MYC and tumor metabolism: chicken and egg

Abstract

Transcription factors of the MYC family are deregulated in the majority of all human cancers. Oncogenic levels of MYC reprogram cellular metabolism, a hallmark of cancer development, to sustain the high rate of proliferation of cancer cells. Conversely, cells need to modulate MYC function according to the availability of nutrients, in order to avoid a metabolic collapse. Here, we review recent evidence that the multiple interactions of MYC with cell metabolism are mutual and review mechanisms that control MYC levels and function in response to metabolic stress situations. The main hypothesis we put forward is that regulation of MYC levels is an integral part of the adaptation of cells to nutrient deprivation. Since such mechanisms would be particularly relevant in tumor cells, we propose that—in contrast to growth factor‐dependent controls—they are not disrupted during tumorigenesis and that maintaining flexibility of expression is integral to MYC's oncogenic function.

Introduction

MYC proteins form a small family of closely related oncoproteins, three of which (MYC, N‐MYC, L‐MYC) have been implicated in the genesis of multiple human tumors (Dang, 2012; Kress et al, 2015). Their expression is tightly controlled by growth factor‐dependent signals in normal cells and is deregulated and enhanced via multiple mechanisms in tumor cells. A large body of evidence demonstrates unequivocally that enhanced MYC expression is a major driving force of tumorigenesis and that both MYC‐driven tumors and tumors driven by other oncogenes, for example, by mutant RAS, continuously depend on elevated MYC levels for growth (Felsher & Bishop, 1999; Shachaf et al, 2004; Soucek et al, 2008; Annibali et al, 2014). Not surprisingly, many biochemical properties appear to be identical among all three proteins, and we will therefore focus on the founding member of the family, MYC, in this review.

For a long time, MYC proteins were considered to be gene‐specific transcription factors. This view has been challenged by recent work demonstrating that MYC proteins bind to virtually all active promoters and many enhancers. Consistently, in some situations MYC proteins globally regulate transcription by RNA polymerase II (RNAPII) and also by RNA polymerases I and III (Wolf et al, 2015). Exactly whether MYC proteins are oncogenic since they regulate—a large number of—specific target genes or whether they have unique biochemical functions during expression of all genes (e.g., during the release of RNAPII from a promoter‐proximal pause position into active elongation) is therefore intensely debated (Dang, 2012; Kress et al, 2015; Wolf et al, 2015). In this review, we will leave this debate aside and use a relaxed definition of a target gene of MYC as any gene for which relative mRNA levels change in a given experimental situation when MYC levels are altered.

Deregulated expression of MYC promotes proliferation and growth of cells and alters intermediary metabolism to match the enhanced demand for anabolic metabolites (Morrish et al, 2009; van Riggelen et al, 2010; Dang, 2013; Cunningham et al, 2014). This “metabolic reprogramming” is observed both in tissue culture and in transgenic models, in which MYC is expressed from constitutive promoters to mimic the release from its growth factor‐dependent control mechanisms (Yuneva et al, 2012; Shroff et al, 2015). In this review, we describe downstream effects of MYC highlighting the ability of this oncogene to globally rewire cellular metabolism. Notably, effects on cellular metabolism depend both on oncogene activity and on the tissue of tumor origin and on interaction with microenvironment components (Yuneva et al, 2012; Davidson et al, 2016; Mayers and Vander Heiden, 2017). Metabolic changes induced by deregulated MYC can therefore vary among different tumor types.

As consequence of metabolic changes, deregulated MYC expression can induce metabolic stress: For example, enhanced expression of MYC causes a decrease in cellular ATP levels and activate AMP‐activated kinase (AMPK) (Liu et al, 2012a; von Eyss et al, 2015). MYC‐induced metabolic stress sensitizes cells toward apoptosis, in part since AMPK phosphorylates p53 and activates its mitochondrial pro‐apoptotic functions (Nieminen et al, 2007, 2013). Similarly, loss of mechanisms that restrain MYC activity following hypoxia can result in MYC‐driven apoptosis (Brunelle et al, 2004). As consequence, cells expressing deregulated MYC can be dependent on a continuous and high supply of nutrients. For example, MYC‐driven tumor cells are dependent on a constant supply of glutamine and glutamine deprivation or inhibition of glutaminase can selectively kill cells with enhanced levels of MYC (Yuneva et al, 2007; Wise et al, 2008; Qing et al, 2012; Nieminen et al, 2013; Wiese et al, 2015). This phenomenon has been termed “glutamine addiction” and provides the rational basis for exploring the therapeutic value of glutaminase inhibitors for MYC‐driven tumors. Similarly, rat fibroblast cells overexpressing MYC undergo apoptosis when deprived of glucose cells (Shim et al, 1998) and MYC‐driven lymphoma cells are particularly sensitive to inhibition of lactate export (Doherty et al, 2014). An attractive concept is to exploit these dependencies for tumor therapy.

