support the hypothesis that certain early drivers of transformation suppress autophagy, perhaps to accommodate the need for a rapid increase in cell mass to fuel cell proliferation. One notable exception to oncogene-induced suppression of autophagy is mutation of K-Ras. Expression of mutant K-Ras enhances aerobic glycolysis (Racker et al., 1985; Vizan et al., 2005), owing, in part, to increased expression of GLUT-1 (Yun et al., 2009) and PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6bisphosphatase) (Telang et al., 2006). The dependence of K-Ras-mutated cells on glucose partially results from the need for increased carbon flux through the pentose phosphate pathway (Weinberg et al., 2010). Although previous work showed suppression of autophagy with expression of mutant K-Ras (Furuta et al., 2004), a number of recent studies have illustrated the ability of mutant K-Ras to promote basal autophagy (Guo et al., 2011; Kim et al., 2011b; Lock et al., 2011). In these studies, suppression of autophagy limited the ability of mutant K-Ras to promote anchorage-independent growth in vitro and tumorigenesis in vivo. Autophagy may sustain transformation induced by mutant K-Ras through the maintenance of bioenergetics. Cells harboring K-Ras mutations that were autophagy-defective had decreased glycolytic capacity (Lock et al., 2011) and abnormal mitochondria owing to suppressed mitophagy, resulting in reduced tricarboxylic acid (TCA) intermediates and reduced oxygen consumption (Guo et al., 2011). Thus, not only is autophagy stimulated when glycolysis is upregulated owing to mutations in K-Ras, but autophagy may also be necessary to maintain metabolic homeostasis after a shift to glycolysis. This notion is also supported by studies showing that enhancements to the glycolytic pathway can stimulate autophagy (Figure 1), as discussed below. Loss of the tumor suppressor p53 promotes glycolysis through enhanced glucose transport (Kawauchi et al., 2008), enhanced phosphoglycerate mutase expression (Kondoh et al., 2005) and suppression of the mitochondrial enzyme SCO2 (synthesis of cytochrome c oxidase 2) Oncogene Influence of metabolic reprogramming on tumor autophagic activity CH Eng and RT Abraham 4690 Glucose Glucose-6-phosphate Pentose phosphate pathway NADPH Fructose-6-phosphate Glutathione ROS TIGAR p53 Fructose-1,6-bisphosphate (2) Glyceraldehyde-3-phosphate mTOR Autophagy GAPDH (2) 1,3-bisphosphoglycerate Atg12 Pyruvate Figure 1 Regulation of autophagy by glycolysis. Glycolysis positively regulates autophagy through various mechanisms. Expression of the glycolytic enzyme GAPDH promotes the transcription of Atg12, which stimulates autophagy. GAPDH, by binding to Rheb, also prevents the activation of mTOR. Conversely, inhibition of glycolysis by TIGAR re-routes glycolytic intermediates toward the pentose phosphate pathway, resulting in the suppression of autophagy by glutathione-dependent reduction of ROS. Atg, autophagy-related genes; GAPDH, glyceraldehyde-3phosphate dehydrogenase; mTOR, mammalian target of rapamycin; ROS, reactive oxygen species; TIGAR, TP53-induced glycolysis and apoptosis regulator. (Matoba et al., 2006). Another target of p53, the TP53induced glycolysis and apoptosis regulator (TIGAR), a fructose 1,6-bisphosphatase, directly inhibits both glycolysis and autophagy. TIGAR inhibits glycolysis by reducing the levels of fructose 1,6-bisphosphate and redirecting glucose metabolism toward the pentose phosphate pathway (Bensaad et al., 2006). The pentose phosphate pathway produces NADPH, which is required for the regeneration of reduced glutathione; thus expression of TIGAR enhances glutathione levels and increases the antioxidant capacity of the cell (Figure 1). Production of glutathione decreases the levels of reactive oxygen species (ROS), resulting in the suppression of autophagy (Bensaad et al., 2009). Thus, loss of the p53 tumor suppressor would decrease TIGAR levels, resulting in the upregulation of both glycolysis and autophagy. Another key glycolytic enzyme is glyceraldehyde-3phosphate dehydrogenase (GAPDH), which catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3bisphosphoglycerate (Figure 1). Expression of GAPDH stimulates the transcription of Atg12, and both increased glycolysis and Atg12 expression by GAPDH are required to protect cells from caspase-independent cell death (Colell et al., 2007). GAPDH expression induces a transient decrease in mitochondrial mass and maintenance of ATP levels after mitochondrial insults, suggesting