generate a proton gradient (ΔΨM) and to establish the protomotive force necessary to drive ATP generation *via* Complex V. Excess cytoplasmic reducing equivalents in the form of NADH may be transferred to the mitochondria *via* the glycerol-3-phosphate and malate–aspartate shuttles and can potentially support OXPHOS when AcCoA levels are low. During hypoxia, glucose uptake, glycolytically derived ATP synthesis, and the "A" isoform of LDH (LDH-A)-catalyzed conversion of pyruvate to lactate are accelerated due to an inability of the ETC to reduce molecular oxygen to water and generate TCA cycle-derived ATP. Most pyruvate will be converted to lactate, thereby generating more NAD+ and sustaining anaerobic glycolysis. This positive feedback loop ensures a constant and balanced source of ATP as an energy source and the regeneration of NAD+ to serve as an electron acceptor for continued glycolysis. By contrast, dividing cells have "divided loyalties" with regard to the disposition of glycolytic intermediates, with some being used to generate pyruvate and others being used as anabolic building blocks. Just as glycolysis and lactate production engage in a positive feedback loop to provide a balanced amount of NAD+ and NADH in support of one another's activity, the pentose phosphate pathway (PPP) and the biosynthesis of fatty acids and sterols, generate NADPH and NADP+, respectively, to support each others' pathways. The pyruvate dehydrogenase complex (PDC), which includes pyruvate dehydrogenase, is the key enzymatic complex that connects glycolysis to the TCA cycle. It catalyzes the conversion of the last product of glycolysis, pyruvate, to the entry-level substrate for the TCA cycle, AcCoA. In doing so, it contributes one-fourth of the NADH that is produced by a single round of the TCA cycle.

The PKM1 and PKM2 isoforms differ from one another as a result of mutually exclusive alternate mRNA splicing such that the PKM1 transcript encodes exon 9 but not exon 10 and PKM2 encodes exon 10 but not exon 9. The splicing decision is governed by at least three heterogeneous nuclear ribonucleoproteins, hnRNP1, hnRNP2, and hnRNP, which bind to intron regions flanking exon 9 and negatively regulate its splicing. Interestingly, hnRNP1 and hnRNP2 are positively regulated by Myc (141, 142). However, the switch to aerobic glycolysis, by whatever means, hardly signals an irreversible commitment; indeed, not only is the Warburg effect plastic but many tumors-even those of similar types are more dependent upon OXPHOS than glycolysis for their energy requirements and/or can rapidly balance these two processes to suit their needs (143). In addition to regulating glycolysis, Myc also exerts significant influence over mitochondrial structure and function. Initially relegated to a metabolic backwater following the realization that many cancer cells rely on the Warburg effect at the expense of OXPHOS, mitochondria do in fact play important roles in cancer metabolism (144, 145). Indeed, certain cancers remain highly reliant on OXPHOS as a means of energy production and show little predilection for Warburg-type metabolism (146). Analogous to glycolysis, however, both mitochondria and the energy producing pathways they encompass undergo significant structural and functional revisions in response to Myc deregulation.

Among the first studies to examine Myc's effect on mitochondrial structure and function were those of Li et al. (147). Utilizing P493-6 human B cells transfected with a tetracycline-inducible human Myc transgene, they showed that mitochondrial mass increased approximately twofold within 72 h of Myc induction, as measured by nonyl acridine orange staining, MitoTracker staining and mitochondrial DNA (mtDNA) content. Oxygen consumption increased to a similar extent thereby indicating a close relationship between structure and function. Transcriptional profiling also identified approximately 200 Myc-responsive genes encoding mitochondrial proteins, most of which were upregulated. Most notable among these was the transcript encoding Tfam, a nuclear-encoded mitochondrial transcription factor that also participates in mtDNA replication (148–150). The *TFAM* gene proximal promoter was also shown to contain a Myc-binding E-Box element. In serum-stimulated fibroblasts, endogenous Myc was shown to bind the same site (147). Although no functional studies were performed, it was assumed that this positive effect of Myc on mitochondrial biogenesis was accompanied by a parallel increase in TCA cycle activity and OXPHOS.

Elucidating the role of endogenous Myc in the regulation of metabolic pathways was hampered for quite some time by the lack of a suitable knockout model. This is because even shortterm Myc depletion in virtually all cell lines is accompanied by cell cycle arrest, apoptosis, or differentiation, thereby severely compromising long-term studies (50, 151, 152). Similarly, whole body inactivation of the *myc* gene is an embryonic lethal (153). Thus, the generation of a *myc−/−* cell line from rat fibroblasts by Mateyak et al. in 1997 (154) provided a tremendous technical advance despite the fact that it remains unresolved as to how these cells survive and replicate.

Although viable, the *myc−/−* fibroblasts described by Mateyak et al. (154) are extremely abnormal. For example, they divide only once every 2–3 days versus every 18–24 h for the *myc+/+* parental line, display an extremely flattened morphology and are highly contact inhibited. Cell cycle regulation is severely compromised at multiple points, with *myc−/−* cells showing a *ca.* 12-fold reduction in the expression of CyclinD–Cdk4 and CyclinD–Cdk6 complexes during the G0/G1 transition and delayed activation of CyclinE–Cdk2 and CyclinA–Cdk2 complexes (145, 155). That most of these abnormalities can be rescued at least partially with retrovirally expressed Myc, the Myc homologs N-Myc and L-Myc, or the Myc-target genes MYCT1/MT-MC1, HMG-IY and SHMT (156, 157) indicates that the various phenotypes of *myc−/−* fibroblasts are directly related to *myc* gene inactivation and do not represent compensatory and Myc-independent growthenhancing adaptations.

The absence of Myc was also reflected in the mitochondrial structure and metabolism of these cells. Graves et al. (158) showed that, compared with *myc+/+* fibroblasts, the mitochondria of *myc−/−* fibroblasts were smaller, fewer in number, deficient in cristae and poorly interconnected. The activation of a Mycestrogen receptor fusion protein (MycER) by 4-hydroxytamoxifen (4OHT) complemented these defects, although, surprisingly, it required 4–5 weeks before maximal mitochondrial mass was restored. The extremely low ΔΨM of these cells (*myc−/−* MycER) cells also increased in parallel over this same time period. This provided indirect evidence that Myc was likely controlling some aspect of the TCA cycle and/or the availability of reducing equivalents needed to drive the ETC. Following 4OHT's removal and the silencing of Myc, mitochondrial mass, ΔΨM and interconnectivity returned to near the baseline levels of *myc−/−* cells over another prolonged period exceeding 10 days (158).

In addition to the above major structural defects, which confirmed and extended those of Li et al. (147), mitochondrial function was also severely compromised in *myc−/−* cells in a manner that mirrored their structural defects. For example, the basal oxygen consumption rate (OCR) of *myc−/−* cells was about half that of *myc+/+* cells and only 15% that of *myc−/−* cells stably reconstituted with a lentiviral vector that drove high level, constitutive Myc expression (*myc−/−* Myc cells). The most prominent effect of Myc on OCR was seen in *myc−/−* Myc cells where the maximum respiratory capacity in response to the de-polarizing agent FCCP was >20 times higher than that of *myc−/−* cells and 6 times higher than that of *myc+/+* cells. Because Myc positively regulates virtually all glycolytic genes (96, 123, 124, 159), the basal rate of glycolysis in *myc−/−* cells was also about half that of *myc+/+* cells and one-third that of *myc−/−* Myc cells (158).

Consistent with their markedly impaired OXPHOS and glycolysis, *myc−/−* cells showed a nearly 70% reduction in basal ATP levels, which normalized following Myc re-expression. In all three cell lines (*myc−/−*, *myc+/+*, and *myc−/−* Myc), exposure to 2-DG caused a more pronounced ATP depletion than did the inhibition of OXPHOS with rotenone. Taken together, these results suggested that at least half the energy in these cells was derived from glycolysis. Consistent with their ATP deficient state, *myc−/−* cells expressed high levels of activated (phosphorylated) AMP-activated protein kinase (AMPK), a serine/threonine kinase that responds to ATP depletion (or more precisely to a high AMP:ATP ratio) by upregulating ATP-generating pathways and downregulating ATP-consuming pathways (160–162). However, since many of the energy-sparing and energy-generating effects of AMPK rely on the upregulation of Myc (160, 163, 164), AMPK seems to be unable to achieve a state of true energy equilibrium in *myc−/−* cells, thereby leading to its constitutive activation in the face of a chronic energy deficit. The restoration of Myc in *myc−/−* Myc cells did lead to AMPK dephosphorylation that correlated with the normalization of ATP levels (160).

The observation that *myc+/+* and *myc−/−* Myc cells contained identical ATP levels could not initially be reconciled with the finding that the latter cells had significantly higher rates of glycolysis and OXPHOS. This discrepancy was resolved by showing that the ATP half-life in the latter cells was nearly 50% shorter (2.6 versus 3.6 min) (158). This was consistent with the previous finding that *myc−/−* WT cells had significantly faster growth rates than *myc+/+* cells and thus likely utilized more ATP (165).

To further understand the role of endogenous Myc in maintaining basal rates of glycolysis and OXPHOS in transformed cells, Graves et al. (158) utilized the conditional, doxycyclineregulatable expression of a short hairpin RNA directed against Myc to silence the oncoprotein's expression in A549 human small cell lung cancer cells, which normally express high levels of Myc. Conforming to the findings in the above-discussed rat fibroblast studies, the knockdown of Myc was associated with marked growth inhibition, a flattened cellular morphology, reduced mitochondrial mass, and the collapse of ΔΨM.

Relative to *myc+/+* and *myc−/−* Myc cells, *myc−/−* cells demonstrated abnormalities in overall structure and function of the ETC (158). Among these were reduced amounts of the so-called "supercomplexes" (SCs) between Complexes I, III, and IV, which allow for more efficient electron transfer (166, 167). Consistent with their atrophic mitochondrial cristae, which are believed to serve as a platform for the formation and accretion of SCs (168, 169), *myc−/−* mitochondria also contained lower levels of Complex I, II, and III as well as both the monomeric and dimeric forms of Complex V ATPase (Vm and Vd, respectively) relative to *myc+/+* cells. In general, SC function in *myc−/−* cells, as measured by *in situ* enzymatic activity of individual complexes separated by non-denaturing blue native gel electrophoresis (BNGE), closely matched Coomassie Blue staining patterns. *myc−/−* fibroblast mitochondria also contained significant levels of an enzymatically inert complex (Complex "X") that was shown by mass spectroscopy to be comprised of multiple subunits from Complexes II–V. It was speculated that during periods of relative oxidative quiescence, Complex X functions as a reservoir for certain mitochondria proteins, which can be rapidly summoned and assembled into their respective ETC complexes in response to increased metabolic needs. This seems like a logical cellular strategy in that cells with depleted energy levels as a result of ETC dysfunction might be better served by utilizing preexisting ETC components for rapid assembly and resumption of ETC function rather than expending even more energy by synthesizing them anew. On the other hand, the surprisingly long time it takes to restore normal mitochondrial structure and function in *myc−/−* cells (158) raises questions as to whether this is the true function of this complex.

Interestingly, *myc−/−* Myc cells showed only a partial normalization of ETC structure and function as measured by the above methods, despite their high-level Myc expression. Relative to *myc+/+* cells, *myc−/−* Myc cells contained only about half the levels of SCs, two-thirds the level Complex V monomers (Vm) and ~15% the level of Complex V dimers (VD) as measured by both enzymatic activity and BNGE. While both VD and VM possess ATP synthase activity, the dimer appears to be more important for dictating the shape of mitochondrial cristae (170). Because the high-level re-expression of Myc also greatly increased glycolysis, it was surmised that this failure to entirely normalize ETC structure and function was due to a combination of factors including structural changes to the mitochondria and their cristae, differences in the relative contribution of glycolysis and OXPHOS to the energy landscape, subtle nuances relating to the control of Myc protein expression, and differential cellular growth rates and their resulting anabolic requirements (158).

The normalization of mitochondrial morphology by the enforced re-expression of Myc in *myc−/−* Myc cells (158) suggested that Myc might influence mitochondrial fusion and/ or fission. The former process is regulated by the so-called "mitofusin" proteins such as Mfn1, Mfn2, and Opa1 whereas the latter process is regulated by the proteins Fis1 and Drp/ Dlp (171–175). Fusion is believed to maximize mitochondrial energy production by allowing old and/or damaged organelles to be "rejuvenated" by combining their contents with those of younger ones, thereby extending their life span and functional integrity and capacity (176, 177). By contrast, fission provides a mechanism by which mitochondrial mass can be reduced during periods of relative metabolic quiescence or Warburg-type respiration or by which defective and/or aged mitochondria can be eliminated. Both fusion and fission can exert significant influence upon mitochondrial energy production and cell survival (176, 178, 179).

Virtually, all the above mitochondrial fission and fusion proteins were expressed at higher levels *in* Myc replete cells relative to *myc−/−* cells making it unclear how, as an integrated group, they affected mitochondrial biogenesis and, if so, which of the processes this favored. The question was answered by experiments in which 4OHT-treated *myc−/−* MycER cells were separately transfected with mitochondrially targeted green or red fluorescent proteins (GFP or RFP, respectively). Cells from the two populations were then mixed and fused by exposure to polyethylene glycol and the rate of GFP+ and RFP+ mitochondrial fusion into "yellow" merged organelles was quantified either in the continued presence of 4OHT or following its removal. Cells actively expressing Myc showed nearly twofold higher rates of mitochondrial fusion compared with cells in which Myc had been silenced for 2 days by removing 4OHT. The faster rate of mitochondrial turnover in the former cells suggested that they were under constant pressure to maintain only the youngest and healthiest mitochondria to meet the increased metabolic needs of this energetically more demanding and faster growing population. Thus, along with affecting the levels of key mitochondrial transcription factors such as Tfam (123, 147), Myc also influences mitochondrial biogenesis and lifespan by modulating the levels of fission/fusion proteins.