The aim of this review is to summarize the opportunities for targeting the metabolism of MYC‐driven tumors for therapy and to identify the critical challenges that need to be addressed for such an approach to be successful. One central argument we put forward is that multiple mechanisms link MYC expression to the metabolic status of tumor cells and that modeling approaches need take to them into account in order not to overestimate the metabolic liabilities caused by enhanced MYC expression.

Oncogenic MYC promotes anabolic reactions: the role of glucose

Cancer cells typically display high glucose uptake, glycolytic metabolism, and lactate production even in the presence of oxygen (“aerobic glycolysis”), a phenomenon termed the Warburg effect (Vander Heiden et al, 2009). This seemingly wasteful use of glucose is most likely due to the higher capacity of glycolysis to produce ATP compared to the oxidative phosphorylation; hence, aerobic glycolysis may satisfy the high demand of rapidly growing cells for ATP (Vander Heiden et al, 2009; Liberti & Locasale, 2016). Metabolic labeling using ¹³C‐glucose reveals that oncogenic levels of MYC promote high consumption of glucose, as assessed both in Burkitt's lymphoma (Le et al, 2012; Murphy et al, 2013) and in MYC‐driven liver carcinoma (Yuneva et al, 2012). MYC exerts its effects on glucose metabolism by increasing the expression of the glucose transporter GLUT1 and by upregulating the expression of glycolytic enzymes, including hexokinase 2 (HK2), phosphofructokinase‐M 1 (PFKM1), and enolase 1 (ENO1; Osthus et al, 2000; Kim et al, 2004). Aerobic glycolysis is also driven by the expression of the M2 isoform of the pyruvate kinase (PKM2), which is expressed in virtually all tumors (Israelsen & Vander Heiden, 2015). MYC enhances expression of PKM2 by promoting the expression of hnRNP splicing factors, as demonstrated in glioma (David et al, 2010; Luan et al, 2015; Fig 1).

Figure 1. Overview of metabolic pathways promoted by MYC .

MYC increases the uptake and the metabolism of glucose and glutamine, the major nutrients of cancer cells, by upregulating the expression of genes encoding for membrane transporters and metabolic enzymes (shown in turquoise; see text). MYC globally promotes anabolic reactions (indicated in magenta) in order to sustain the biosynthetic needs of proliferating cells. Full arrows show direct reactions, and dashed ones indicate the presence of metabolic intermediates. GLUT1: glucose transporter 1; HK2: hexokinase 2; PKM2: pyruvate kinase M2; LDH: lactate dehydrogenase; MCT1: monocarboxylate transporter 1; SHMT: serine hydroxymethyltransferase; ACLY: ATP citrate lyase; FASN: fatty acid synthase; CAD: carbamoyl‐phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase; PRPS2: phosphoribosyl pyrophosphate synthetase 2; IMPDH2: inosine‐5′‐monophosphate dehydrogenase 2; SLC1A5: solute carrier 1A5; GLS: glutaminase; TCA: tricarboxylic acid; glucose‐6‐P: glucose 6‐phosphate; fructose‐6‐P: fructose 6‐phosphate; 3‐P‐glycerate: 3‐phosphoglycerate; ribose‐5‐p: ribose 5‐phosphate; α‐KG: α‐ketoglutarate; NADPH: nicotinamide adenine dinucleotide phosphate; NADH: nicotinamide adenine dinucleotide.

During glycolysis, NAD⁺ is reduced to NADH⁺ + H⁺ and cells need to regenerate NAD⁺ to maintain the glycolytic flux. This depends on LDHA, encoding for lactate dehydrogenase A, which utilizes pyruvate as substrate derived from both the glycolytic and the glutaminolytic pathway and converts it into lactate (Shim et al, 1998; Wise et al, 2008). Overexpressing MYC enhances LDHA expression and typically results in extracellular acidification due to the increased production of lactate (Shim et al, 1998; Lewis et al, 2000; Fan et al, 2010; Maya‐Mendoza et al, 2015). MYC also promotes the secretion of lactate through the expression of the bidirectional monocarboxylate transporter MCT1 (Doherty et al, 2014). Lactate is not simply a waste product of tumors, but an active role of this metabolite in promoting multiple pro‐oncogenic functions emerged over time (San‐Millan & Brooks, 2016). An indirect mechanism through which MYC promotes the uptake of glucose is via blocking the transcriptional function of MondoA, which in turn results in the inhibition of the thioredoxin‐interacting protein (TXNIP), a negative regulator of glycolysis (Shen et al, 2015). Intriguingly, deregulated MYC expression also blocks the induction of mRNAs encoding gluconeogenesis enzymes in response to a high‐fat diet in liver, arguing that repressive functions of MYC contribute to alterations in cellular metabolism (Riu et al, 2003).