that autophagy may protect cells by eliminating damaged mitochondria, and enhanced glycolysis provides enough ATP necessary for mitochondrial regeneration and cell survival. Interestingly, GAPDH Oncogene may also promote autophagy by negatively regulating mTOR activity. GAPDH competes with mTOR for binding to the mTOR activator Rheb, and under lowglucose conditions, expression of GAPDH, through binding to Rheb, prevents the activation of mTOR (Lee et al., 2009), which would promote autophagy. Enhanced glycolysis directly correlates with stimulation of autophagy (Figure 1). This correlation occurs not only in tumor cells, but also in activated T cells, another highly proliferative cell type. Similar to tumor cells, activation of T cells results in a metabolic switch from oxidative phosphorylation to aerobic glycolysis (Fox et al., 2005). Autophagy is required for T-cell activation and functions to meet their increased bioenergetic needs (Hubbard et al., 2010). Thus, induction of autophagy may be necessary to maintain cellular bioenergetics after a shift to glycolytic metabolism, not only in tumor cells but in proliferative normal cells as well. Effects of metabolic reprogramming on autophagy: hypoxia and ROS Although upregulation of aerobic glycolysis supports the increased biosynthetic demands of tumor cells under normoxic conditions, glycolysis is also enhanced if access to oxygen is limited owing to inadequate tissue perfusion. Hypoxic tumor tissues show markers of increased autophagic activity (Rouschop et al., 2010). Hypoxia also generates ROS (Semenza, 2010), which directly induce autophagy (Djavaheri-Mergny et al., 2006; Chen et al., 2009; Huang et al., 2009). Generation of ROS also occurs with nutrient starvation (Scherz-Shouval et al., 2007) and mitochondrial electron transport. The master transcriptional regulators of hypoxic responses are the hypoxia-inducible factors (HIFs). Active HIF is a heterodimer comprising HIF-1a or HIF-2a partnered with the constitutively expressed HIF-1b subunit. The expression of the HIF-1/2a subunits is tightly controlled through oxygen-dependent hydroxylation by prolyl hydroxylases. In the presence of oxygen, HIF-1/2a is targeted for degradation. Under low-oxygen conditions, or in the presence of ROS, HIF1/2a becomes stabilized, partners with HIF-1b and enters the nucleus, where the HIF complexes orchestrate hypoxia-induced gene expression (Semenza, 2010). Stabilization of HIF-1a promotes mitochondrial autophagy (mitophagy) through transcription of the Bcl-2 homology-domain-3-containing proteins BNIP3 and BNIP3L. BNIP3 stimulates autophagy by preventing the inhibitory binding of Bcl-2 to Beclin-1, which frees Beclin-1 to participate in autophagosome formation (Figure 2). Mitophagy induced by BNIP3 results in the clearance of damaged mitochondria, which are major sources of cell-damaging ROS (Zhang et al., 2008). Inhibition of autophagy sensitizes cells to hypoxic cell death (Bellot et al., 2009; Rouschop et al., 2009) and ROS-induced cell death (Huang et al., 2009). Collectively, these results suggest that ROS-induced autophagic activity limits cellular damage by removing damaged macromolecules and dysfunctional mitochondria. Influence of metabolic reprogramming on tumor autophagic activity CH Eng and RT Abraham 4691 Mitophagy LC3-PE Atg4A Nutrient Starvation ROS ATM TSC2 mTOR REDD1 Hypoxia Bcl-2 Autophagy Beclin HIF-1 BNIP3/BNIP3L PERK ATF4 Atg5 CHOP LC3 Figure 2 Stimulation of autophagy by ROS and hypoxia. ROS, which are produced during nutrient starvation or hypoxia, stimulate autophagy through multiple signaling pathways. Oxidation and inactivation of Atg4A by ROS allows the buildup of LC3 conjugated to phosphatidylethanolamine and subsequent promotion of autophagy. ROS also inhibit mTOR through activation of TSC2 by ATM. Stimulation of autophagy by hypoxia occurs through suppression of mTOR through HIF-1a-dependent activation of REDD1. HIF-1a also induces the transcription of BNIP3/ BNIP3L, which prevents the binding of Bcl-2 to Beclin, allowing Beclin to stimulate autophagy. Finally, hypoxia promotes autophagy through an HIF-1-independent pathway involving transcriptional induction of Atg5 and LC3 through the PERK-responsive transcription factors ATF4 and CHOP. The activation of autophagy by hypoxia and ROS results in a quality-control feedback loop by elimination of damaged mitochondria through mitophagy, thereby reducing ROS. Atg, autophagy-related genes; HIF, hypoxia-inducible factor; mTOR, mammalian