Subsequent work demonstrated that alterations in mitochondrial structure and function can reciprocally impact the function of both endogenous and overexpressed Myc. In these studies, Sarin et al. (180) enforced the expression of the mitochondrial fission protein Drp/Dlp in Rat1a-MycER cells, leading to a state of non-stop, fission-induced mitochondrial fragmentation and a pronounced reduction in overall mitochondrial size, mass and interconnectivity. Accompanying this was a nearly 15-fold higher rate of mitochondrial fusion relative to control cells suggesting that Drp1/Dlp1-overexpressing cells constantly upregulate fusion in a futile compensatory attempt to offset their excessive Drp1/Dlp1-driven fission. Despite the fact that these cells expressed normal levels of Myc, their mitochondria were both structurally and functionally reminiscent of those from *myc−/−* cells. Structurally, their ETC complexes were defective; BNGE revealed a 28% reduction Complex I, a 45% reduction in Complex V and a 38% reduction in SCs. Furthermore, Complex "X," the proposed repository for certain ETC subunits in *myc−/−* cells (158), now appeared. Thus, enforced and uncorrected mitochondrial hyper fission leads to a loss of mitochondrial structural integrity resembling that of *myc−/−* cells in the face of otherwise normal Myc levels.

Further characterization of Drp1/Dlp1-overexpressing cells showed that, like *myc−/−* cells, they too had a critical energy shortage, with a >80% reduction in ATP levels and impaired glycolysis and OXPHOS. This energy-depleted state, coupled with the failure to adequately compensate for it, was evidenced by a *ca.* 30% decrease in mean cell volume and a >10-fold increase in phosphoAMPK (180). Collectively, these findings suggested that the price for such energy-conserving processes was a reduction in energetically demanding biomass accumulation. Most likely as a result of their abnormal ETC structure and/or their ATP deficit, Drp1/Dlp1 overexpressing cells, like *myc−/−* cells, expressed higher levels of ROS than control cells (181, 182). No obvious growth differences between control and Drp1/Dlp1-overexpressing cells were observed under standard conditions although the latter were significantly more resistant to apoptosis in response to Myc overexpression or serum deprivation and this was supported by the less pronounced release of cytochrome c from mitochondria. Treatment with 5-amino-1-b-d-ribofuranosyl-imidazole-4-carboxamide, an AMP analog that activates AMPK and increases ATP pools (161, 162, 183) doubled the ATP content of Drp1/Dlp1 overexpressing cells, normalized their size and increased their sensitivity to apoptotic stimuli (180). The prolonged survival of Drp1/Dlp1-overexpressing cells may reflect the fact that ATP

depletion tends to protect against apoptosis, perhaps by inhibiting caspases 3 and 8 and Apaf-1, and that, in some circumstances AMPK activation can restore or promote apoptosis (184–188). Collectively, these results show that enforced mitochondrial fission driven by Drp1/Dlp1 can override Myc's role in maintaining normal mitochondrial integrity and adequate ATP levels. Whether this is due to a lack of response of mitochondria as a result of their inability to fuse in response to Myc (as seems likely) or another effect of Drp1/Dlp1 remains to be determined.

The above studies showed that, at least in proliferating fibroblasts propagated *in vitro*, where nutrient supplies and oxygen concentrations are high and non-rate-limiting, both glycolysis and OXPHOS are subject to positive regulation by endogenous Myc. Moreover, they establish that the overexpression of Myc, to the levels required to drive proliferation and transformation, continues to exert a simultaneous positive effect on both glycolysis and OXPHOS. The effects are quite heterogeneous, are both direct and indirect and involve changes in transcripts encoding glycolytic enzymes and mitochondrial structural and functional components. These studies show that the Warburg effect and OXPHOS are by no means mutually exclusive. Rather they are better viewed as being complementary, with neither one being entirely dispensable (**Figure 2**).

FIGURE 2 | The balance between glycolysis and oxidative phosphorylation (OXPHOS) in normal and tumor cells. The sizes of the triangles are meant to indicate the relative metabolic activity under each of the depicted conditions. (A) Under aerobic conditions, glucose in quiescent, non-proliferating cells is converted in a linear fashion into pyruvate, which is then consumed by the TCA cycle to produce the bulk of ATP (Figure 1). (B) Under hypoxic periods in these same cells, OXPHOS is no longer able to furnish ATP. Energy production then switches rapidly to glycolysis, which can temporarily maintain energy supplies. Both glucose uptake and glycolysis can be highly upregulated although at the expense of generating considerable amounts of lactate. (C) In well-oxygenated, rapidly proliferating tumor cells and normal cells, glycolysis and OXPHOS are both upregulated and balanced so as to provide for the most efficient production of ATP, the most rapid biomass accumulation and the most rapid rate of proliferation. The metabolic building blocks for these processes are derived from both the glycolytic pathway and the TCA cycle, thus explaining the need for each. Examples of the first include the diversion of glucose-6-phosphate into the pentose phosphate pathway to provide pentose sugars for nucleotide synthesis (Figure 1), the diversion of 3-phosphoglycerate for the synthesis of purine nucleotides and the diversion of phosphoenol pyruvate for the synthesis of tyrosine, tryptophan, and phenylalanine. Examples of the second include the transport of citrate into the cytoplasm for use in fatty acid and sterol biosynthesis and the diversion of oxaloacetate into the cytoplasm for conversion in aspartate, asparagine, and pyrimidine nucleotides. While the graphic here depicts an equal contribution by both glycolysis and OXPHOS, their relative contribution to overall metabolism can shift in response to proliferative rates (indicated by the small blue boxes), oxygen tension, and the supply of environmentally derived nutrients. In rare cancers, mutations in select TCA cycle genes can impart increased, and less reversible, reliance on aerobic glycolysis as a means of generating energy, thus confirming to Warburg's original explanation of his eponymous effect (41–43).

The role of endogenous Myc in sustaining glycolysis and mitochondrial function has received additional support from studies in other cell types with several structurally and mechanistically distinct small molecule Myc inhibitors (189–191). HL60 promyelocytic cells exposed for only 2–5 days to these compounds dramatically reduced their ATP content, activated AMPK, accumulated neutral lipids and downregulated Myc as they underwent terminal myeloid differentiation. Because the manipulation of Myc had long been know to exert profound effects on the differentiation of hematopoietic and other cell types (68, 72, 77, 87, 192), Wang et al. repeated these experiments using two mechanistically distinct inhibitors of Complex I, metformin and rotenone (191). Decreased ATP, AMPK activation and myeloid differentiation were again noted, but Myc levels were unaffected. These studies supported the idea that ATP levels are a strong and Myc-independent determinant of differentiation, at least in myeloid cells (193). They further implied that a major role of Myc in differentiation is to maintain ATP levels, most likely with the purpose of allowing for the continued accumulation of biomass. This is also likely aided by Myc's ability to induce the expression of many genes involved in cell cycle progression (101, 194, 195). Differentiation may therefore represent one possible means of maintaining viability in response to energy-depleted states.

In other studies using a transgenic mouse model of neuroblastoma driven by the highly related Myc homolog N-Myc, Zirath et al. (196) showed that the treatment of tumor-bearing animals with the small molecule Myc inhibitor 10058-F4 (197), which also binds to and distorts the structure of N-Myc (198), inhibited tumor growth and promoted tumor differentiation as evidenced by neurite outgrowth. It also caused the accumulation of high levels of intracellular neutral lipid (196).

Activated T-cells are among the most rapidly dividing metazoan cells. Following antigen stimulation, they accumulate biomass for approximately 24 h and then enter a phase of rapid proliferation and clonal expansion, with cell division occurring as frequently as every 4 h (199). It has long been known that this replicative phase is associated with markedly increased glycolysis and glutaminolysis although the precise pathways needed to effect this metabolic reprogramming remain ill defined (200–202). Wang et al. (203) investigated Myc's contribution to the biomass accretion and proliferative expansion following *ex vivo* stimulation of murine T-cells with anti-CD3+ anti-CD28. Immediately following their initial 24 h growth period, control cells entered the expected rapid proliferative phase during which time they were subject to metabolomic profiling using mass spectroscopy. Wang et al. (203) found that these cells accumulated metabolites during the initial growth phase and then activated glycolysis and directed glucose into the PPP. Concurrently, FAO declined as did the delivery of pyruvate into the TCA cycle. By contrast, OXPHOS and glutaminolysis increased, with a significant amount of the glutamine-associated carbon and nitrogen ultimately being incorporated into α-ketoglutarate and nucleotides, respectively. This latter finding indicated that exogenous glutamine was directed along two distinct pathways, the first being the TCA cycle in which glutamine was converted to glutamate and then to α-ketoglutarate and the second being the purine synthesis pathway in which both the N3 and N6 positions of the purine ring are derived from the glutamine amide moiety. Thus, not unlike the case of Myc-overexpressing fibroblasts discussed earlier (158), T-cell activation was accompanied by increases in both glycolysis and OXPHOS although the source of substrates for the latter pathway shifted from fatty acids and glucose to glutamine. Impaired proliferation was observed when the cells were deprived of either glucose or glutamine or when glycolysis and glutaminolysis, but not FAO, were blocked pharmacologically.

To investigate the molecular basis for the above-described metabolic reprogramming, Wang et al. (203) excised Myc from *mycflox/flox* T cells following conditional activation of a 4OHTinducible CreER transgene and compared their *ex vivo* response to anti-CD3+ anti-CD28 activation to the above control *mycflox/flox* cells. They noted a severe impairment of both the initial mass accretion (growth) phase and the subsequent expansion phase. *In vivo* testing of these cells following their stimulation with staphylococcal enterotoxin B revealed a blunted response similar to that observed *ex vivo*, with a decrease in both the growth and activation phases. Metabolomic profiling showed that the accumulation of amino acids, nucleotides and lipids in these cells was lower than that measured in similarly activated *mycflox/flox* cells. Of note was that *myc−/−* cells still activated both ERK and AKT pathways at levels commensurate with those seen in control *mycflox/flox* cells. This supported the idea that the observed effects were not a consequence of an inability to respond to signals upstream from Myc but rather to the lack of Myc itself. Further metabolomic inquiry showed that *myc−/−* T cells had impaired FAO and glucose flux during both the growth and proliferative phase, accumulated less lactate and directed less glucose-derived carbon into the PPP. Consistent with these findings, the induction of several glycolytic enzymes and glucose transporters were also suppressed as was glutaminolysis. The PKM2 isoform was also less highly induced in *myc−/−* T-cells.

#### THE ROLE OF GLUTAMINOLYSIS IN METABOLIC REPROGRAMMING

It has been known since the mid-1950s that both normal and transformed cells share a particular predilection for exogenous glutamine and sometimes even prefer it to glucose as an energygenerating substrate (204–207). Indeed, some tumors have such exaggerated demands for this amino acid that they can deplete host plasma glutamine levels despite its being the most abundant amino acid (208, 209). Recent evidence supports the idea that glycolysis and glutaminolysis cooperatively support high rates of cell proliferation (210, 211).

Glutaminolysis offers several advantages that explain its ability to complement and/or replace glucose as an energy source (**Figure 3**). First, it can be used directly for *de novo* protein synthesis as can the amino acids derived from it including glutamate, proline, histidine, alanine, aspartic acid, and arginine. Second, it may facilitate the uptake of other amino acids, thereby regulating and coordinating their availability for protein synthesis as well (212–214). Third, it serves as the starting point for the biosynthesis of purine nucleosides, thereby linking protein and nucleic acid synthesis. It is noteworthy that

and pyrimidine synthetic pathways. In addition, glutamine can undergo reductive carboxylation to supply citrate, which can be converted by ATP citrate lyase to supply cytosolic acetyl-CoA (AcCoA) for *de novo* lipid synthesis (216, 217). Finally, glutamine can be catalyzed by glutamine synthase 1 (Gls1)/2 and then to α-ketoglutarate by the mitochondrial enzyme glutamine dehydrogenase generating a molecule of NADH (or NADPH) in a glycolysis-independent manner. α-Ketoglutarate can also be furnished *via* the action of glutamine pyruvate transaminase (GPT) or glutamate oxaloacetate transaminase (GOT), which utilize pyruvate or oxaloacetate, respectively, in reactions that also yield alanine and aspartic acid. Taken together, glutaminolysis thus coordinates amino acid, lipid, and nucleoside biosynthesis while concurrently providing energy to support these processes and contributing to the anaplerotic substrate supply.

the NADPH derived from the diversion of glycolytic substrates into the PPP can also positively impact glutamine uptake and sustain reactions involved in lipid synthesis (215, 216). Fourth, the glutamate dehydrogenase-mediated conversion of glutamate to α-ketoglutarate generates an additional molecule of NADH. This, together with the NADH generated by the α-ketoglutarate dehydrogenase and malate dehydrogenase reactions and the FADH2 generated by the SDH reaction, ensures that nearly normal levels of reducing equivalents can be supplied in a manner that is independent of AcCoA, glycolysis and FAO. Fifth, glutamine-derived α-ketoglutarate can also participate in a reverse carboxylation reaction to furnish citrate for the generation of cytoplasmic AcCoA for use in *de novo* fatty acid and cholesterol biosynthesis (216, 217). Finally, during periods of oxidative stress as commonly occur in many tumors, high levels of ROS can inhibit the aconitase-catalyzed conversion of citrate to isocitrate, thus limiting the supply of glycolytically derived α-ketoglutarate. Glutaminolysis provides a means of overcoming metabolic roadblocks such as this and thereby ensuring a

more stable and consistent supply of α-ketoglutarate and the downstream reducing equivalents derived from it (215, 218).