The high K_m of PKM2 for phosphoenolpyruvate increases concentrations of glycolytic intermediates and channels them into two biosynthetic pathways (Israelsen & Vander Heiden, 2015), Fig  1. The pentose phosphate pathway (PPP) consists of two branches: the oxidative phase and the non‐oxidative phase. The first one uses glucose 6‐phosphate to produce both ribose sugars for nucleotide biosynthesis and NADPH for sustaining anabolic reactions; the non‐oxidative phase allows for the recycling of glycolytic substrates, like fructose 6‐phosphate and glyceraldehyde 3‐phosphate, which can be rechanneled into the oxidative arm depending on the cellular metabolic needs (Patra & Hay, 2014). Serum‐induced MYC activation in rat fibroblasts increases the flux of glucose toward ribose sugars (Morrish et al, 2009). In activated T cells, MYC increases the expression of G6PDH, encoding for glucose‐6‐phosphate dehydrogenase, which catalyzes the first reaction of the oxidative phase, and TKT, which encodes for transketolase, an enzyme of the non‐oxidative branch (Wang et al, 2011).

The serine synthesis pathway (SSP) represents a second important branch of glycolysis, and the activity of the SSP is often upregulated in cancer. Serine‐derived glycine fuels the one‐carbon metabolism, constituted by two interconnected pathways: the folate and the methionine metabolism. Such reactions are in turn needed for the biosynthesis of macromolecules like purines and glutathione (Yang & Vousden, 2016). The conversion of serine to glycine is catalyzed by the serine hydroxymethyltransferase (SHMT). Both the cytosolic and the mitochondrial isoforms of this enzyme (SHMT1 and SHMT2, respectively) are targets of MYC, as first demonstrated in rat fibroblasts (Nikiforov et al, 2002). Increased glycine synthesis resulting from the upregulation of enzyme involved in the SSP is dependent on the oncogenic activity of MYC, as demonstrated in both hepatocellular carcinoma cell lines (Sun et al, 2015) and in a MYC‐driven liver tumor (Anderton et al, 2017).

Glucose also serves as sugar donor for posttranslational modifications of proteins. This occurs through the hexosamine biosynthetic pathway (HBP) in which both glucose and glutamine are metabolized to produce UDP‐N‐acetylglucosamine (UDP‐Glc‐NAc). The N‐acetylglucosamine is in turn transferred from UDP‐Glc‐NAc to serine or threonine residues of substrate proteins, a reaction catalyzed by the enzyme O‐GlcNAc transferase (OGT). The HBP is hyperactivated in a variety of cancers and O‐GlcNAcylated proteins regulate multiple oncogenic processes (Ferrer et al, 2016). In addition to promoting the uptake of glucose and glutamine and to channeling these substrates into the HBP (Morrish et al, 2009), in breast cancer cells MYC increases the expression of the chaperone protein HSP90, which stabilizes OGT, thus promoting the modification of target proteins (Sodi et al, 2015). MYC itself is an OGT target and MYC O‐GlcNAcylation on Thr58 increases its stability and competes with the phosphorylation on the same site, which primes MYC for degradation (Itkonen et al, 2013).

Oncogenic MYC promotes anabolic reactions: the role of glutamine

Consistent with the increased demand of tumor cells for glutamine, several transporters have been shown to be upregulated in many types of cancer (Bhutia & Ganapathy, 2016). SLC1A5 (ASCT2) and SLC38A5 represent the best characterized glutamine transporters; both of them are upregulated by MYC in glioma cell lines (Wise et al, 2008), and a positive correlation between N‐MYC amplification and ASCT2 expression has been observed in neuroblastoma cells (Qing et al, 2012; Ren et al, 2015). Such membrane proteins transport also additional amino acids, like serine and alanine (Bhutia & Ganapathy, 2016), that can be channeled into biosynthetic pathways.

A major function of glutamine in cancer is to enter the TCA cycle in the form of α‐ketoglutarate (α‐KG), through a process termed glutaminolysis. MYC promotes the glutaminolytic pathway by upregulating the expression of GLS, encoding for the enzyme glutaminase, which catalyzes the conversion of glutamine to glutamate (Wise et al, 2008; Gao et al, 2009). GLS expression is upregulated by MYC both directly or indirectly, through MYC‐mediated inhibition of miR‐23 expression, which in turn suppresses GLS translation (Gao et al, 2009). Another study demonstrated that MYC controls GLS levels in an mTOR‐dependent way (Csibi et al, 2014). In vivo studies confirmed that MYC impacts on the metabolism of glutamine mainly by enhancing the expression of GLS1, as demonstrated in both liver and renal carcinoma models (Yuneva et al, 2012; Shroff et al, 2015; Xiang et al, 2015). Glutamine‐derived glutamate enters the TCA cycle as α‐KG either by direct deamination catalyzed by glutamate dehydrogenase (GLUD) or through the action of transaminases, which catalyze the transfer of the amino group to oxaloacetate (glutamate oxaloacetate aminotransferase or GOT) or pyruvate (glutamate pyruvate aminotransferase or GPT) with the simultaneous production of the non‐essential amino acids aspartate and alanine, respectively. Increased levels of both aspartate and alanine have been detected in MYC‐driven renal carcinoma (Shroff et al, 2015) and in pre‐tumorigenic liver expressing high MYC levels (Hu et al, 2011). Decreased levels of GPT and GOT have been observed in MYC‐dependent osteosarcoma cells upon suppression of MYC expression (Anso et al, 2013). Dependency of cancer cells on either GLUD or transaminases activity has been correlated with high levels of MYC (Yang et al, 2009; Qing et al, 2012; Korangath et al, 2015).