target of rapamycin; ROS, reactive oxygen species; TSC2, tuberous sclerosis complex-2. Hypoxia-induced autophagy also occurs in cells deficient in BNIP3. The unfolded protein response in the endoplasmic reticulum is activated by hypoxia, and part of this response includes activation of PERK (protein kinase regulated by RNA (PKR)-like endoplasmic reticulum kinase). Through the PERK-responsive transcription factors CHOP (CCAAT-enhancerbinding proteins (C/EBP) homologous protein) and ATF4 (activating transcription factor 4), the autophagy genes Atg5 and MAP1LC3B are transcriptionally induced. The induction of MAP1LC3B transcription is necessary to maintain autophagy as LC3 is consumed by the autophagic processes in hypoxic cells (Rouschop et al., 2010). As mentioned previously, mTOR is a critical regulator of autophagy and also functions as a signal transducer for decreased oxygen availability. Hypoxia signals to mTOR through the transcriptional target REDD1, a protein, which is activated by HIF-1a and represses mTORC1, resulting in increased autophagy (Brugarolas et al., 2004). Inhibition of mTOR also occurs with increased ROS generated during hypoxia. ROS activates ataxia telangiectasia mutated (ATM), leading to the subsequent phosphorylations of LKB1, AMPK and finally TSC2, resulting in the stimulation of TSC2 activity and subsequent inhibition of mTOR (Alexander et al., 2010). Thus, inhibition of mTOR is another mechanism by which hypoxia and ROS promote autophagy (Figure 2). Although high levels of ROS are deleterious to cells, lower concentrations of ROS may be beneficial owing to their enhancement of autophagy. During nutrient starvation, accumulation of ROS results in the maintenance of autophagy through oxidation of Atg4A (Scherz-Shouval et al., 2007). The Atg4 isoforms A–D are cysteine proteases that drive autophagosomal maturation by cleaving LC3 prior to conjugation with phosphatidyl-ethanolamine (PE), and subsequently de-lipidate LC3 after autophagosome– lysosome fusion. Oxidation of the catalytic cysteine in Atg4A inhibits its activity, thereby suppressing the delipidation of LC3, promoting the accumulation of LC3-PE. Expression of a non-oxidizable Atg4A or Atg4B mutant prevents autophagosome formation. Thus, ROS-mediated inhibition of Atg4 is required for the maintenance of autophagy during nutrient starvation. In at least one instance, induction of autophagy increases ROS and results in cell death. Upon caspase inhibition, stimulation of autophagy results in the degradation of the ROS scavenger, catalase. In this setting, autophagy enhances ROS accumulation by removing a major ROS-detoxifying enzyme (Yu et al., 2006). However, this finding may be specific for the context of caspase inhibition, as the majority of the studies detailed above show that autophagy induced by hypoxia or ROS enables cellular survival through clearance of dysfunctional mitochondria and reduction of ROS (Figure 2). Effects of metabolic reprogramming on autophagy: glutaminolysis Although Warburg initially hypothesized that tumor cells switch from oxidative phosphorylation to aerobic glycolysis because of defective mitochondria, it has become clear that mitochondrial function is vital to support anabolic metabolism in rapidly proliferating tumor cells. One such mitochondrial process, the TCA cycle, is essential for generating both NADPH and precursors of fatty acid synthesis. The consumption of glucose through glycolysis at the expense of oxidative phosphorylation necessitates the use of an alternative fuel for mitochondrial processes. Glucose and glutamine are the two most highly metabolized nutrients in tumor cells, and glutamine has emerged as a dominant carbon source for the TCA cycle in highly proliferative cells. Many proteins involved in glutamine metabolism are upregulated in tumor cells, including glutamine transporters and enzymes involved in glutamine breakdown. Overexpression of Myc promotes the expression of the glutamine transporters ASCT2 and SN2, and the mitochondrial enzyme glutaminase, which converts glutamine to glutamate (Wise et al., 2008; Gao et al., 2009). These events cause cells transformed with Myc to be dependent on glutamine for survival (Yuneva et al., 2007). Oncogenic induction of glycolysis also creates a dependence on glutamine, as shown by the requirement of K-Ras-transformed cells for glutamine metabolism in anchorage-independent growth (Weinberg et al., 2010) and re-entry into the cell cycle (Gaglio et al., 2009). Oncogene