Given the central role of glutaminolysis for sustaining cellular proliferation, coupled with a previous finding that the apoptosis mediated by the absence of glutamine is Myc dependent (219), it was not surprising that Myc regulates this process at several levels. Wise et al. (220) first reported that glioma cells consumed large amounts of glutamine to drive OXPHOS and that the shRNAmediated suppression of Myc reduced glutamine consumption. They further observed that glioma cells were unable to survive in glutamine-deficient medium, even when supplied with glucose, and that the cell-permeable α-ketoglutarate analog dimethyl-αketoglutarate could substitute for glutamine, thereby providing strong evidence that the shunting of exogenous glutamine into the TCA cycle was directly responsible its effect on survival. Moreover, they showed that glycolysis and glutaminolysis, while both Myc regulated, were under distinct forms of control by virtue of the fact that PI3K/Akt signaling regulated the former but not the latter. Myc was subsequently found to bind selectively to E-box-containing promoter regions of the glutamine transporter genes *SLC1A5/ASCT2* and *SN2*. shRNA-mediated suppression of Myc resulted in lowered expression of both transporters' transcripts and reduced glutamine consumption. Finally, the enforced transient expression of high levels of Myc in MEFs upregulated SLCA15/ASCT2 as well as glutamine synthase 1 (Gls1) and diverted glucose away from its oxidative metabolism by the TCA cycle and into the Warburg-type aerobic glycolysis instead. The mechanism by which Myc upregulated Gls1 appeared to involve increased transcription and/or stabilization of its mRNA.

Gao et al. (221) extended these results by showing that the levels of Gls1 protein in P-493 B cells and PC3 prostate cancer cells varied in direct proportion to the degree of Myc expression. However, and in contrast to Wise et al. (220), this was not true for Gls1 transcript levels leading the investigators to eventually determine that Myc regulated Gls1 at the posttranscriptional level by inhibiting the expression of two microRNAs, miR23a and miR23b (miR23a/b). Both miRNAs were noted to have homology to potential "seed" binding sequences in the Gls1 mRNA 3′-untranslated (3′ UT) region. A luciferase reporter vector containing the Gls1 3′ UTR was shown to be responsive to these miRNAs. Thus, the Myc-mediated upregulation of Gls1is indirect by virtue of its inhibition of at least two miRNAs, which inhibit Gls1 mRNA translation.

In a variation of the above theme, Qing et al. (222), extended these findings to include several human neuroblastoma cell lines and 80 primary human neuroblastomas with varying degrees of N-Myc overexpression. As with the studies of Wise et al. and Gao et al. (221), they found that the cell lines underwent apoptosis in an N-Myc-dependent manner when deprived of glutamine. They also found higher levels of expression of Gls2 (but not Gls1), glutamate oxaloacetate transaminase (GOT2), SLCA15/ASCT2 and several other amino acid transporters that correlated with N-Myc expression levels. Apoptosis in cell lines in response to glutamine deprivation could also be inhibited or delayed by providing the cell-permeable TCA substrate dimethyl α-ketoglutarate in place of glutamine.

Pérez-Escuredo et al. (211) showed that the uptake of 3 H-glutamine and its utilization by highly oxidative cervical cancer cells was enhanced by lactate, which also accelerated tumor growth. They also showed that Matrigel-embedded tumor cells grown subcutaneously in immunocompromised nu/nu mice in the presence of high local concentrations of extracellular lactate upregulated Slca1A5/ASCT2 and Gls1 at the protein level. shRNA-mediated knockdown of the lactate transporter MCT1 abolished these effects indicating that actual transport of extracellular lactate was mediating the effects on glutaminolysis. The upregulation of Slca1A5/ASCT2 and Gls1 appeared to be mediated by Myc, whose levels were significantly increased by as little as a 6-h exposure to lactate. Further investigation found that the conversion of lactate to pyruvate blocked the activity of prolyl hydroxylases, which are negative regulators of hypoxia-inducible factors (HIFs) 1α and 2α (223). It was suggested that HIF-2α stabilizes Myc *via* its intranuclear binding to Myc-Max heterodimers (224) and indeed, this tripartite interaction was observed in co-immunoprecipitation experiments. Furthermore, the silencing of HIF-2α abolished the upregulation of Myc, Slc1A5/

ASCT2 and Gls1. Although HIF-1α was not shown to interact with Myc or Max, it is known to collaborate with Myc to induce the expression of glycolytic genes, thus potentially contributing to the intracellular lactate burden and further stabilizing Myc (224). These effects may have been further aided by the stabilization of HIF-1α by Myc itself (225).

Wang et al. (203) also examined glutamine dependency in the previously mentioned model of normal T-cell activation discussed earlier. They determined that glutamine deprivation resulted in impaired T-cell activation as well as decreased lipid and protein biosynthesis and led to an eventual G0/G1 arrest without affecting viability.

Although the Warburg effect and glutaminolysis are typically associated with high levels of proliferation (1, 2, 5, 226), they have also been observed in response to hypertrophy in otherwise non-dividing cells. Piao et al. (227), showed that the heart, which relies primarily on glycolysis and FAO for energy, reverts to using glutminolysis and also increases glucose utilization when subject to conditions that induce hypertrophy. Using two different models of right ventricular hypertrophy, they found variable degrees of Myc induction and increased expression of glutamine receptors SLC1A5/ASCT2 and Slc7A5 as well as increases in the mRNAs encoding the Glut1 glucose transporter and hexokinase (HK) 1. Consistent with the former of these findings, the investigators also noted increased 14CO2 production derived from 14C-labeled glutamine. In response to the glutamine antagonist 6-diazo-5-oxo-l-norleucine a decrease in glutaminolysis was noted and was associated with a compensatory increase in glucose oxidation and elevated cardiac output. These studies strongly implicate glutaminolysis as being directly involved in the biomass accumulation that accompanies active proliferation but not in proliferation *per se*.

#### DISTINCT *IN VIVO* METABOLIC ROLES FOR MYC: NOT ALWAYS THE SAME FUNCTION IN NORMAL AND NEOPLASTIC TISSUES

Myc's unequivocal role in integrating normal mass accretion and proliferative signals with altered metabolism in fibroblasts, myeloid cells, T-cells, and other cell types *in vitro* as discussed earlier contrasts sharply with recent studies in hepatocytes where Myc was found to be entirely dispensable for the long-term regeneration of normal liver parenchyma (228). Several previous studies had indicated that mice with a conditional, hepatocytespecific knockout of the *myc* gene could regenerate hepatic mass following two-thirds partial hepatectomy (PH) (229–232). Less clear was whether this was achieved as rapidly as occurred in control livers. To some extent, this uncertainty was the consequence of different groups having used different and mostly indirect techniques to measure hepatocyte proliferation and liver regeneration. Further compounding this was the fact that the PH model is a relatively crude and suboptimal way to measure longterm regenerative potential given that the average hepatocyte must divide only about 1.6 times to replace the resected liver mass and that the entire regenerative process is complete within 7–10 days (233). Moreover, as many as 30–40% of the hepatocytes in the regenerating liver remnant remain quiescent following PH, and about the same amount of "regeneration" can be attributed to hypertrophy rather than actual cell division (234, 235). Thus, none of these reports actually addressed the question of whether Myc was necessary to support sustained, long-term hepatocyte proliferation as might occur during the course of normal hepatocyte turnover or repair from chronic injury, both of which are processes of much longer duration (234). It further left open the questions of whether subtle but nonetheless significant differences in regeneration rates might have escaped detection using the PH model and what, in fact, was actually being measured in these other reports.

Edmunds et al. (228) addressed all of these issues by capitalizing on an elegant, robust, and sensitive murine model of Type I hereditary tyrosinemia (236, 237). In these mice, as in humans, inactivation of the fumarylacetoacetate hydrolase (FAH) gene, which encodes the final enzyme in the pathway for tyrosine catabolism, leads to the accumulation of toxic levels of the upstream tyrosine catabolites maleylacetoacetate and fumarylacetoacetate, eventually causing hepatocyte death, fibrosis, and hepatic failure (238). This ultimately fatal outcome can be blocked with the drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (Nitisinone or NTBC), a reversible inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) (239). HPPD is a more proximal enzyme in the pathway and converts the first tyrosine catabolite 4-hydroxyphenylpyruvate to homogenistate. In this way, *fah−/−* mice can be maintained in a healthy state simply by providing NTBC in their drinking water and thereby blocking tyrosine catabolism and the accumulation of the deleterious metabolites.

*Fah−/−* mice can also be cured by the intrasplenic injection of as few as 105 *fah+/+* hepatocytes followed by the intermittent discontinuation and resumption of NTBC (236, 237). Animals initially lose weight as they accumulate the toxic tyrosine catabolites. However, as endogenous *fah−/−* hepatocytes are gradually replaced by the *fah+/+* donor population over 4–5 months, the recipient mice are eventually rendered NTBC-free and their livers are comprised of 50–80% donor hepatocytes (228, 236, 237, 240). As a way of monitoring hepatocyte proliferative potential, this model offers several advantages over PH. First, because the donor cells must divide 50- to 100-fold during recipient liver repopulation, they undergo many more population doublings than do post-PH hepatocytes, thereby providing a more demanding and long-term replicative challenge. Second, donor hepatocytes from different sources can be used in "competitive" repopulation assays, analogous to those used for decades in bone marrow transplantation studies (241, 242). Provided that the recipient and donor populations can be distinguished, the ultimate contribution of each to the steady-state transplanted liver can be assessed with exquisite precision and even quite small deviations from the input donor ratios can be easily quantified (228, 240). Third, if so desired, the input ratio of the competing donor populations can be varied to reveal even more dramatic differences in regenerative potential. Finally, because the competing donor populations replicate in identical environments, differences in regeneration rates can be ascertained with many fewer animals than are required with PH-based experiments.

Edmunds et al. (228) exploited the FAH model to assess the regenerative capacities of mixed *fah+/+* populations of *myc+/+* and *myc−/−* hepatocytes. Surprisingly, the ratio of the two donor hepatocyte populations recovered from the fully reconstituted recipient livers more than 4 months after their co-transplantation was identical to that of the input donor populations. Thus, even under the most demanding of circumstances, Myc's absence did not impair the long-term regenerative potential of hepatocytes in this particular model.

In addition to being at odds with the above-discussed role for Myc in the proliferation of fibroblasts and T-cells and numerous other cell types (154, 158, 191, 196, 203), these results also differ from studies in *Drosophila* and some cancer lines showing that cells expressing higher levels of Myc tend to outcompete those with low levels (243–245). Similarly, the conditional deletion of *myc* or its dominant-negative inhibition in intestinal crypt cells or bone marrow cells is associated with severe proliferative defects although these may be ameliorated over time (49, 246–248). Thus, the elimination of endogenous Myc seems to have highly variable and tissue-specific effects, with liver representing an atypical although perhaps not unique example.

Ultimately, while the metabolic consequences of endogenous Myc loss are tissue specific and variable, it seems reasonable to conclude that, in most cases, Myc is responsible for maintaining context-appropriate levels of ATP and anabolic substrates by regulating the uptake and oxidation of nutrients that furnish glycolysis and the TCA cycle. In Myc's absence, as noted above, many tissue types appear to adapt to the associated nutrient and energy deficits *via* various strategies that include variable reductions in cell mass, proliferative rate and anabolic activity (49, 158, 180, 228, 249). Interestingly, while Edmunds et al. (228) did not observe any significant differences in cell size, ATP levels or AMPK phosphorylation in the livers of mice following transplantation with *myc+/+* or *myc−/−* hepatocytes, these studies were performed on hepatocytes that had already re-populated the liver and reached a non-proliferating equilibrium state. It is certainly possible that more profound energy deficits might have been observed had the actual proliferating population been assessed at an earlier time following transplant. Nonetheless, these studies clearly demonstrated that *myc−/−* hepatocytes remain as fully capable as their wild-type counterparts at contributing to the long-term repopulation of the liver irrespective of whatever defects they may harbor.

Although *myc−/−* hepatocytes demonstrated no obvious proliferative impairment in the above-described repopulation assay, they nevertheless showed several abnormalities that were evident even prior to transplantation (228). First, despite body weights identical to those of *myc+/+* mice, juvenile mice with hepatocyte-specific deletion of *myc* had smaller livers, consistent with a previous observation that *myc* hypomorph mice tend to have smaller numbers of otherwise normal-sized cells in some organs, including liver (249, 250). At first glance, this would seem to be inconsistent with fact that *myc+/+* and *myc−/−* hepatocytes competed equally in repopulation studies (228). However, it is possible that the requirements for Myc in the developing liver versus the fully developed liver are different. There may thus exist a phase early in development, but not beyond, during which Myc is required for hepatocyte expansion. Alternatively, the smaller size of *myc−/−* livers may more reflect an unappreciated role of Myc in regulating organ size (251, 252) than in limiting the proliferative potential of its individual constituent cells, which is what is measured in hepatocyte transplant studies.

In contrast to the above findings, adult *myc−/−* livers actually weighed *more* than *myc+/+* livers. The former possessed a significantly higher neutral lipid and triglyceride content, which likely accounted for their increased mass (228). This implied either that *myc−/−* livers take up and store greater amounts of these lipids and/or utilize less of them. Arguing against the latter point was the finding that *myc−/−* livers showed variable but significant increases in FAO. This suggested that, in the absence of Myc, hepatocytes both take up and utilize more fatty acids, with the former process outpacing the latter, eventually culminating in an increased storage pool, not unlike that seen in *myc−/−* fibroblasts or following short-term pharmacologic Myc or N-Myc inhibition in other cell types (191, 196, 228).