The TCA cycle is used as a source of multiple precursors, which are diverted into biosynthetic pathways in order to sustain the requirements of highly proliferative cells. Hence, the anaplerotic function of glutamine, that is, the replenishment of TCA cycle intermediates, is important for supporting anabolic reactions (DeBerardinis et al, 2008). For example, de novo synthesis of fatty acids uses acetyl‐CoA as a substrate and enzymes belonging to this pathway are often upregulated in cancer. Acetyl‐CoA can be derived from both glucose and glutamine, through the cytosolic conversion of TCA‐produced citrate by ATP citrate lyase (ACLY). In the cytosol, acetyl‐CoA is converted by acetyl‐CoA carboxylase (ACC) to malonyl‐CoA, which is in turn used for the biosynthesis of palmitate by fatty acid synthase (FASN). Several studies suggest that MYC may promote the synthesis of fatty acids, although deeper mechanistic investigations will be needed to corroborate such evidences. MYC promotes fatty acid biosynthesis in rat fibroblasts through increasing the expression of all three enzymes (Morrish et al, 2009, 2010; Edmunds et al, 2014). Metabolic analysis performed on a combination of engineered cells, in vivo models, and human tumor samples showed that deregulated MYC in prostate cancer specifically enhances lipid metabolism (Priolo et al, 2014). Specific lipid production associated with the oncogenic activity of MYC has been observed also in lymphomas. Increased levels of phosphatidylglycerol, a precursor of cardiolipin, are found on the outer mitochondrial membrane, in MYC‐driven lymphoma tumor models compared to normal thymus (Eberlin et al, 2014). The same lipid is also increased in in vivo models of MYC‐driven hepatocellular carcinoma and renal cell carcinoma (Perry et al, 2013; Shroff et al, 2015).

Glutamine‐derived glutamate can also be used as substrate for proline synthesis. Although the conversion between the two amino acids is reversible, MYC preferentially suppresses the proline to glutamate production in favor of the opposite reaction, as demonstrated in Burkitt's lymphoma and prostate cancer (Liu et al, 2012b, 2015). The first effect is due to MYC‐induced expression of miR‐23b*, which in turn posttranscriptionally suppresses the translation of PRODH, encoding for the enzyme proline oxidase (or proline dehydrogenase). Interestingly, miR‐23b* is produced from the same transcript of miR‐23, which suppresses GLS translation; thus, oncogenic MYC exerts a coordinated effect in promoting the catabolism of glutamine, rather than its synthesis, as demonstrated also by the inverse correlation between MYC and GS levels (Liu et al, 2012b, 2015). At the same time, MYC enhances the expression of enzymes involved in the biosynthesis of proline, namely P5C synthase (P5CS) and P5C reductase (PYCR) (Liu et al, 2012b, 2015). Blocking proline biosynthesis inhibited cell growth in a way independent from exogenous proline supplementation. The synthesis of proline yields NAD(P)⁺ through the conversion of 1‐pyrroline‐5‐carboxylate (P5C) to proline catalyzed by PYRC, which is in turn required for sustaining both glycolysis and the oxidative arm of the PPP (Liu et al, 2012b, 2015).

Importantly, in vivo studies document a remarkable flexibility in the metabolic features of MYC‐driven tumors. For example, MYC‐induced liver tumors consume glutamine, while lung tumors produce it. This difference is mainly due to difference in expression of glutamine synthetase (GS), which catalyzes the cytosolic production of glutamine from glutamate and ammonia, and is expressed in lung, rather than in liver tumors (Yuneva et al, 2012). Expression of GS expression makes cancer cells independent of the external glutamine supply (Kung et al, 2011). Interestingly, oncogenic MYC can also drive and increase in GS expression. Bott et al showed that MYC induces the expression of thymine DNA glycosylase in breast cancer cell lines. This in turn promotes the demethylation of the GS promoter, thus enhancing GS expression. Increased glutamine production supports cell proliferation through increased nucleotide biosynthesis (Bott et al, 2015). Thus, it is likely that MYC can differentially promote the synthesis of glutamine or its metabolism through the TCA cycle, depending on the context and the metabolic needs of the tumor. Similarly, while MYC often promotes the biosynthesis of lipids, MYC overexpression in triple‐negative breast cancer correlates with an increased dependency on mitochondrial fatty acid oxidation, whose inhibition has been proposed as a possible therapeutic approach (Camarda et al, 2016). The divergence of effects mediated by oncogenic MYC highlights the importance of in vivo studies to better understand the metabolic requirements of tumors, since both the interaction with the tumor microenvironment and an intact organ structure affect metabolic dependencies. This has been clearly demonstrated using K‐RAS‐driven lung tumor cells, which are glutamine dependent in vitro, but not in vivo (Davidson et al, 2016).