Influence of metabolic reprogramming on tumor autophagic activity CH Eng and RT Abraham 4692 After transport into the cell, glutamine is metabolized to glutamate by mitochondrial glutaminase. This reaction liberates one molecule of ammonia. The further conversion of glutamate to a-ketoglutarate leads to the production of a second molecule of ammonia if the reaction is mediated by glutamate dehydrogenase. Ammonia is also produced through the de-amidation of other amino acids, although in tumor cells it is thought that glutamine is the main source of free ammonia. Although ammonia has nutritive properties in plants and fungi, a large body of evidence suggests that it is primarily a toxic waste product in mammalian cells. Indeed, organisms have developed efficient means of ammonia detoxification, through concentration in the liver where it is converted to urea, which is excreted by the kidneys. However, in localized regions, such as the interstitial space of tumors that may not be readily accessible to the bloodstream, ammonia can accumulate to low millimolar levels (Eng et al., 2010). Recent findings suggest that the ammonia present in tumor interstitial space might be beneficial to tumor cell survival, through stimulation of autophagy. Although higher concentrations of ammonia block autophagy by raising the lysosomal pH, lower concentrations (2–4 mM) of ammonia paradoxically promote autophagy in cancer cells (Eng et al., 2010). Basal autophagy, which maintains cellular viability, is decreased in cells cultured in glutamine-deficient medium, whereas increasing glutamine concentrations upregulates basal autophagy (Sakiyama et al., 2009). Ammonia-induced autophagy protects cells from apoptosis induced by tumor necrosis factor-a and limits cell proliferation under metabolic stress (Eng et al., 2010). Cell death induced by long-term culture in glutamine-free medium is also rescued by low concentrations of ammonia (Meng et al., 2010). As these autophagy-promoting concentrations of ammonia are also found in tumor interstitial fluids, ammonia may act as a diffusible autophagy-inducing signal that protects tumor cells located in poorly vascularized regions from metabolic stress (Eng and Abraham, 2010). Thus, glutamine enhances tumor cell proliferation and survival not only through the anabolic effects of replenishment of TCA cycle intermediates, but also through the catabolic protection from metabolic stress offered through autophagy. Effects of metabolic deregulations on autophagy: lipogenesis Upregulation of de novo lipogenesis is another metabolic alteration associated with tumor aggressiveness. In contrast to normal cells, where the primary sources of fatty acids are exogenous dietary lipids, tumor cells synthesize the majority of their fatty acids. This lipogenic phenotype may be related to the Warburg effect, as a significant portion of glucose taken up by tumor cells is used for the production of fatty acids (DeBerardinis et al., 2007; DeBerardinis et al., 2008). Three enzymes (ATP citrate lyase, acetyl-CoA carboxylase-A and fatty acid synthase) involved in the Oncogene conversion of the TCA cycle intermediate, citrate, to fatty acids are upregulated in tumor tissues (Menendez and Lupu, 2007), and expression of fatty acid synthase is correlated with poorer prognosis in breast cancer (Kuhajda et al., 1994). Upregulation of fatty acid synthesis during tumorigenesis might be necessary for increased membrane synthesis and production of lipid messengers such as phosphatidylinositol-3,4,5-triphosphate and lysophosphatidic acid, and indeed, inhibition of fatty acid synthase decreases tumor cell proliferation (Lupu and Menendez, 2006). In addition, transcriptional profiling of an isogenic cell line panel manifesting increasing levels of malignant behavior showed correlations with lipid metabolism (Hirsch et al., 2010). Although little is known about the relationship between cancer-mediated abnormalities in lipogenesis and autophagy, recent work suggests that autophagy regulates lipid metabolism in normal tissues. Triglycerides and other lipids are substrates for autophagic degradation, and inhibition of autophagy leads to increased triglyceride lipid stores and decreased triglyceride breakdown in hepatocytes (Singh et al., 2009). Fatty acids are stored in lipid droplets in the form of triglycerides. As inhibition of autophagy promotes triglyceride storage, it may counteract the effects of increased fatty acid synthesis owing to overexpression of lipogenic