Edmunds et al. (228) studied the structure and function of isolated mitochondria from *myc+/+* and *myc−/−* livers using BNGE and noted no obvious differences in the stoichiometries of the protein subunits of ETC Complexes I–IV or the ATP synthase (Complex V). Mass spectroscopic quantification of over 400 mitochondrial proteins, including all 93 subunits of the ETC, also showed no significant quantitative differences between the two groups. However, Complex I and Complex II activities were modestly but significantly reduced in mitochondria from *myc−/−* livers in response to ADP and succinate whereas the activity of Complex V was increased by about the same amount. Coupled with the finding that ATP levels in *myc+/+* and *myc−/−* livers were comparable, this suggested that the loss in ETC function in *myc−/−* livers was compensated for by a more efficient generation of ATP *via* Complex V and/or by the more inherently efficient process of FAO (253).

Collectively, these findings suggest a form of metabolic reprogramming by the *myc−/−* liver only partially resembling that seen in *myc−/−* fibroblasts, which may reflect the different tissues under consideration as well as their different proliferative rates. For example, ATP and AcCoA levels were markedly diminished in *myc−/−* fibroblasts but were maintained at normal levels in *myc−/−* livers (158, 228) (**Figure 4**). This appears to have been due to the employment of FAO as an alternative energy source by hepatocytes as well as to an increase in neutral lipid accumulation that has been previously reported in *myc−/−* fibroblasts, in hematopoietic cells following short-term inhibition of Myc, and in neuroblastomas following treatment with small molecule Myc inhibitors (158, 191, 196). The neutral lipid accumulation by *myc−/−* hepatocytes was even more striking following their transplantation into *fah−/−* recipient mice. Oil Red O staining of fully reconstituted livers confirmed their significantly higher triglyceride content as well as larger and more numerous neutral lipid droplets (228). Indeed, the lipid within these livers was so abundant that much of it was extracellular. This was likely responsible for the significant inflammatory cell infiltrate that was observed as well as for the upregulation of numerous transcripts involved in acute and chronic inflammation, leukocyte signaling and fibrosis. Immunohistochemical staining for 4-hydroxynonenal, a by-product of ROS-mediated lipid peroxidation, was also detected as was evidence for dysregulated mitochondrial structure and function as previously reported in mice maintained on high fat diets (254–256). Other findings included the downregulation of 19 of the 44 transcripts encoding subunits of Complex I and seven of the 20 transcripts encoding subunits of Complex V. In the latter group, 10 of the remaining 13 transcripts were upregulated. Taken together, these studies suggested that the loss of Myc expression in resting hepatocytes is initially associated with the gradual accumulation of neutral lipid, not unlike that seen in non-alcoholic fatty liver disease (NAFLD) in association with ETC dysfunction (257, 258). When coupled with the metabolic stress imposed by regeneration-associated proliferation and chronic inflammation, this progressed to a phenotype closely resembling non-alcoholic steatohepatitis (NASH), a long-term consequence of NAFLD that is associated with high levels of oxidative stress, inflammation, fibrosis and eventual hepatic failure (257, 259). It is tempting to speculate that the loss of Myc in these hepatocytes, particularly during times of proliferation and high energy demand, leads to an attenuated glycolytic response and the dysregulation of mitochondrial structure and function. The resulting energy depletion and mitochondrial stress is accompanied by an increased reliance on FAO coupled with unbalanced fatty acid uptake and storage, intracellular ROS generation, inflammation, and long-term parenchymal damage that mimics NAFLD and NASH (228).

The above findings do not provide simple explanations for how endogenous Myc affects metabolism during normal growth. They do suggest, however, that these changes occur at multiple levels and that the compensation by *myc−/−* hepatocytes ultimately affects neither ATP generation, AcCoA levels nor proliferation. It remains to be determined precisely how ATP levels in *myc−/−* livers are maintained but it appears to be dependent on several different pathways including an increased efficiency of ATP production as suggested by the Complex V assays and the provision of alternate sources of AcCoA mainly from enhanced FAO. An alternative source might originate with increased glutaminolysis, which could conceivable circumvent the need to rely on glycolysis- or FAO-derived AcCoA by providing reducing equivalents in the form of both NADH and FADH2. Whatever the cumulative corrective mechanisms are that help to normalize hepatocyte energy generation and proliferative function, they come at considerable metabolic cost in the form of increasingly severe lipid storage and utilization defects.

Metabolic changes have also been studied in *myc−/−* hepatocytes in response to oncogenic signaling following the induction of hepatoblastoma (HB). HB is the most common pediatric liver cancer, almost invariably arising in children under the age of 3 years and is associated with somatic mutations of the β-catenin gene in >80% of cases (260, 261). A useful mouse model of HB has recently been described in which tumors arise with nearly 100% penetrance following the hydrodynamic tail vein injection-mediated delivery of "Sleeping Beauty" plasmids (262, 263) encoding patient-derived mutant forms of β-catenin and yes-associated protein (YAP) (264–266). A major transcriptional target for β-catenin is Myc (267) and, not unexpectedly, Myc is among the most highly upregulated genes in HBs (Monga and Prochownik, unpublished data).

Given that Myc plays no role in normal hepatocyte repopulation as discussed earlier (228), it was of interest to determine whether Myc is needed to support the more rapid growth and increased metabolic demands of HBs. Indeed, Wang et al. (125) found that, although tumors arose in *myc−/−* livers at the same frequency as they did in *myc+/+* livers, their growth rates were significantly impaired. Thus, while Myc was dispensable for normal hepatocyte proliferation and for the induction of HBs, it was clearly required for determining the rate of tumor growth (125, 228).

To understand the basis for Myc's selective role in HB proliferation (228), the metabolic properties of *myc+/+* and *myc−/−* HBs were compared. An initial assessment of mitochondrial function showed both types of HBs had lower OCRs and Complex II activity compared with corresponding livers. However, less suppression of these activities was seen in *myc−/−* HBs. It is likely that this reflected their slower growth rates and thus a reduced tendency to rely on Warburg-type aerobic glycolysis for energy generation. Consistent with this, glycolytic transcripts as a group were upregulated in *myc+/+* HBs to a significantly greater extent than they were in *myc−/−* HBs (11.2-fold versus 8.9-fold, *P* < 0.0004). The presumptive lower glycolytic rate of the latter likely accounted for their lower AcCoA levels. Despite these differences, both tumor types showed elevated levels of ATP and a downregulation of phosphoAMPK relative to their respective livers. This suggested that, despite differences in their metabolic activities, the slower growth rates of *myc−/−* HBs were not the result of any obvious energy deficit. On the other hand, it is conceivable that *myc−/−* HBs were energetically constrained and that they maintained a rate of growth that was compatible with these limitations while still allowing normal levels of ATP to be maintained. The presumptive reduced rate of glycolysis by *myc−/−* HBs may also have restrained the supply of anabolic precursors necessary for sustaining rapid tumor growth. In addition, these tumors were also less able than *myc+/+* tumors to upregulate the group of transcripts encoding the ~80 ribosomal protein genes (3.6-fold versus 5.2-fold, *P* < 10−4 ) (125). Thus, rather than being severely deprived of energy, as in the case for *myc−/−* fibroblasts, *myc−/−* HBs may instead be deprived of anabolic precursors and the ability to increase protein synthesis rates, despite adequate energy supplies. Protein synthesis rates and ribosomal protein content are known to be rate-limiting factors in the growth of many cancers (108, 268).

In further pursuit of an explanation for the overall downregulation of OXPHOS by tumors, Edmunds et al. (228) quantified mtDNA using qPCR to amplify two distinct regions of the mitochondrial genome. They documented a ~60–80% reduced mitochondrial mass in both *myc+/+* and *myc−/−* tumors, thus providing a structural explanation for the Warburg effect. Results published at about the same time by Reznick et al. (269) showed a similar loss of mtDNA from a wide variety of human cancers whose genomic data had been compiled in *The Cancer Genome Atlas* (https://cancergenome.nih.gov).

In HBs arising in *myc+/+* and *myc−/−* livers, eight of the 14 most deregulated pathways identified by Ingenuity Pathway Analysis involved lipid biosynthesis. Importantly, transcripts encoding several key enzymes involved in fatty acid synthesis (FAS) such as ATP citrate lyase (ACLY), fatty acid synthase, and AcCoA carboxylase were markedly upregulated, although not in a Myc-dependent manner. By contrast, transcripts encoding enzymes involved in FAO such as trifunctional protein, carnitine palmitoyltransferase-2 and very long-chain acyl-CoA dehydrogenase were markedly downregulated. Indeed, even transcripts whose encoded enzymes participate in peroxisomal FAO such as the fatty acid transporter ATP-binding cassette-D3, peroxisomal biofunctionalized protein, and acyl-CoA oxidase-1 were downregulated. Functional assays of the FAO pathway were consistent with these findings and showed marked downregulation of FAO activity in both *myc+/+* and *myc−/−* tumors (125). Taken together, these studies imply that, unlike the case in fibroblasts, where the loss of Myc leads to a near cessation of both glycolysis and OXPHOS and an upregulation of FAO as an alternate energy source, in HBs, glycolysis and OXPHOS are much less affected and rely less on FAO to supply energy.

#### METABOLIC LINKS BETWEEN NORMAL AND NEOPLASTIC STATES

As discussed at length above, proliferating tumor cells require a continual supply of the basic cellular building blocks consisting of amino acids, nucleic acids, and lipids. In this regard, tumor cells are not very different from the progenitor cells that give rise to our various organs during embryogenesis, or the regenerative stem cells now known to reside within many of our adult tissues. Furthermore, differentiated cells may dedifferentiate and then proliferate to repair an injury, followed by re-differentiation. The metabolic similarities between these different types of proliferating cells are striking: all rely heavily upon glucose and glutamine to meet their steep anabolic needs (270–272). Not coincidentally, Myc maintains stem cell pluripotency and self-renewal, perhaps through its effects on driving glucose/glutamine uptake and metabolism (273). On the other end of the spectrum are nonproliferative, differentiated cells that are often metabolically reliant upon FAO. Several fetal tissues that rely upon glucose during growth and development undergo a metabolic switch to FAO after birth. The best-known example of this is the heart (274) and we have made similar observations in human kidney (Goetzman, unpublished data). Senescence is also characterized by a switch to FAO while lipid synthesis is turned off (275, 276). With some notable exceptions (i.e., prostate cancer) most cancers are glucose/glutamine dependent and not FAO dependent (277, 278).

The glucose/glutamine versus FAO dichotomy is maintained in both normal and neoplastic tissues by a key metabolic phenomenon called the Randle cycle, sometimes simply referred to as the glucose–fatty acid cycle. The Randle cycle refers to the reciprocal nature of mitochondrial rates of pyruvate oxidation and FAO (279, 280). The Randle cycle centers on pyruvate dehydrogenase (PDH) and carnitine palmitoyltransferase 1 (CPT1), which are the rate-limiting enzymes of pyruvate oxidation and FAO, respectively. When glucose is abundant and glycolysis is high, pyruvate concentrations rise and inhibit pyruvate dehydrogenase kinase (PDK1), the kinase responsible for phosphorylating and silencing PDH. PDK1 inhibition activates PDH and increases mitochondrial pyruvate oxidation to generate AcCoA and NADH. Increases in intramitochondrial AcCoA and NADH have two consequences for FAO. First, the intramitochondrial FAO machinery is directly inhibited by high NADH/NAD+ and AcCoA/CoA ratios (281). Second, the entry of pyruvate-derived AcCoA into the TCA cycle produces citrate, which is exported from mitochondria to the cytosol where it is reconverted to AcCoA *via* the action of ACYL. Cytoplasmic AcCoA is then converted to malonyl-CoA by AcCoA carboxylase (ACC). Malonyl-CoA in turn is a potent inhibitor of CPT1. CPT1 is the gatekeeper for FAO *via* its role in regulating fatty acid transport across the mitochondrial membrane; thus, the result of sustained PDH flux is the sequestration of fatty acids in the cytosol, away from the degradative FAO machinery. Cytosolic fatty acids are then either converted to triglyceride droplets to fuel the cell should glucose become unavailable, or in the case of proliferating cells, incorporated into phospholipid acyl-chains for synthesizing new membranes. The same malonyl-CoA which silences CPT1 also feeds into the *de novo* FAS pathway to further support the anabolic needs of the cell (282). Because of this cycle, the rate of PDH flux has been shown to highly correlate with lipogenesis in many cell types. In the liver, for example, insulin increases PDH activity and lipogenesis from glucose-derived AcCoA, while starvation rapidly inhibits PDH activity and thus limits glucose-derived lipogenesis (283).

The Randle cycle also works in the opposite direction, i.e., high rates of FAO suppress glucose utilization. In the same way that glucose-derived AcCoA and NADH inhibits the FAO machinery, FAO-derived AcCoA and NADH potently inhibit PDH *via* activation of PDK1. There is also some evidence that fatty acid-derived citrate exported to the cytosol can suppress glycolysis at the level of phosphofructokinase-1 (284–286). The resulting accumulation of glucose-6-phosphate feeds back to inhibit hexokinase (HK) and glucose uptake (**Figure 5**) (286). Overall, the net result of these various points of negative feedback control are a downregulation of glucose utilization by FAO. A key theme of the Randle cycle is that lipogenesis sides with glucose oxidation, and that cells cannot simultaneously conduct FAO and FAS. This is because activation of FAS, by virtue of cytosolic conversion of citrate to AcCoA to malonyl-CoA, would inhibit FAO at the level of CPT1. This maybe the reason why proliferating cells, be they cancerous or not, suppress FAO such that they can drive their absolute requirement for FAS and membrane synthesis. Likewise, it explains how terminally differentiated and senescent cells are able to sustain high rates of FAO for their energetic needs. In short, because of FAO suppression of glucose-derived FAS, it is difficult for cells to proliferate while conducting a high rate of FAO. Exogenous fatty acids can be incorporated into membranes directly, but in FAO-dependent cells the phospholipid synthesis pathways would be in continual competition with mitochondria for incoming exogenous fatty acids.

