Sirtuins and the Metabolic Hurdles in Cancer

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Natalie J. German and Marcia C. Haigis Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA *Correspondence: [email protected] (N.J.G.), [email protected] (M.C.H.) http://dx.doi.org/10.1016/j.cub.2015.05.012

The nutrient demands of cancer cannot be met by normal cell metabolism. Cancer cells undergo dramatic alteration of metabolic pathways in a process called reprogramming, characterized by increased nutrient uptake and re-purposing of these fuels for biosynthetic, bioenergetic or signaling pathways. Partitioning carbon sources toward growth and away from ATP production necessitates other means of generating energy for biosynthetic reactions. Additionally, cancer cell adaptations frequently lead to increased production of reactive oxygen species and lactic acid, which can be beneﬁcial to cancer growth but also are potentially toxic and must be appropriately cleared. Sirtuins are a family of deacylases and ADP-ribosyltransferases with clear links to regulation of cancer metabolism. Through their unique ability to integrate cellular stress and nutrient status with coordination of metabolic outputs, sirtuins are well poised to play pivotal roles in tumor progression and survival. Here, we review the multi-faceted duties of sirtuins in tackling the metabolic hurdles in cancer. We focus on both beneﬁcial and adverse effects of sirtuins in the regulation of energetic, biosynthetic and toxicity barriers faced by cancer cells.

Introduction Here, we review the roles of sirtuins in the metabolic hurdles of It is more relevant than ever to understand how metabolism cancer. We will first overview sirtuin enzymatic activity and links influences tumor growth. Bioenergetic and biosynthetic depen- to cancer incidence and severity. Then we will discuss sirtuin- dencies of cancer cells are increasingly being realized as prom- mediated control of metabolic pathways with a focus on glucose ising candidates for therapeutic interventions in cancer [1–3]. metabolism, refilling of the tricarboxylic acid (TCA) cycle and A vast number of studies validate the notion that metabolic in defense against reactive oxygen species (ROS) in cancer. dysfunction is not just a consequence of cancer growth but rather Sirtuins coordinate many other processes linked to cancer, a driving factor in tumor progression [4,5]. Indeed, altered nutrient including DNA repair, metastasis, apoptosis and translation: for utilization enables tumor cells to fuel a number of processes, such reviews more comprehensively assessing those roles, we refer as amassing a pool of biosynthetic precursors, constructing the reader to other sources (such as [13,14,19]). signaling molecules, generating molecules for post-translational or epigenetic modifications, and maintaining pH and redox Connections between Sirtuin Activity and the Metabolic homeostasis [6,7]. Furthermore, metabolic dysfunction has posi- State tioned itself at the forefront of cancer research with the recogni- Sirtuin Enzymatic Activity tion of the undeniable connection between increased cancer inci- SIRT1–SIRT7 are a family of deacylases and ADP-ribosyltrans- dence and the background of obesity and metabolic disease, ferases that share a conserved catalytic core domain but vary pathologies that have reached epidemic proportions in the in subcellular localization and preferred substrates [20]. The dif- United States and much of the world [8–11]. It is critical to fully un- ferences between sirtuins lead to variations in the ultimate meta- derstand how tumor cells alter fuel usage and to identify path- bolic effect that is coordinated by each sirtuin [15]. SIRT1, SIRT6 ways that might promote or oppose this metabolic dysfunction. and SIRT7 are primarily nuclear and regulate transcription fac- Sirtuins are a highly conserved family of regulatory enzymes tors and histone modifications to coordinate gene expression that are well poised to play pivotal roles in tumor metabolism. programs that can direct the cellular metabolic state [21]. Cyto- The seven mammalian sirtuins (SIRT1–SIRT7) have the unique solic functions of SIRT1 have also been identified. SIRT2 is ability to integrate the cellular stress response with the coordina- largely cytosolic and coordinates microtubule dynamics as well tion of metabolic fitness and homeostasis [12,13]. The role of as the activity of transcription factors residing outside the nu- sirtuins as post-translational modifying enzymes may have orig- cleus [22,23]. Localization of SIRT3, SIRT4 and SIRT5 in the inated to allow survival under stress and low nutrient conditions, mitochondrial matrix enables these sirtuins to directly alter the and many of these functions have now been linked to growth activity of many metabolic enzymes [24]. regulation in the harsh conditions experienced by cancer cells Sirtuins catalyze nicotinamide adenine dinucleotide (NAD+)- [14–16]. In recent years, a number of studies have shown that sir- dependent deacylation or ADP-ribosylation reactions with vary- tuins not only coordinate cancer cell growth and survival, but ing degrees of substrate versatility [20]. Although originally also regulate the nutrient state of a tumor [17,18]. There is referred to as deacetylases, sirtuins are now classified more growing interest in pinpointing metabolic regulatory nodes that broadly as deacylases. This term accounts for the ability of can be targeted in cancer treatment and determining whether certain sirtuins to remove not only acetyl groups from lysine sirtuins specifically may be promising biomarkers or therapeutic residues, but also other acyl modifications, including propionyl, targets in cancer. butyryl, malonyl, succinyl and the lengthy fatty-acid-derived