enzymes in tumor cells. Conclusions In order to maintain cellular homeostasis in the setting of rapid cellular proliferation, transformed cells alter numerous metabolic pathways. Increased glucose uptake through glycolysis hastens ATP production and this altered utilization of glucose necessitates an upregulation of glutamine metabolism in order to fuel mitochondrial pathways. Anerobic glycolysis is also necessary for cells to adapt to a hypoxic microenvironment, which is associated with both nutrient deprivation and increased levels of ROS. All of these metabolic perturbations result in a stimulation of autophagy. Autophagy appears to have dual roles in tumorigenesis. Suppression of autophagy may be a requirement for the early stages of transformation, as evidenced by the observations that autophagy-defective cells are more tumorigenic than their wild-type counterparts. In addition, autophagy might be diminished during oncogeneinduced transformation (such as by activation of PI3K), although there are clear exceptions (such as mutation of K-Ras). If one could intervene early on in the tumorigenic process, stimulation of autophagy might promote growth suppression and counter ROS generation, organelle dysfunction and genomic instability, thereby interfering with tumor formation. However, at the time of diagnosis, tumors are generally well-established. Metabolic alterations such as increased glycolysis, glutaminolysis and hypoxia/ ROS accumulation that accompany the establishment of tumors results in the upregulation of autophagic flux. Thus, therapeutic inhibition of autophagy at this stage Influence of metabolic reprogramming on tumor autophagic activity CH Eng and RT Abraham 4693 Tumor Cell Survival Proliferative and survival signals Autophagy Inhibition Proliferative and survival signals Autophagy Autophagy NH3 NH3 tumor stromal cells metabolically challenged tumor cells nutrients, oxygen metabolically replete tumor cells nutrients, oxygen ROS damaged mitochondria misfolded proteins cellular homeostasis Tumor Cell Death Figure 3 Effect of autophagy inhibition within the tumor landscape. The tumor microenvironment contains a gradient of oxygen and nutrients (increasing from green to purple), within which reside tumor cells themselves and supporting stromal cells. Autophagy is stimulated in tumor cells owing to metabolic deregulations and glutaminolytic production of ammonia, which acts as a diffusible stimulator of autophagy in the neighboring, metabolically challenged cells (green). Tumor cells also stimulate autophagy in adjacent stromal cells (brown), which allows the stromal cells to impart proliferative and survival signals back to the tumor cells. Inhibition of autophagy would block both autocrine- and paracrine-mediated survival pathways, resulting in tumor cell death. would result in a bioenergetic imbalance, and render cancer cells unable to cope with their metabolically stressed microenvironment and promote their demise. The altered metabolic program and persistent proliferation of tumor cells also results in misfolded proteins, damaged mitochondria, ROS and excess lipids, and without autophagy to alleviate the effects of these harmful agents, tumor cells might be coaxed into poisoning themselves with the byproducts of their own metabolic alterations. Additionally, inhibition of autophagy might block pathways used by tumor cells to survive chemotherapeutic stress, and thus sensitize cells to cytotoxic chemotherapy. Finally, communication between tumor cells and tumor stroma also contributes to the metabolic and autophagic landscape of the tumor microenvironment (Figure 3). The high rate of glutaminolysis in tumor cells results in the production of ammonia, which could diffuse to the nutrient- and oxygen-limited regions of the tumor to combat metabolic stress by stimulating autophagy (Eng and Abraham, 2010). Tumor-associated stromal cells also reside in the same metabolically stressed environment as tumor cells, and contribute to the survival and proliferation of the tumor cells themselves (McAllister and Weinberg, 2010). Part of this survival mechanism is because of autophagy induced in adjacent cancer-associated fibroblasts (Martinez-Outschoorn et al., 2010). Thus, inhibition of autophagy would not only promote tumor cell death in a cell-autonomous manner, but also interfere with cellular survival that results from heterogeneous cell–cell interactions within the diverse tumor landscape. 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