The Randle cycle and connection between glucose utilization and FAS would suggest that tumors would tend to have low FAO and high FAS. Indeed, this has been observed in many cancers, and inhibition of the FAS pathway has been shown to slow cancer growth in many instances (287, 288). In the HB mouse model discussed at length above, FAO was found to be markedly lower than in normal control liver while PDH activity was increased

several-fold (240). This occurred both in the presence and absence of Myc suggesting that there are multiple mechanisms responsible for driving the glucose–fatty acid cycle in tumors. A similar reciprocal relationship between FAO and PDH activity was also seen in a model of hepatocellular carcinoma induced by the deregulated, doxycycline-regulated induction of Myc (126). Of course, as is true with all aspects of cancer metabolism, there have also been multiple studies demonstrating cancers with the opposite metabolic phenotype, i.e., high FAO and low PDH activities (289–294). It is interesting to note that prostate cancer, the best-characterized example of a cancer with high FAO, is well known for its typically slow growth rate. Some studies have shown that maximizing pyruvate flux through PDH by inhibiting PDK with dichloroacetate can sensitize cells to other chemotherapies, presumably by promoting mitochondrially driven apoptosis (295). As with many metabolic pathways, too much of any one thing may lead to cell death. In the case of PDH, it has been shown in normal liver that most PDH capacity is not being used, even under fed, insulin-stimulated conditions (283). Only about 10% of the PDH in fed rodent liver is in the active (unphosphorylated) state; acute insulin exposure doubles this to 20%. Dichloroacetate increased the active state to 100%. Therefore, it remains possible that either supraphysiological activation or inhibition of PDH may be beneficial in crippling cancer growth, with the usual caveat that it depends upon the cancer being treated.

With regard to liver cancers, the commonality of high PDH activity in both HBs and HCC (125, 240) led to the hypothesis that eliminating PDH may slow tumor growth. First, Jackson et al. examined the consequences of conditional inactivation of the catalytic PDHA1 subunit on normal hepatocyte proliferation in murine liver (240). Using the above-described competitive hepatocyte re-population assay, it was determined that wildtype and PDH knockout hepatocytes contributed equally to the long-term reconstitution of *fah−/−* recipient livers. Thus, despite the severing of the direct line of communication between glycolysis and the TCA cycle, and between glucose utilization and FAS, proliferation remained unaffected. That PDH is profoundly linked to lipogenesis was demonstrated by the striking loss of lipid stores in PDH KO liver (**Figure 6**). Based on this observation it is tempting to speculate that PDH inhibition could represent a novel therapeutic approach for the treatment of NAFLD. What remains to be determined is the magnitude of PDH suppression needed to achieve this outcome, the length of time PDH suppression must continue, and how rapidly lipid re-accumulates following its reactivation.

Two approaches were next taken to ascertain how inactivation of PDH altered the properties of HBs induced by mutant β-catenin + YAP (125, 240). In the first approach, both the resultant tumors and the adjacent, non-transformed parenchyma were rendered PDH-negative ("pan KO"), whereas in the second, only tumors and not the adjacent non-transformed liver tissue were rendered PDH-negative ("restricted KO"). Survival of the pan KO tumor group did not differ significantly from that of the control group with normal levels of PDH expression in all tissues whereas survival of restricted KO tumor group was significantly prolonged (mean survival ~125 versus 90 days). Further investigation showed that pan KO HBs tended to be smaller than control tumors despite the similar survival of the two groups. By contrast, HBs from the long-surviving restricted KO group were about the same size as control tumors. These apparent discrepancies were ultimately resolved by showing that mice in the pan KO group had a severe lactic acidosis at the time of their demise. It was concluded that, in the face of the absence of PDH activity in pan KO hepatocytes, HB-derived lactate could not be metabolized back to pyruvate by the untransformed hepatocytes as it could by the PDH-expressing hepatocytes of restricted KO mice. The demise of the pan KO animals was thus due to a combination of tumor burden and lactic acidosis. The normal respiratory compensation that attempts to correct metabolic acidosis was almost certainly compromised in these mice as well due to the restricted diaphragmatic mobility imposed by the tumor burden.

The most notable metabolic defect in PDH KO tumors was a *ca.* 80% reduction in total AcCoA levels. Surprisingly, only a slight, statistically non-significant increase in compensatory FAO was observed (240). Importantly, while the Randle cycle shows that driving one pathway will limit the other, these studies suggest that directly limiting one pathway does not necessarily stimulate the other (i.e., eliminating PDH may not necessarily stimulate high FAO). Interestingly and unexpectedly, the PDH KO studies indicated that despite the profound inability to maintain normal levels of AcCoA, KO hepatocytes were able to sustain normal or near-normal rates of growth even following their transformation into highly proliferative tumor cells. It is possible that reductive carboxylation of glutamine, which was not examined, could supply just enough cytosolic AcCoA to drive the fatty acid synthesis needed to supply lipids for new membranes. In this scenario, the minimal induction of compensatory FAO might be explained by glutamine-derived malonyl-CoA, which would limit the capacity for FAO by inhibition of CPT1. In short, this series of studies with PDH KO mice highlight the amazing metabolic plasticity of both normal and neoplastic livers, and stress the difficulty of targeting metabolic pathways to treat cancers.

#### THERAPEUTIC STRATEGIES AIMED AT TARGETING MYC AND ITS EFFECTORS

Myc has long been viewed as an exciting but challenging oncologic target (51, 52, 296). The enthusiasm stems from the fact that Myc is among the most commonly deregulated oncoproteins in all of cancer (24, 51, 52, 97). Even in those neoplasms where Myc deregulation is not immediately appreciable, other oncogenic signaling pathways that *are* deregulated invariably converge upon Myc which then carries out their transcriptional bidding. In these cases, inhibiting Myc still leads to cell cycle arrest, the collapse of ATP production and apoptosis (50, 191). Unfortunately, Myc's lack of enzymatic activity or prominent structural features makes it extremely difficult to target with small molecules (51, 52, 296). The means by which Myc inhibition has been attempted and descriptions of the successes and (mostly) failures along the way have been recently reviewed (297). These attempts included strategies such as altering the structure of Myc DNA-binding sites, inhibiting the structure and/or function of Myc-Max heterodimers, and inhibiting Myc's engagement with and activation

of the transcriptional activation machinery (297). A second challenge to inhibiting Myc is that the plethora of target genes whose expression is altered, either directly or indirectly, in response to Myc deregulation includes many that are transforming in their own right. Thus, an obvious alternate strategy, such as targeting one of these rather than the intimidating Myc itself, is likely to fail because of their sheer number and oncogenic functional redundancy. A final challenge is that many of the early Myc inhibitors that have been described to date, even those which function well *in vitro*, have demonstrated disappointing therapeutic efficacy *in vivo* due to poor tissue penetration, low potency, or rapid metabolism (51, 52, 296, 297).

While these issues are clearly important when considering the targeting of oncogenic pathways, some become less so when contemplating the employment of inhibitors in non-neoplastic settings where Myc is a potential target that may not require systemic inhibition. For example, the intimal hyperplasia that accompanies atherosclerotic stenosis has long been known to be accompanied by and to be dependent upon Myc overexpression (52, 298, 299). We have proposed that drug-eluting stents, which released Myc inhibitors continuously and over short distances, would provide a means by which high-local compound concentrations could be achieved thus overcoming some of the pharmacologic shortcomings that plague their systemic delivery (52). Myc antisense oligonucleotides have shown efficacy is this regard even though their ability to reduce Myc levels has been somewhat modest (300, 301).

Non-neoplastic settings also now permit the consideration of downstream Myc effectors that would not otherwise represent tractable targets due to their neoplastic redundancy as mentioned earlier. An excellent example of this is PDH, which is neither an oncoprotein nor even a direct Myc target although it is dramatically upregulated and activated in response to activation of the Myc pathway (**Figure 6**) (125, 126, 240). Inhibiting PDH *via* more traditional approaches than are available for Myc could prove to be of enormous therapeutic benefit for the treatment of NAFLD, a common condition associated with considerable nonneoplastic morbidity and mortality as well being a significant predisposing factor for the development of cancer (257). Time will tell whether the inhibition of Myc and its partners in crime will be of greater therapeutic benefit for cancer, for metabolic disorders, or for those diseases harboring overlapping features of both.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

This review manuscript was written and edited by EP and EG. EP designed and prepared the figures.

#### FUNDING

This work was supported by NIH grants R01 DK090242 to EG and RO1 CA174713 to EP.

function at acidic pH. *Nat Chem Biol* (2017) 13:494–500. doi:10.1038/ nchembio.2307

oncogenes in B cells. *Proc Natl Acad Sci U S A* (1983) 80:4822–6. doi:10.1073/ pnas.80.15.4822

reduced fertility in heterozygous female mice. *Genes Dev* (1993) 7:671–82. doi:10.1101/gad.7.4.671

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Goetzman and Prochownik. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## ANRIL: A lncRNA at the CDKN2A/B Locus With Roles in Cancer and Metabolic Disease

#### Yahui Kong, Chih-Heng Hsieh and Laura C. Alonso\*

*Department of Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, United States*

The *CDKN2A/B* genomic locus is associated with risk of human cancers and metabolic disease. Although the locus contains several important protein-coding genes, studies suggest disease roles for a lesser-known antisense lncRNA encoded at this locus, called *ANRIL*. *ANRIL* is a complex gene containing at least 21 exons in simians, with many reported linear and circular isoforms. Like other genes, abundance of ANRIL is regulated by epigenetics, classic transcription regulation, splicing, and post-transcriptional influences such as RNA stability and microRNAs. Known molecular functions of *ANRIL* include *in cis* and *in trans* gene regulation through chromatin modification complexes, and influence over microRNA signaling networks. Polymorphisms at the *ANRIL* gene are linked to risk for many different cancers, as well as risk of atherosclerotic cardiovascular disease, bone mass, obesity and type 2 diabetes. A broad array of variable reported impacts of polymorphisms on *ANRIL* abundance, splicing and function suggests that *ANRIL* has cell-type and context-dependent regulation and actions. In cancer cells, *ANRIL* gain of function increases proliferation, metastasis, cell survival and epithelial-mesenchymal transformation, whereas *ANRIL* loss of function decreases tumor size and growth, invasion and metastasis, and increases apoptosis and senescence. In metabolic disease, polymorphisms at the *ANRIL* gene are linked to risk of type 2 diabetes, coronary artery disease, coronary artery calcium score, myocardial infarction, and stroke. Intriguingly, with the exception of one polymorphism in exon 2 of *ANRIL*, the single nucleotide polymorphisms (SNPs) associated with atherosclerosis and diabetes are non-overlapping. Evidence suggests that *ANRIL* gain of function increases atherosclerosis; in diabetes, a risk-SNP reduced the pancreatic beta cell proliferation index. Studies are limited by the uncertain relevance of rodent models to *ANRIL* studies, since most *ANRIL* exons do not exist in mouse. Diverse cell-type-dependent results suggest it is necessary to perform studies in the relevant primary human tissue for each disease. Much remains to be learned about the biology of *ANRIL* in human health and disease; this research area may lead to insight into disease mechanisms and therapeutic approaches.

#### Edited by:

*Che-Pei Kung, School of Medicine, Washington University in St. Louis, United States*

#### Reviewed by:

*Subrata Chakrabarti, University of Western Ontario, Canada Tania Lee Slatter, University of Otago, New Zealand*

\*Correspondence: *Laura C. Alonso<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cancer Endocrinology, a section of the journal Frontiers in Endocrinology*

Received: *07 May 2018* Accepted: *29 June 2018* Published: *24 July 2018*

#### Citation:

*Kong Y, Hsieh C-H and Alonso LC (2018) ANRIL: A lncRNA at the CDKN2A/B Locus With Roles in Cancer and Metabolic Disease. Front. Endocrinol. 9:405. doi: 10.3389/fendo.2018.00405*

Keywords: ANRIL, CDKN2A, CDKN2B, long noncoding RNA, diabetes, pancreatic islet, cancer, metabolic disease

### INTRODUCTION

The discovery of functional noncoding RNAs has opened a kaleidoscopic world of unanticipated mechanisms extending far beyond the DNA-RNA-protein paradigm; noncoding RNAs may in fact outnumber coding RNAs (1). Long noncoding RNAs (lncRNAs) have been discovered throughout the genome; scientists are working to explore their functions in health and disease. The ANRIL lncRNA was first identified in a melanoma kindred with a large (403 kb) deletion at the CDKN2A/B locus (2). ANRIL has attracted broad attention because it is located at a genomic hotspot for disease heritability, the CDKN2A/B locus. Although protein coding genes at this locus have important well-studied roles in cell cycle regulation, data suggest that some locus disease-associated single nucleotide polymorphisms (SNPs) act through effects on ANRIL itself. Intriguingly, studies suggest ANRIL not only impacts the biology of cancer, but also has celltype-specific roles in metabolic disease. Although ANRIL has been reviewed in the past (3, 4), knowledge has exponentially increased in recent years. Here we review advances in ANRIL SNPs, gene regulation, cell biology, and disease roles of ANRIL.