Oncogenic MYC enhances the biosynthesis of nucleotides

Increased nucleotide biosynthesis is required to sustain the enhanced transcription and replication driven by MYC. The biosynthesis of purine and pyrimidine nucleotides requires substrates whose metabolism is controlled by MYC, for example, the amino acids aspartate and glycine, in addition to glutamine, while glucose metabolism provides both ATP and the ribose moiety necessary for the biosynthesis of phosphoribosylpyrophosphate (PRPP), through the PPP. MYC directs the channeling of these substrates toward the biosynthesis of nucleotides and promotes their usage by enhancing the expression of enzymes involved in virtually all steps of these pathways (Liu et al, 2008; Mannava et al, 2008). In addition, MYC promotes the reduction in ribonucleotides to deoxynucleotides through the upregulation of RRM2, encoding for the small subunit of the enzymatic complex ribonucleotide reductase (Mannava et al, 2008). Thus, high MYC levels result in an increase in both ribo‐ and deoxyribonucleotide levels, which is observed in multiple cellular systems (Bester et al, 2011; Cunningham et al, 2014). Phosphoribosylpyrophosphate is a key substrate for both the purine and the pyrimidine biosynthesis. In the first case, the purine ring is assembled around a PRPP molecule, while in the second pathway PRPP is attached to the synthetized pyrimidine ring. Oncogenic MYC upregulates the expression of PRPS2, encoding for the enzyme, which catalyzes PRPP synthesis from ribose 5‐phosphate and ATP (Mannava et al, 2008; Cunningham et al, 2014). Cunningham et al showed that MYC exerts a translational control on PRPS2, mediated by pyrimidine‐rich translational elements present in the 5′‐UTR of PRPS2 and not of its related isoform PRPS1. Effects on the translation of specific mRNA are achieved by MYC through the upregulation of the eukaryotic translation initiation factor 4E (eIF4E), the limiting factor of the initiation complex eIF4F (Ruggero et al, 2004). This mechanism may coordinate protein and nucleotide biosynthesis in an oncogenic setting. Interestingly, PRPS2 is dispensable for the development of normal B cell but its inhibition is synthetic lethal with MYC overexpressing cells, possibly by limiting the amount of nucleotides available for assembly of ribosomes and the consequent protein synthesis (Cunningham et al, 2014).

Polyamine biosynthesis

A possible use of glutamine is the biosynthesis of polyamines (putrescine, spermidine, and spermine). Polyamine are polycations able to interact with negatively charged molecules such as nucleic acids and protein. They have multiple functions in the regulation of cell growth, modulation of histone acetylation levels, and intracellular signaling (Minois et al, 2011). Increased polyamine levels are associated with a variety of cancers, and therapeutic approaches targeting the metabolism of polyamine have been developed (Murray‐Stewart et al, 2016). The biosynthesis of polyamine begins from ornithine and requires decarboxylated S‐adenosyl‐methionine produced from methionine for the subsequent steps. Although glutamine‐derived glutamate or proline represent alternative substrates for the production of ornithine, compared to the mostly used arginine, in activated T lymphocytes MYC has been observed to drive the biosynthesis of polyamines through the increased expression of enzymes, which mediate both the conversion of glutamate and proline to ornithine [aldehyde dehydrogenase 18 family member A1 (Aldh18a1), proline dehydrogenase (Prodh) and ornithine aminotransferase (OAT)] and the biosynthesis of polyamines [ornithine decarboxylase (ODC), spermine synthase (SRM) and spermidine synthase (SMS)] (Bello‐Fernandez et al, 1993; Trubiani et al, 1999; Nilsson et al, 2005; Wang et al, 2011; Funakoshi‐Tago et al, 2013; Ruiz‐Perez et al, 2015). The coordinated expression of genes encoding polyamine biosynthetic enzymes is also observed in a mouse model of MYC‐driven B‐cell lymphoma (E_μ‐Myc). Increased expression of ODC, the rate‐limiting enzyme in the polyamine biosynthesis, is a critical mediator of MYC‐induced tumorigenesis, since genetic or drug‐mediated inhibition of ODC results in an inability of MYC to downregulate the cyclin‐dependent kinase inhibitors p21 and p27, thus impairing the progression through the cell cycle. Intriguingly, the activity of ODC and its metabolic products appear to be necessary for tumor development rather than maintenance, before the occurrence of additional genetic events like the loss of the tumor suppressor (Nilsson et al, 2005).