### THE CDKN2A/B LOCUS

ANRIL, or CDKN2B-AS1, is located at the human CDKN2A/B locus at 9p21.3. This gene cluster, extending over a nearly 350 kb genomic region housed within a single topologically associated domain (TAD) (5), contains three protein coding genes and, antisense to them, the ANRIL lncRNA (**Figure 1**). The protein coding genes include S-methyl-5′ -thioadenosine phosphorylase (MTAP), CDKN2A, which encodes splice variants p16INK4A and p14ARF, and CDKN2B, which encodes p15INK4B (9, 10). MTAP lies at one end of the locus, 192 kb telomeric to the 5′ start of ANRIL. At the centromeric end of the locus, the ANRIL gene contains 19–21 reported exons over a 126 kb region. CDKN2A lies between MTAP and ANRIL, near the first exon of ANRIL; CDKN2B is located within the first intron of ANRIL, in an antisense direction. The proteins encoded by CDKN2A and CDKN2B are tumor suppressors with well-established roles in cell proliferation, apoptosis, senescence and aging (11, 12). p16INK4A and p15INK4B are cyclin dependent kinase (CDK) inhibitors, inhibiting retinoblastoma phosphorylation by CDK4/6. The p14ARF protein, a splice variant of CDKN2A which due to a frame shift has no amino acid homology to the principal other CDKN2A splice variant, p16INK4A, modulates p53 activity. ANRIL is transcribed by RNA polymerase II and spliced into multiple linear and circular isoforms in a tissue-specific manner. In general, ANRIL roles, explored in detail below, include gene regulation in cis and in trans through interaction with polycomb repressive complex (PRC) histone modifiers, as well as RNA-RNA interactions such as microRNA (miRNA) sponge activity (3, 13). Known biological impact of ANRIL activities include modulation of proliferation, apoptosis and cellular adhesion pathways (14).

### EVOLUTION OF THE ANRIL GENE

The evolutionary development of the human ANRIL gene has been studied by comparative analysis of the genomes of 27 organisms including non-mammalian vertebrates, non-placental mammals, non-primate placental mammals, and primates (15). ANRIL originated in ancestors of the Eutherian (placental) mammalian clade. Initially the gene contained only a few exons; over time, ANRIL underwent clade-specific evolution, adding exons in many mammals but losing exons in rodents. The full 21 exon gene is present only in simians. ANRIL genes contain many repeat elements, both intronic, and exonic; evidence suggests that transposon activity has mediated many of the observed evolutionary changes in exon presence or absence, location, sequence, conservation, and structure, as well as introduction of splice sites (15).

Early ANRIL variants were likely not transcribed or functional (15). One hallmark of functional RNAs is splice signals at intron/exon boundaries. In simians, 191 intron/exon boundaries contained canonical splice signals, while 20 did not. In lower mammals, however, only about half of intron/exon junctions contained identifiable splice signals (15). This finding suggests that as ANRIL gained exons, and exon sequences became more conserved across species, it also increased the number of splice signals and gained functionality. Taken together, data suggest that ANRIL may be functional only in simians, and that functionality may have been introduced by transposon activity (15).

## ANRIL ISOFORMS AND STRUCTURE

With at least 21 exons (new exons discovered as recently as 2017 (7)), the ANRIL gene can potentially generate a large number of splice variants. In fact, many ANRIL isoforms have been reported (**Figure 1**) (16). Exon numbering has changed over time as new exons were discovered. Studies observe multiple isoforms in any given cell type, mostly at low abundance. A different range of isoforms may be identified from one cell type to another, but tissue-dependent isoform expression in primary cells or tissues has not yet been comprehensively quantified using the same reagents and techniques. Intriguingly, many studies have now identified both linear and circular ANRIL isoforms (6–8). The longest open reading frame identified in any ANRIL variant is 86 codons, supporting the concept that functionality of this gene is through RNA activity (16).

#### Linear and Circular Isoforms

Many conventional linear polyadenylated ANRIL isoforms are detected in different cell types. Circular ANRIL (circANRIL) isoforms, without polyadenylation, have also been described. Circular RNAs, which are formed by "back-splicing" in which a downstream splice donor site is joined to an upstream splice

**Abbreviations:** CAD, coronary artery disease; CAC, coronary artery calcium; CDK, cyclin dependent kinase; circANRIL, circular ANRIL; circRNA, circular RNA; GWAS, genome-wide association study; lncRNA, long noncoding RNA; miRNA, microRNA; MI, myocardial infarction; MTAP, S-methyl-5′ -thioadenosine phosphorylase; PBMC, peripheral blood mononuclear cell; PBTL, peripheral blood T-lymphocytes; PRC, polycomb repressive complex; SNP, single nucleotide polymorphism; VSMC, vascular smooth muscle cells.

locus, the *ANRIL* lncRNA is antisense to the protein coding genes. The p15INK4B gene is contained within intron 1 of *ANRIL*. (B) To date, *ANRIL* has 21 reported exons. (C,D) Numerous linear (C) and circular (D) exons have been reported. Due to the discovery of additional exons, distal exons in some circular isoforms have been renumbered based on the current 21 reported exons. \*Exons 15-16 refer to exons 14-15 in Holdt et al. (6). \*\*Exon 16 refers to exon 14 in Sarkar et al. (7) and Burd et al. (8).

acceptor site, were discovered in 2012 to be a broadly occurring phenomenon across developmental stages and tissues, arising from at least 14% of human transcribed genes (17, 18). CircRNAs enjoy distinct properties from linear RNAs, including, in general, enhanced stability and longevity, cytoplasmic localization, and lack of translation (although if an IRES is engineered, circRNAs can support translation) (19). Traditional PCR using antisenseoriented primers cannot distinguish between linear and circular isoforms; other methodology, such as PCR using "outwardfacing" primers directed away from each other, detection of specific exon-exon junctions, or protection from RNAse R digestion can quantify circular RNAs.

Careful examination of melanoma cell lines showed that the abundance of individual ANRIL exons is non-uniform, supporting the presence of different isoforms (7). In both transformed cell lines and in human brain derived cells, abundance of ANRIL exons was lower than exons from locus protein-coding genes CDKN2A and CDKN2B (8). Linear isoforms tend to include proximal exons (1-2), whereas isoforms with only central exons (4-16) are more likely to be circular (8). In melanoma cells, proximal exons (exon 1 and exon 5-6) were more highly expressed than distal exons. This suggests that short isoforms of ANRIL, which tend to include proximal exons, are more abundant than longer isoforms in this cell type (7). In human peripheral blood mononuclear cells and a monocyte cell line, four major groups of ANRIL transcripts were found, all with common proximal exons including exons 1, 5, and 6 but with different distal exons, of various lengths (8). Multiple circular ANRIL isoforms have been detected. A circANRIL isoform with an exon 14-5 head-to-tail junction was reported to be the predominant form in both an immortalized fibroblast cell line (8) and in a majority of melanoma cell lines (7). Other non-canonical back-spliced junctions observed in melanoma samples included exon 14-5, 7-4, 10-5, and 14-4 (7). The exons most commonly observed in circANRIL in melanoma cells were 4, 5, 6, 7, 10, 13, and 14; in varied human cell types, the majority of circANRIL species were exon 5-6-7 containing (6). ANRIL exons 1, 2, 3, 8, 9, 11, and 12 were rarely included in circular RNA products (8). In melanoma lines, no correlation was observed between abundance of linear and circANRIL (7). However, circANRIL expression was inversely correlated with linear ANRIL expression in peripheral blood mononuclear cells in a cardiovascular cohort (6). CircANRIL was found to be resistant to RNAse R digestion compared with linear ANRIL, and an actinomycin D time course confirmed enhanced stability of the 14-5 circANRIL isoform compared with linear isoforms (7, 8).

#### Secondary Structure

Structure and function of lncRNAs is of high interest in the scientific community, given the increasing recognition of lncRNA roles in cancer and the normal biology of higher organisms. As such, prediction of lncRNA structures is an important computational challenge. One approach is to identify structural elements through comparison of related lncRNAs. The MONSTER tool was used to compare ANRIL to two lncRNAs with similar biological function: HOTAIR and COLDAIR (20). MONSTER identifies sequence-predicted secondary structure, such as regions likely to be single stranded RNA, double stranded RNA, hairpin loops, interior loops and bulges. Comparing predictions of two lncRNAs with similar functions is proposed as a mechanism to identify structural motifs. When HOTAIR, COLDAIR, and ANRIL were compared, several common structures were identified, putative structural motifs related to their common function in epigenetic regulation, which could lead to a molecular understanding of mechanism of action in future studies (20). Another study identified the region of ANRIL that interacts with CBX7, a polycomb repressor component; secondary structure analysis revealed hairpin structural motifs with significant binding affinity to CBX7. Fluorescence anisotropy suggested a ternary complex between a particular loop of ANRIL, CBX7 and a H3K27me3 methylated histone peptide (21).

#### Cellular Localization

RNA localization impacts function. In melanoma cells, linear ANRIL species containing proximal (exon 1) and distal (exons 13b, 19) exons were predominantly found in the nucleus. However, middle exons (exons 5, 6, and 7), which are found in both linear and circANRIL, were observed in cytoplasmic fractions, suggesting that circANRIL species may be predominantly cytoplasmic (7). Nuclear localization suggests linear isoforms may be responsible for the known ANRIL function of regulating gene transcription via chromatin modulation (see below). Conversely, cytoplasmic localization suggests circANRIL forms may participate in post-transcriptional functions. In gastric (22), prostate (21), and urothelial (23) cancer cells, ANRIL was predominantly nuclear. In a beautiful high-resolution analysis of single-molecule lncRNA localization, ANRIL was found to be mostly localized to cell nuclei, in one or several bright foci. Like other lncRNAs analyzed, ANRIL nuclei foci were lost in mitotic cells (24). Physiological stimuli that change ANRIL localization may provide clues as to ANRIL functions. Intriguingly, in a retinal cell line ANRIL isoforms were observed by fluorescence in situ hybridization to localize to the peri-nuclear cytoplasmic space. ANRIL abundance was induced by glucose, but ANRIL localization did not change with high glucose exposure (25). On the other hand, a study in HUVEC cells, using primers predicted to detect both linear and circular isoforms, found ANRIL to be mostly nuclear; nuclear ANRIL was increased after exposure to TNF-α (26). At least one study has used ANRIL as a nuclear positive control to test localization of other transcripts (27). Future cell type specific studies of ANRIL localization under basal, stimulated, and stress conditions may lead to clues as to ANRIL roles in tissue health and disease.

## REGULATION OF ANRIL ABUNDANCE

Abundance of ANRIL species is determined by promoter transcriptional activity, splicing decisions, and RNA stability (**Figure 2**). Like other genes, ANRIL promoter activity is influenced by epigenetic control and transcription factor occupancy. Intriguingly, epidemiological findings suggest that epigenetic regulation of ANRIL, through promoter methylation, has important long-lasting consequences for tissue function (29– 31). As such, ANRIL regulation is one mediator of the impact of early life environmental signals on adult human health.

### ANRIL Promoter Methylation

Epidemiological and experimental findings demonstrate that methylation of the ANRIL promoter region regulates ANRIL gene expression and has functional importance. The first exons of ANRIL and p14ARF are separated by only 300 bp, in head-tohead antisense orientation; the intergenic region between them is a bidirectional promoter (4, 32, 33). In silico analysis of ENCODE ChromHMM data (34) revealed that this region is enriched for both promoter and enhancer activity, and DNAse I hypersensitivity, across multiple cell types, suggesting this is a regulatory region. This region is bound by CTCF, usually considered to be a transcriptional repressor, insulating promoters from enhancer activity. Oddly, CTCF binding at the ANRIL promoter was associated with active-chromatin mark histone H3K4 trimethylation (35). CTCF binding, and ANRIL and p14ARF expression, were inhibited by methylation of local CpG islands and increased by demethylation. Knockdown of CTCF prevented the demethylation-induced expression of ANRIL and p14ARF, confirming that CTCF is a methylation-sensitive positive regulator of ANRIL promoter activity (35).

Additional evidence supports the functional importance of CpG sites for ANRIL promoter activity, locus gene expression and transcription factor binding. Mutagenesis of the CpG sites affects both ANRIL and p14ARF promoter activity (29, 30). Methylation status of several CpG differentially methylated regions at ANRIL in umbilical cord tissues was positively associated with abundance of linear but not circular ANRIL, but inversely associated with p14ARF and p16INK4a expression (29). CpG methylation also affects other transcription factors binding at the ANRIL promoter to regulate downstream gene expression, such as interferon gamma, SMAD3/4 and ERα (29, 30). Methylation status of CpG islands around the p16INK4A transcription start site was also shown to coordinate transcription of ANRIL and p16INK4A in human cells (36). Given the multiple protein complexes binding across different CpG sites, and variable quantitative impact of individual CpG region mutagenesis on ANRIL isoforms and locus gene expression, regulation appears to be complex.

### Transcription Factors Regulate ANRIL Production

ANRIL expression is influenced by cellular processes such as genotoxic stress, tumorigenesis, senescence, and inflammation. Activity at the bidirectional promoter region upstream of both ANRIL and p14ARF genes is influenced by the critical cell cycle regulator E2F1 (32, 33). In response to the genotoxic stress of DNA damage, E2F1 transcriptionally activates ANRIL in an ATM-dependent manner (33, 37). In this case, ANRIL is thought to promote cell growth by suppressing locus INKfamily inhibitors after DNA repair is complete, allowing reentry into cell cycling (33). The ANRIL promoter was also responsive to E2F1 in cancer cells (32). In addition to E2F1, several known potent oncogenes regulate ANRIL expression in various cancers. In lung cancer, c-MYC binds to an E-box in the ANRIL promoter and induces ANRIL expression (38). In nasopharyngeal carcinoma, transcription factor SOX2 was

shown to bind directly to the ANRIL promoter and activate transcription of ANRIL and its downstream effector β-catenin (39). In liver cancer, SP1 binds the ANRIL promoter and positively regulates ANRIL transcription (40). On the other hand, TET2, a tumor suppressor in human gastric cancer, binds to the promoter region of ANRIL and regulates expression of ANRIL as well as p16INK4a, p15INK4b, and p14ARF (41). Transcription regulation of ANRIL is involved not only in cell DNA damage and oncogenesis, but also in disparate processes such as cell senescence and inflammation. In senescence, oncogenic Ras was found to reduce expression of ANRIL (13, 21, 42). In inflammation, STAT1 activates the ANRIL locus in vascular endothelial cells has been reported; CAD-associated ANRIL SNP rs10757278, located in a known downstream enhancer region, disrupts the STAT1 binding site and modulates IFN-γ induced ANRIL expression via stimulation (43). Intriguingly, the binding of STAT1 at this enhancer exerts cell-type specific regulation of ANRIL expression: repression in lymphoblastoid cells lines, but activation in HUVEC cells (43). In sum, data support an important role for cell-type specific transcriptional regulation of the ANRIL lncRNA in a range of cellular processes and outcomes.

### Regulation of ANRIL Splicing

Cell type dependent variation in abundance of different ANRIL isoforms suggests that splicing may be a point of regulation (8, 44). Almost nothing is known about ANRIL splicing decisions. Disease-associated ANRIL gene polymorphisms have shed light on this process. In lymphocytes, the coronary artery disease (CAD) associated SNP rs10757278 (intron 12) correlates with abundance of certain circular (14-5 and 4-6) and linear (exon 1-2, but not 18-19, containing) isoforms (8). The rs10757278 A allele was found to inhibit skipping of exon 15, promoting circANRIL species ending in exon 14 (8). Mechanisms regulating ANRIL splicing require further study.

### Post-Transcriptional Regulation

Determinants of ANRIL transcript longevity and stability remain uncertain, but miRNAs can participate. ANRIL, downregulated following Kaposi's sarcoma associated herpesvirus (KSHV) infection, contains multiple seed matches for KSHV miRNAs. Forced miRNA expression decreased ANRIL abundance, and miRNA pull-down experiments confirmed a direct interaction. In addition, KSHV latency associated proteins vFLIP and vCyclin also decreased ANRIL abundance, suggesting posttranscriptional miRNA-dependent and independent regulation (45).

## FUNCTIONS OF THE ANRIL LNCRNA

### Transcription Regulation via Chromatin Modifying Complexes

Many studies show that ANRIL functions in cells to regulate gene expression via chromatin modification. Acting in cis, ANRIL interacts with both PRC-1 and−2 to mediate epigenetic transcriptional repression of neighboring genes CDKN2A and CDKN2B, through mechanisms involving histone modification and chromatin remodeling (13, 21, 33). ANRIL interacts with PRC1 component CBX7 to recruit PRC1 to the p14ARF and p16INK4A loci, silencing the CDKN2A locus by H3K27 trimethylation (21). At CDKN2B, ANRIL was shown to recruit SUZ12, a subunit of the PRC2 (13). ANRIL also interacts with PRC-associated protein YY1 (46). Intriguingly, the structural conformation of the methyl-lysine binding pocket in the chromodomain of CBX7, which interacts with H3K27 trimethylation to cause chromatin compaction, is influenced by allosteric RNA-protein binding with ANRIL (47). However, despite this well-documented repression of other locus genes by ANRIL, a positive correlation between ANRIL (both short and long isoforms), CDKN2A and CDKN2B RNA abundance has been frequently reported, suggesting transcriptional coregulation of these genes predominates in many tissues (8, 10, 14, 16, 44, 48–51).

ANRIL also acts in a PRC1/2 dependent mechanism to repress distant genes in trans (32, 46). Trans regulation by ANRIL may be dependent on Alu motifs, which are found both in ANRIL transcripts and in the promoters of ANRIL target genes (46). This mechanism was shown to regulate the CARD8 gene in endothelial cells (52). Polycomb group proteins, which are highly enriched near Alu motifs across the genome, are recruited to target gene promoters upon ANRIL over-expression. In support of this concept, silencing ANRIL impacts expression of a large number of genes across the genome (14). Separate from chromatin modification, ANRIL is reported to regulate Wnt signaling by binding to SOX2, increasing transcriptional activity of the WNT/β-catenin pathway (39).

#### miRNA Abundance and Activity

ANRIL also influences gene expression via miRNA networks. ANRIL regulates miRNAs both at the epigenetic level, through regulation of miRNA transcription, and through direct binding to miRNAs, acting as a miRNA "sponge." In gastric cancer cells, ANRIL epigenetically silences miR-99a/miR-449a through a PRC2 mechanism (22). In general, expression of ANRIL and its target miRNAs are negatively correlated in tissues and cell lines (22, 53–57). ANRIL has been described as having pro-oncogenic effects by sponging miRNAs (see below for more details). On the other hand, circANRIL containing exons 5-6-7 was found to lack miRNA sponge activity (6). Inhibition of miRNAs can reverse the effects of ANRIL knockdown.

#### Cellular Outcomes of ANRIL Activity

ANRIL has broad impacts on cell biology, including influence over proliferation, senescence, apoptosis, extracellular matrix remodeling, and inflammation (14). In cancer, ANRIL-miRNA interactions regulate networks of downstream targets of miRNAs, promoting an oncogenic role for ANRIL in cell proliferation, metastasis, invasion, radio-resistance, drug-induced cytotoxicity and apoptosis, involving many different signaling pathways (22, 53–57). Specifically, repression of cell cycle inhibitors p14ARF, p15INK4B, and p16INK4A increases proliferation, decreases senescence, and contributes to the DNA damage response (13, 21, 33). PRC-mediated epigenetic repression of Kruppellike factor 2 (KLF2) influences proliferation and apoptosis (40, 58). Cooperation between ANRIL and PRC-associated YY1 increases TNF-alpha dependent inflammatory mediators (IL-6, IL-8) through NF-kB (26). ANRIL influencesthe cellular response to oxidative stress through a miR-125a regulation of MCL-1 (59). Circular ANRIL species were found to regulate ribosome biogenesis in vascular smooth muscle cells (6).

### THE ANRIL GENE IS ASSOCIATED WITH HUMAN DISEASE

A primary driver of interest in ANRIL is the large body of genomic data linking the ANRIL gene with risk of human disease. Genome-wide association studies (GWAS) have identified many disease-associated SNPs in or near the ANRIL gene (60). The CDKN2A/B locus is remarkable for the large number of associated diseases, ranging from aging and frailty to cancer to metabolic disease. Perhaps surprisingly given the validated importance of the products of the CDKN2A and CDKN2B genes in cell biology, in some cases ANRIL expression shows stronger phenotype association than protein-coding CDKN2A/B locus genes (4, 48), linking ANRIL itself to a range of important human diseases.

### ANRIL SNPs and Disease Risk

Studies indicate that SNPs in the ANRIL gene can impact ANRIL expression and function. The CDKN2A/B locus is associated with risk of cancer, atherosclerotic disease, type 2 diabetes, stroke, aneurysm, periodontitis, Alzheimer's disease, aging, frailty, glaucoma, endometriosis, multiple sclerosis, hypertension (10, 61). Reviewed here are only SNPs within or downstream of the ANRIL gene; broader CDKN2A/B locus disease associations have been reviewed previously (10, 62). Integrating information from published observations and the NCBI linkage disequilibrium database (63), we find that disease-associated SNPs in the ANRIL gene that modulate locus gene expression fall into approximately six groups (**Table 1**) defined loosely by linkage block and reported effects. Exceptions outnumber the rules, however; for nearly all groups there are reports of SNPs with different or even opposite effects. Summarized here is a generalized synopsis of the majority of reports. Group A SNPs, while located in ANRIL introns, generally impact CDKN2A/B but not ANRIL biology (48, 50, 64). All other SNP groups have reported impacts on ANRIL itself, but reports often describe conflicting direction of change. Some SNPs are reported to fall in enhancer regions (43, 49, 64) or to impact ANRIL splicing (8) or secondary structure (69, 74). The data are incomplete. A particular weakness of the field is that although tissue-specific effects are likely to determine how polymorphisms impact disease risk, in many cases the relevant primary tissue has not been tested.

### Disease-Associated SNPs May Influence ANRIL Abundance

There is no consistent global pattern with respect to SNP impact on ANRIL abundance. For most SNP groups, risk-SNPs are reported that both increase and decrease ANRIL levels in different studies. Variability may be related to differences in technique used to detect ANRIL that favor one isoform over others, cell type studied, acute and chronic biology and genetic origin of the cellular material studied, and of course the individual biology of each polymorphism. Most ANRIL SNPs fall in large linkage blocks, which are variable among different human genetic groups; in many cases the SNP tested may not be the causative SNP in the linkage block, and published linkage blocks may not apply to the material tested if not carefully matched by

TABLE 1 | Disease associated SNPs in/near the *ANRIL* gene that modulate locus gene expression.

*Groups A–F are defined loosely based on linkage disequilibrium (defined as LD*>*0.8 in Caucasian population in LDHap) and by predicted or tested impact on ANRIL expression or structure. Intron and exon numbers are based on 21 exons.*

origin. It is entirely possible that all conflicting results are correct; for example, a CAD risk-SNP could increase pro-proliferative ANRIL isoforms in endothelial, macrophage or vascular smooth muscle cells to drive atherosclerosis, whereas a diabetes risk-SNP at the same position could decrease proliferative ANRIL isoforms in beta cells to limit beta cell mass. The complexity of the human system necessitates testing the relevant ANRIL isoforms in the relevant cell type, preferably in primary cells, in tissue- and disease-specific manner.

A comprehensive review of all SNP effects is beyond the scope of this review. Some ANRIL located disease-associated SNPs impact both ANRIL expression and CDKN2A/CDKN2B expression (14, 48); others impact ANRIL but not CDKN2A or CDKN2B (14, 48, 60), and still others impact CDKN2A/CDKN2B but not ANRIL (48, 50, 64). Some SNPs are located within predicted or proven enhancer regions (10, 43, 48, 49, 64, 75) or miRNA binding sites (65, 66), providing possible mechanisms of cell type specific gene regulation.

### Disease-Associated SNPs May Influence ANRIL Structure or Function

Beyond regulation of ANRIL transcription, polymorphisms could impact ANRIL function by influencing relative abundance of different isoforms through RNA splicing or stability, or through altering the secondary structure or interactions of any given isoform. Several studies have identified ANRIL isoform-specific effects (50, 53, 67, 71, 72); for example, four SNPs forming an atherosclerosis risk haplotype were associated with increased expression of some, but not all, ANRIL isoforms (44). SNPs may influence the relative abundance of linear compared to circular isoforms (8). Several SNPs are reported to impact ANRIL free energy of folding, resulting in a predicted change in secondary structure, with implications for function and stability (48, 69, 74).

### ANRIL IN CANCER

ANRIL was initially identified in a kindred of familial melanomaneural system tumor with a germ-line deletion of the entire CDKN2A/B locus (2). Although the CDKN2A/B locus is deleted or silenced in approximately 40% of human cancers, related to the tumor suppressive actions of CDKN2A and CDKN2B (76), ANRIL itself has pro-oncogenic properties. ANRIL is implicated in many malignancies, including cancers of the bladder (77), ovary (78, 79), lung (38, 58, 80–82), liver (40, 54, 83), stomach (22), breast (57, 84, 85), esophagus (86), nasopharyngeal cavity (39, 87, 88), thyroid (89), bone (90), cervix (91), colon (92), prostate (21, 56), glioma (55), and others (76). High tissue abundance of ANRIL in cancers is associated with aggressive clinicopathologic features such as high histological grade tumor size, advanced tumor-node-metastasis stage, and poor overall survival (22, 38, 40, 58, 78, 79, 83, 87, 89, 91–93). Certain SNPs within the ANRIL gene are associated with ANRIL and CDKN2A/B locus gene expression and clinical parameters (4, 48, 70, 94–96). ANRIL may be useful as a prognostic biomarker and a therapeutic target for clinical cancer management.

#### Molecular Mechanisms of ANRIL in Cancer

Accumulating evidence suggests that ANRIL participates in tumorigenesis by influencing cell proliferation, apoptosis and metastasis. Depletion or overexpression of ANRIL changes expression levels of many genes involved in proliferation, cellular adhesion and apoptosis (14, 32, 46). ANRIL overexpression promotes proliferation, migration, invasion, and epithelialmesenchymal transformation but inhibits cell apoptosis; ANRIL loss-of-function represses tumor size and growth rate, cell proliferation, migration, invasion, metastasis, and enhances apoptosis and senescence (22, 38, 55, 56, 58, 77, 80, 81, 84, 89–92). Suppression of ANRIL is required for Ras-induced senescence (13, 21, 42). High ANRIL levels are associated with resistance to chemotherapy, and ANRIL knockdown may promote chemosensitivity (37, 79, 88, 97–99). On the other hand, ANRIL mediated anti-oncogenic effects of phospholipase D in lung cancers (82).