Metabolic controls of MYC activity

MYC is both upstream and downstream of mTORC1

Expression of MYC is downstream of multiple control mechanisms that are regulated by nutrient levels and respond to metabolic stress (Fig 2). A paradigm example is the mTOR pathway, the nutrient‐sensing pathway in mammalian cells. The activity of mTORC1, a central kinase in the pathway, depends on both the availability of nutrients and on the cellular energetic status (Gonzalez & Hall, 2017). Glutamine positively controls mTORC1 activity. Glutamine activates mTORC1 by either promoting the uptake of leucine, which in turn directly activates mTORC1 (Nicklin et al, 2009) or through the production of α‐KG via the glutaminolytic pathway (Duran et al, 2012). The first mechanism is promoted by the amino acid transporter LAT1, which exchanges intracellular glutamine with extracellular leucine. Both MYC and N‐MYC enhance the expression of SLC7A5, which encodes for a subunit of LAT1 (Gao et al, 2009; Hayashi et al, 2012; Qing et al, 2012); thus, MYC collectively promotes both the uptake of glutamine and its exchange with leucine. Leucine is also an activator of the enzyme glutamate dehydrogenase (GDH), which converts glutamate to α‐KG (Couee & Tipton, 1989). Thus, by enhancing the uptake of leucine, MYC potentially contributes to stimulate the glutaminolytic pathway. In addition to enhancing the uptake of nutrients, MYC sustains the function of mTORC1 also by driving the synthesis of macromolecules, like translation factors and ribosomal components, necessary for protein biosynthesis (Lin et al, 2008; van Riggelen et al, 2010; Pourdehnad et al, 2013).

Figure 2. Metabolic control of MYC protein levels.

Sufficient levels of nutrients like glucose, glutamine, and their derived metabolites maintain high levels of MYC. The graph summarizes mechanisms discussed in the text.

Conversely, mTORC1 controls the translation of MYC (West et al, 1998; Shahbazian et al, 2010; Csibi et al, 2014; Leu et al, 2016). An exemplar mechanism indicating the tight connection between MYC and mTORC1 activity is provided by the work of Csibi et al (2014), who observed that the mTORC1 target S6K1 promotes MYC translation by acting on its 5′‐UTR and that MYC, in turn, increases the protein levels of GLS, likely in a posttranscriptional way since no changes in the GLS mRNA levels were observed. MYC and mTORC1 coordinate multiple processes that promote cellular growth and proliferation and overlapping metabolic functions between these two nodes have been demonstrated, for example, both drive the expression of nucleotide biosynthetic enzymes (Liu et al, 2008; Mannava et al, 2008; Ben‐Sahra et al, 2013, 2016).

FoxO proteins antagonize MYC function

A second example of the interplay between MYC expression and metabolism is provided by the forkhead box O (FoxO) family of transcription factors. FoxO proteins are activated in response to various kinds of stress, including metabolic and oxidative stress (Eijkelenboom & Burgering, 2013). For example, activation of AMPK‐activated protein kinase (AMPK) stimulates FoxO activity (Greer et al, 2007). Following increase in the AMP/ATP ratio, indicator of changes in the cellular energetic status, activated AMPK phosphorylates FoxO3 on multiple sites and promotes its transcriptional activity. Such effect supports cell survival under stress conditions due to the FoxO3‐mediated activation of genes involved in stress resistance, for example, by promoting the metabolism of alternative sources in absence of glucose, maintaining intracellular energetic homeostasis through autophagy or reducing oxidative stress (Greer et al, 2007; Chiacchiera & Simone, 2009; Li et al, 2009).

FoxO proteins antagonize activation of multiple target genes by MYC via several different mechanisms (Bouchard et al, 2004). On multiple MYC target genes, non‐phosphorylated FoxO blocks the loading of RNAPII on the promoter, thereby blunting the ability of MYC to promote transcriptional elongation by RNAPII (Bouchard et al, 2004). In hypoxia, HIF‐1‐α activates FoxO3A, which displaces MYC from the promoter of mitochondrial genes (e.g., MRPL12 (mitochondrial ribosomal protein L12), ACO2 (aconitase 2), LARS2 (mitochondrial leucyl‐tRNA synthetase), and OXNAD1 (oxidoreductase NAD‐binding domain containing 1)) (Jensen et al, 2011). Thus, by disabling the MYC‐mediated induction of mitochondrial genes, FoxO3A promotes the shift toward a glycolytic metabolism. MYC function can also be antagonized by FoxO3A through the increased expression of Mad/Mxi family proteins, particularly Mxi1‐SRα that binds to MAX and prevent the formation of the MYC/MAX heterodimers (Delpuech et al, 2007). Activation of FoxO3A can also destabilize MYC and induce its proteasomal degradation. The decrease in MYC protein levels diminishes the production of reactive oxygen species (ROS) by reducing mitochondrial activity, thereby preventing harmful effects associated with increased ROS levels such as genomic instability due to ROS‐induced DNA damage. (Ferber et al, 2012). The 3′‐UTR of MYC is also targeted by FoxO‐dependent stress‐responsive circuits. FoxO3A can limit MYC levels by inducing the expression of miR‐34b/c, which bind to the 3′‐UTR of MYC, thereby inhibiting its translation (Kress et al, 2011). Different stimuli can mediate the induction of specific miRNAs through FoxOs. In glioblastomas, for example, FoxOs induce both miR‐34c and miR‐145 to repress MYC (Masui et al, 2013).