ANRIL may promote carcinogenesis through a number of mechanisms. Canonical ANRIL transcriptional mechanisms may play a role, such as by in cis suppression of the CDKN2A/CDKN2B tumor suppressor genes (80, 81, 100), or through PRC-mediated in trans gene regulation (40, 58, 80). ANRIL miRNA regulation has been implicated in cancers as well, including mechanisms involving let-7a and miR-125a in nasopharyngeal and oral carcinoma (56, 88, 101), miR-99a/miR-449a in gastric cancer (22), miR-122-5p in hepatocellular carcinoma (54), miR-186 in cervical cancer (91), and miR-199a in breast cancer (57), miR-34a in glioma (55), and miR-323 in pediatric medulloblastoma (102). Transcription factors affected by ANRIL in cancers include KLF2 (40, 58), SMAD (56, 86, 89) and β-catenin. ANRIL interacts with signal transduction pathways in cancers such as PI3K/AKT, p38 MAPK, TGF-β, ATM-E2F1, and MTOR (33, 55, 56, 86, 89, 99, 103). ANRIL can also drive cancer progression by increasing glucose uptake for glycolysis (87), through lymphangiogenesis via LYVE-1, VEFG-C, and VEGFR-3 (92), and through invasion and metastasis via MET and MMP3 (78). An intriguing but mostly unexplored phenomenon is breakpoint fusion transcripts including exons from ANRIL fused with exons from MTAP, a neighboring protein-coding gene, which were identified in 20% of screened melanoma cell lines (104).

### ANRIL IN METABOLIC DISEASE

In addition to cancer, genome-wide association studies have repeatedly and confidently identified links between the genomic region containing ANRIL and risk of developing cardiometabolic disease, including type 2 diabetes and manifestations of atherosclerosis such as CAD and stroke (10, 62). This locus influences risk not only of classic type 2 (obesity-related) diabetes, but also with related syndromes such as gestational diabetes, transplant-associated diabetes, and cystic fibrosis related diabetes, but not risk of type 1 (autoimmune) diabetes (10). Although diabetes is a clinical risk factor for atherosclerosis, the genetic influence for these conditions at the ANRIL locus is mostly non-overlapping, with atherosclerosis SNPs located throughout the ANRIL gene, and T2D SNPs located distal to the last ANRIL exon (7). One exception is a SNP located in ANRIL exon 2, rs564398, which is associated with both T2D and CAD (105). Since CDKN2A/B locus genes are known for their roles in cell cycle regulation and cancer, and not metabolism, many questions remain as to how this locus impacts metabolic disease.

#### ANRIL and Atherosclerotic Disease

Since ANRIL locus SNPs influence risk of atherosclerosis, many studies have now tested whether ANRIL gene expression is related to atherosclerosis-associated diseases. In subjects with angiographically confirmed CAD in the Leipzig heart study, specific ANRIL isoforms were positively correlated with CAD risk SNP haplotype in PBMCs, whole blood, and atherosclerotic plaque tissue (44). In the Framingham heart study, ANRIL SNPs were associated with multiple CAD-related outcomes, and showed isoform-specific ANRIL correlation in leukocytes, with short isoforms predicted to contribute to CAD pathogenesis (50). CAD risk-SNPs may regulate the relative abundance of linear and circular ANRIL isoforms (8). Intriguingly, abundance of ANRIL in circulating plasma was positively correlated with in-stent restenosis (53), but in PBMCs harvested at the time of angioplasty/reperfusion, ANRIL levels were lower in subjects with myocardial infarction, but higher in subjects with older age, diabetes, hypertension. In this cohort, ANRIL levels in PBMCs improved model prediction of subsequent left ventricular dysfunction (106). ANRIL promoter methylation may mediate an epigenetic influence on future cardiac risk; higher CpG methylation at birth was associated with higher pulse wave velocity, a marker for increased arterial stiffness indicating greater cardiovascular risk, at 9 years of age (31).

Mechanisms by which ANRIL impacts atherosclerotic disease remain debated. In aortic smooth muscle cells, knockdown of ANRIL using siRNA targeting exon 1 or exon 19 revealed altered gene expression networks impacting cell proliferation, apoptosis, extracellular matrix, and inflammation (14). Atherogenic gene expression networks were regulated by ANRIL via the Alu mechanism, in which Alu motifs target ANRIL to particular gene locations, recruiting PRC complexes and altering gene methylation status (46). ANRIL may impact risk of ischemic stroke by regulating the Caspase recruitment domain 8 (CARD8) gene in endothelial cells (52). A known CAD-associated miRNA, miR-92a, may mediate some ANRIL effects; ANRIL targets GATA2, MAP1B, and ARG1 were found to require miR-92a, placing this miRNA downstream of ANRIL for some atherogenic effects (69). Finally, ANRIL is related to inflammation: ANRIL is increased by pro-inflammatory factors NF-κ B and TNF-α in endothelial cells, and ANRIL was found to bind directly to the YY1 transcription factor to mediate TNF-a induction of cytokines IL-6 and IL-8 (26).

### ANRIL and Obesity, Bone Mass, and Estrogen Signaling

Although GWAS studies do not suggest a link between CDKN2A/B locus SNPs and obesity risk in adult populations, intriguingly, ANRIL may be a genomic site of environmental epigenetic influence on obesity. The ANRIL promoter contains CpG methylation sites that are differentially regulated across samples. In human tissues taken at birth, lower CpG methylation in infancy predicted higher fat mass at 6 years of age, as well as increased bone size, mineralization and density (29, 30). ANRIL promoter methylation was also negatively correlated with BMI in contemporaneous samples of peripheral blood from adolescents and in adipose tissue from adults (29). Methylation of these CpG sites increased tissue abundance of ANRIL RNA, in a mechanism that might include increased activity of an estrogen response element. Functional studies in a liposarcoma cell line showed that transcription factor binding to an adjacent ERE was enhanced by methylation, and estradiol increased ANRIL expression (29).

### ANRIL and Type 2 Diabetes

Multiple SNPs in different linkage blocks at the CDKN2A/B locus are associated with T2D risk; evidence in human populations suggests these SNPs impact pancreatic islet mass or function (10). Despite the fact that the T2D risk SNPs are located in or near the ANRIL gene, the field has largely assumed the effect was mediated by the protein coding genes at the locus, due to extensive published work implicating p16INK4A in the regulation of beta cell mass (10). However, although studies have found no association between CDKN2A/B T2D SNPs and transcript level of p14ARF, p15INK4B, or p16INK4A in human islets (10, 107), an age-dependent positive association was identified between distal T2D risk-SNPs (group F in **Table 1**) and ANRIL expression (68). On the other hand, a T2D risk-SNP in ANRIL exon 2 (group C in **Table 1**) was associated with reduced ANRIL expression, again with no change in p14ARF, p15INK4B, or p16INK4A expression (14, 48); however, these studies were carried out in blood rather than islets. In human islets, this exon 2 SNP was shown to remove a CpG methylation site; risk allele was associated with reduced islet insulin content but no change in locus gene expression (108). Risk allele at this SNP was associated with impaired beta cell proliferation response to high glucose (68). In a study relevant to diabetic retinopathy, high glucose exposure increased ANRIL expression in human retinal epithelial cells (25). In ANRIL was found to increase expression of VEGF, a critical element of the neovascularization that is central to damage from retinopathy, via a mechanism involving PRC2 and miR200b (25, 109).

### SUMMARY

Studies suggest the ANRIL lncRNA influences risk of a number of diseases, including many types of cancer as well as metabolic disease. Current understanding of ANRIL biology indicates the primary function of this lncRNA is to regulate gene expression, both locally at CDKN2A/B as well as across the genome, via mechanisms including chromatin modulation, transcription factor binding, and miRNA regulation. Knowledge concerning ANRIL function in cancers is more solid and advanced than for metabolic tissues. Mechanisms by which SNPs influence ANRIL abundance remain uncertain and require more study; how DNA methylation regulates ANRIL in cancers also will benefit from more study. Much remains to be learned about the structural complexity of ANRIL; how the various identified linear and circular isoforms impact tissue biology to modulate disease risk is mostly unknown. There is an urgent need for deeper understanding of how ANRIL isoforms modulate cellular function in human organs and tissues, and to explore the differing roles of ANRIL in cancer and metabolic disease. Given the advent of RNA therapeutics, and the broad disease relevance of ANRIL, it is possible that these studies may lead to future disease prevention and treatment.

### AUTHOR CONTRIBUTIONS

YK, C-HH, and LA wrote and revised the manuscript.

### FUNDING

This work was supported by NIH/NIDDK: R01DK114686 and NIH/NIDDK DK095140 (LA), the American Diabetes Association grant #1-18-IBS-233 (LA) in collaboration with the Order of the Amaranth, and the George F. and Sybil H. Fuller Foundation.

### ACKNOWLEDGMENTS

We would like to thank the Beta Cell Biology Group at the University of Massachusetts Medical School for helpful discussions.

## REFERENCES

through TGF-β/Smad signaling pathway. Oncotarget (2016) 7:57903–18. doi: 10.18632/oncotarget.11087

in human islets. Cell Metab. (2012) 16:122–34. doi: 10.1016/j.cmet.2012. 06.006

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Kong, Hsieh and Alonso. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Critical Enzymatic Functions of FTO in Obesity and Cancer

Xiaolan Deng1,2, Rui Su1 , Savanna Stanford1 and Jianjun Chen1 \*

1 Department of Systems Biology and The Gehr Family Center for Leukemia Research, Beckman Research Institute of City of Hope, Monrovia, CA, United States, 2 School of Pharmacy, China Medical University, Shenyang, China

Fat mass and obesity-associated protein (FTO) single-nucleotide polymorphisms (SNPs) have been linked to increased body mass and obesity in humans by genome-wide association studies (GWAS) since 2007. Although some recent studies suggest that the obesity-related SNPs in FTO influence obesity susceptibility likely through altering the expression of the adjacent genes such as IRX3 and RPGRIP1L, rather than FTO itself, a solid link between the SNP risk genotype and the increased FTO expression in both human blood cells and fibroblasts has been reported. Moreover, multiple lines of evidence have demonstrated that FTO does play a critical role in the regulation of fat mass, adipogenesis, and body weight. Epidemiology studies also showed a strong association of FTO SNPs and overweight/obesity with increased risk of various types of cancers. As the first identified messenger RNA N 6 -methyladenosine (m6A) demethylase, FTO has been shown recently to play m6A-dependent roles in adipogenesis and tumorigenesis (especially in the development of leukemia and glioblastoma). Given the critical roles of FTO in cancers, the development of selective and effective inhibitors targeting FTO holds potential to treat cancers. This mini review discusses the roles and underlying molecular mechanisms of FTO in both obesity and cancers, and also summarizes recent advances in the development of FTO inhibitors.

#### Edited by:

Che-Pei Kung, Washington University in St. Louis, United States

#### Reviewed by:

Eric Kalkhoven, Utrecht University, Netherlands Luca De Toni, Università degli Studi di Padova, Italy

> \*Correspondence: Jianjun Chen<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cancer Endocrinology, a section of the journal Frontiers in Endocrinology

Received: 04 May 2018 Accepted: 27 June 2018 Published: 30 July 2018

#### Citation:

Deng X, Su R, Stanford S and Chen J (2018) Critical Enzymatic Functions of FTO in Obesity and Cancer. Front. Endocrinol. 9:396. doi: 10.3389/fendo.2018.00396

Keywords: fat mass and obesity-associated protein (FTO), obesity, cancer, mRNA N6 -methyladenosine (m6A), m6A demethylase, AML, GBM, FTO inhibitors

## INTRODUCTION

As the first genome-wide association studies (GWAS)-identified obesity susceptibility gene, the fat mass and obesity-associated gene (FTO) has been well known for the strong association of the multiple single-nucleotide polymorphisms (SNPs) located in its intron 1 with risk of obesity (1–10). Although there are some controversial reports regarding the association between FTO SNPs and FTO expression (11–13), mouse model studies have shown the pivotal role of FTO in the regulation of fat mass, adipogenesis, and body weight (14–20). The link between the SNP risk genotype and increased FTO expression in human fibroblasts and blood cells has also been demonstrated (21–23). Studies have demonstrated that a strong association exists between FTO SNPs and/or overweight/obesity with the increased risk of various types of cancers (24–29), implying a role of FTO in the pathogenesis of cancers. Indeed, the oncogenic role of FTO has been reported in leukemia and glioblastoma (GBM), where FTO is highly expressed (30– 32). More importantly, FTO was reported as the first N 6 -methyladenosine (m6A) demethylase of eukaryotic messenger RNA (mRNA) (33), and the functions of FTO in adipogenesis and tumorigenesis have been linked to its m6A demethylase activity (30–32, 34). As the most abundant internal modification in eukaryotic mRNAs, m6A usually occurs at the consensus motif of RRm6ACH ([G/A/U][G>A]m6AC[U>A>C]); enriched in 3′ untranslated region (UTR), gene coding regions, and especially near stop codons (35, 36). The m6A modification is deposited by the METTL3-METTL14-WTAP methyltransferase complex (i.e., writer) (37–39) and can be removed by m6A demethylases (i.e., erasers) such as FTO and ALKBH5 (33, 40). The m6A modification functions as a post-transcriptional modulator of gene expression by decreasing or increasing mRNA stability, or promoting mRNA translation efficiency through its recognition of different m6A reader proteins (41–48). The roles of m6A modification and the associated machinery in the pathogenesis of various types of cancers have been reported recently (30– 32, 48–59). This review focuses on the functions of FTO in both adipogenesis and tumorigenesis and on the underlying m6A-dependent mechanisms, along with a brief discussion of recent advance in the development of FTO inhibitors and their therapeutic potential to treat cancers.

#### ASSOCIATION OF FTO WITH OVERWEIGHT/OBESITY AND ITS ROLE IN ADIPOGENESIS