Repression of MYC function by FoxOs is antagonized by mTORC2, which promotes FoxOs acetylation, and by FoxOs phosphorylation following PI3K‐AKT activation (Masui et al, 2013). Both pathways are physiologically activated by growth factors stimulation and hyperactivated in a cancer context (Fruman & Rommel, 2014). Consistently, the ability of FoxO proteins to suppress MYC function in response to metabolic stress may be compromised in tumors. For example, regulation of MYC via the 3′‐UTR is disrupted during colon cancer progression, through the silencing of miR‐34b/c expression (Kress et al, 2011). Similarly, the FoxO/Mxi1‐SRα/miR‐145 axis, which antagonizes MYC function and decreases its levels, operates in vivo in Tsc1 knockout (i.e., mTORC1 activation) polycystic kidneys, but is lost in Tsc1 knockout kidney tumors (Gan et al, 2010). Intriguingly, disruption of the FoxO/MYC regulatory circuit can have both positive and negative effects on tumor growth. For example, FoxO3A knockdown impaired the growth of tumors in a xenograft model, likely because they maintain the mitochondrial function under hypoxia (Jensen et al, 2011).

Regulation of MYC levels and function by glucose

Several mechanisms regulate both MYC levels and function in response to glucose availability. Increased degradation of MYC following glucose starvation and oxygen deprivation maintains colorectal cancer cells viable, although metabolically inactive (Okuyama et al, 2010; Wong et al, 2013). MYC is O‐glcNAcylated and stabilized by this posttranslational modification (Itkonen et al, 2013; Sodi et al, 2015). Following glucose deprivation, the decreased OGT activity and consequent increased MYC degradation allow cell survival in hepatocarcinoma cells (Buren et al, 2016). Intriguingly, glucose deprivation not only regulates MYC levels via proteasomal degradation, but also induces calpain‐mediated proteolysis of MYC, which results in the formation of a truncated protein localized in the cytosol (“MYC‐nick”; Conacci‐Sorrell et al, 2014). MYC‐nick comprises the N‐terminal region of MYC and has transcription‐independent functions since it lacks the nuclear localization signal and the DNA binding domain (Conacci‐Sorrell et al, 2010). Strikingly, MYC‐nick protects cells from stress‐induced cell death, since it promotes the acetylation of cytosolic proteins, involved, for example, in sustaining autophagy, via the same domain that recruits histone acetylases in the nucleus (Conacci‐Sorrell et al, 2014).

MYC is part of an extended network comprising MAX, MXD, MLX, and Mondo proteins. Mondo transcription factors (MondoA and chREBP) represent the nutrient‐sensing branch of this network because their activity is directly stimulated by the glycolytic metabolite glucose 6‐phosphate (Kaadige et al, 2010). Reduced MYC protein levels following glucose starvation may alter the existing balance between each component of the network. Since MYC drives the uptake of glucose by competing with MondoA and downregulating TXNIP expression (Shen et al, 2015), one could speculate that in absence of glucose the increased degradation of MYC could positively regulate MondoA and TXNIP function, potentially contributing to maintain the cells in a metabolically inactive state.

Glucose starvation does not uniformly lead to MYC downregulation (Wu et al, 2015). Under this condition, upregulation of MYC protein levels occurs in hepatocellular carcinoma cells and supports the synthesis of glutathione through the upregulation of the serine synthesis pathway. Increased SSP activity, in turn, enables cell survival by maintaining the cellular redox balance (Sun et al, 2015). Glucose deprivation can also induce the unfolded protein response (UPR) since it interferes with protein glycosylation (Xu et al, 2005). Induction of UPR in turn increases MYC levels in multiple myeloma cells via an internal ribosomal entry site (IRES) that is present in the 5′‐UTR of the MYC mRNA (Shi et al, 2016). An increase in MYC levels could therefore be responsible for driving apoptosis under these conditions.

MYC and glutamine: safe or addicted?

The role of glutamine levels as possible upstream regulator of MYC has received less attention. In colorectal cancer cells, translation of MYC is controlled by glutamine via a sequence element in the 3′‐UTR of the MYC mRNA (Dejure et al, 2017). Surprisingly, this regulatory sequence does not respond to TCA cycle intermediates, but to intracellular levels of glutamine‐derived adenosine nucleotides. The decrease in MYC levels following glutamine starvation and the consequent reduction in nucleotide levels reduces transcription by RNAPII. When MYC is ectopically expressed in glutamine‐starved cells, RNAPII stalls during transcription on hundreds of genes and this correlates with formation of R‐loops, which can cause DNA damage and genomic instability (Dejure et al, 2017). Notably, cells undergo apoptosis under these conditions in the absence of a MYC‐dependent induction of pro‐apoptotic genes, suggesting that stalling of RNAPII and R‐loop formation are pro‐apoptotic events. In this sense, coupling of MYC expression to metabolic signals may be critical for cells to maintain genomic stability.

The precise pathway by which adenosine levels regulate MYC translation has not been resolved. The MYC 3′‐UTR is the target of multiple miRNAs, which inhibit the translation of an mRNA. Glutamine starvation can induce the phosphorylation of p53 on Ser15, which results in the stabilization of p53. This is required for allowing cell survival under stress conditions (Reid et al, 2013). p53, in turn, can trigger the activation of stress‐responsive pathways, which can potentially target the 3′‐UTR of MYC (Sachdeva et al, 2009; Cannell et al, 2010; Lezina et al, 2013). Regulation of RNA‐binding proteins (RBPs) targeting the 3′‐UTR of MYC by cellular metabolism has been also extensively characterized (Barreau et al, 2005). For example, a cleavage product of the human antigen R (HuR) (HuR‐CP1) associates with MYC 3′‐UTR, blocking its translation after prolonged hypoxia (Talwar et al, 2011). HuR also regulates MYC stability in presence of polyamine, whose synthesis is also dependent on glutamine (Liu et al, 2009). Finally, multiple metabolic enzymes, for example, glycolytic enzymes, moonlight as RNA‐binding proteins via their nucleotide‐binding motifs (Castello et al, 2012, 2015); hence, the regulation of MYC translation may be directly coupled to the metabolic status of cells.

Restraining MYC can prevent apoptosis and metabolic stress

These mechanisms enable cells to maintain cell viability under metabolically unfavorable conditions, through the regulation of MYC levels. On the other side, multiple studies have linked deregulated MYC activity to the induction of cell death as a form of intrinsic tumor suppression (Lowe et al, 2004). At least in some instances, MYC‐driven apoptosis can be linked to sustained anabolic reaction following nutrient depletion. One of the best characterized examples is provided by the MYC‐mediated glutamine addiction, according to which cells expressing high levels of MYC are sensitive to glutamine deprivation or glutaminase inhibition (Yuneva et al, 2007; Wise et al, 2008; Qing et al, 2012; Nieminen et al, 2013; Wiese et al, 2015). Similarly, rat fibroblast cells overexpressing MYC undergo apoptosis when deprived of glucose (Shim et al, 1998) and MYC‐driven lymphoma cells are particularly sensitive to inhibition of lactate export (Doherty et al, 2014). Since such studies made use of MYC transgenes, one possibility to explain opposing observation is the fact that the absence of UTR elements does not allow posttranscriptional mechanisms of regulation to take place. Several mechanisms by which deregulated MYC levels induce metabolism‐driven apoptosis have been identified (Fig 3).

Figure 3. Metabolic adaptation through the regulation of MYC levels.

Proposed mechanisms by which deregulated MYC causes metabolic stress and apoptosis. See text for details.

• Enhanced expression of MYC causes a decrease in cellular ATP levels and activates AMP‐activated kinase (AMPK) (Liu et al, 2012a; von Eyss et al, 2015). MYC‐induced metabolic stress can sensitize cells toward apoptosis, in part since AMPK phosphorylates p53 and activates its mitochondrial pro‐apoptotic functions (Nieminen et al, 2007, 2013).

• Deregulated MYC can cause transcriptional elongation in the absence of sufficient nucleotides and induce stalling of RNA polymerases and R‐loop formation (Dejure et al, 2017).

• Deregulated MYC can cause a catastrophic metabolic imbalance. MYC is part of extended network of helix‐loop‐helix transcription factors and its effects on metabolism are counterbalances by a nutrient‐sensing member of the network, MondoA. Reduced MondoA levels are tolerated in normal cells; however, deregulated of MYC renders cells dependent on MondoA, since MondoA counterbalances MYC effects on glutaminolysis and lipid biosynthesis. In the absence of MondoA, cells undergo apoptosis, which can be rescued by fatty acid (oleic acid) supplementation (Carroll et al, 2015).

Conclusions

While growth factor‐dependent controls of MYC expression often act via the MYC promoter, the expression of MYC is controlled mainly by posttranscriptional mechanisms in response to nutrient supply and metabolic stress. There is little evidence that metabolic controls of MYC expression and function are disrupted in tumor cells. To the contrary, such controls are likely to be under strong positive selective pressure during tumorigenesis to ensure cell survival under stressed conditions. Currently used transgenic models often bypass these controls, since they use transgenes that express the MYC coding sequence only and/or employ stable alleles of MYC. It is likely that—by bypassing metabolic controls of MYC expression—current tumor modeling strategies overestimate the metabolic liabilities of MYC‐driven tumors.

The EMBO Journal (2017) 36: 3409–3420