A Acyl groups O Acyl-lysine Deacylated O O NAD+ Nicotinamide residue lysine residue Lys Lys Lys O Acetyl Propionyl Butyryl O O NH2 O O O NH2 R NH +NH 3 HO + SIRT N HO Lys Lys ADP N ADP OH O + + Malonyl Succinyl O O N N O O C C OH OH OH O O Lys Lys O 2’-O-acyl- O 6 7 ADP ribose R Myristoyl Palmitoyl

B Arginine ADP-ribosylated NAD+ Nicotinamide residue residue O O ADP O NH H2N NH + + 2 NH2 HN ADP N SIRT OH OH HN N + O + + NH2 HN

OH OH N C N C O Current Biology

Figure 1. Sirtuin-catalyzed reactions. (A) During deacylation reactions, sirtuins direct NAD+ to nucleophilically attack the acylated lysine residue, leading to removal of the acyl modification. NAD+ is cleaved in the process, forming nicotinamide and 2’-O-acyl ADP-ribose. Sirtuins can potentially remove diverse acyl modifications (inset) from lysine residues. (B) During ADP-ribosylation reactions, sirtuins use NAD+ to nucleophilically attack an arginine (shown) or cysteine residue. NAD+ is cleaved, resulting in release of nicotinamide and transfer of the ADP-ribose portion of NAD+ to the substrate residue. myristoyl and palmitoyl groups [25]. SIRT5, for example, is a cell death in response to puromycin. Of note, quantitative studies strong desuccinylase, and SIRT4 was recently reported to func- of OAADPR have been impeded by the rapid hydrolytic degradation as a lipoamidase by removing lipoyl or biotinyl modifications tion of this molecule to ADP-ribose in cells [36]. The role of sir- from lysine residues [26]. Through these processes, sirtuins have tuin-derived metabolites is a promising and little studied area been shown to alter substrate activity, localization, stability and of sirtuin biology. protein–protein interactions [14]. Sirtuins and NAD+ Sensitivity Regardless of which type of substrate moiety is modified by Sirtuins are unique sensors of the metabolic state because their sirtuins, a similar NAD+-dependent reaction mechanism pro- NAD+-dependent enzymatic activity intrinsically couples their ceeds. During sirtuin-catalyzed deacylation (Figure 1A), NAD+ function to the metabolic status of the cell or organism [37–41]. nucleophilically attacks an acyl group of a substrate lysine. The According to the metabolic state of the cell, the ratio of NAD tog- resulting intermediate is cleaved to form 2’-O-acyl-ADP ribose gles between varying amounts of NAD+ and NADH [42]: NADH is (OAADPR) and nicotinamide, and the acyl group is removed a high-energy, reduced form of NAD that can donate electrons to from the lysine residue in the process. In sirtuin-catalyzed the electron transport chain, and NAD+ is the lower energy, ADP-ribosylation (Figure 1B), NAD+ similarly attacks a substrate oxidized counterpart required to fuel glycolysis. When the cell residue, typically an arginine residue [27], although cysteine res- uses oxidative metabolism, NADH generated by the TCA cycle idues are also candidate sites [28–30]. The ADP-ribose portion of and glycolysis donates electrons to complex I of the electron NAD+ is transferred to the substrate residue, yielding nicotin- transport chain (ETC). This contributes to a proton gradient amide as a side product [31,32]. that will ultimately produce ATP. Upon electron transfer, NADH The metabolic by-products of sirtuin activity have potential to is oxidized back to NAD+. In highly glycolytic cells with low accumulate and influence cellular biology. At high concentra- ETC function, NAD+ is alternatively regenerated from NADH via tions, nicotinamide inhibits sirtuin function [33]. Work by Grubi- lactate dehydrogenase (LDH) activity in order to sustain glycol- sha et al. [34] showed that pools of OAADPR generated by ysis. NAD+ can also be synthesized de novo from tryptophan certain sirtuins bind and inhibit the non-selective cation channel and vitamin B3 derivatives, or via salvage pathways using nico- TRPM2. This channel is normally activated by oxidative or nitra- tinamide or nicotinamide riboside. Thus, the NAD+:NADH ratio is tive stress as well as by the drug puromycin, which directly tar- affected by the cellular metabolic state, and changes in this ratio gets TRPM2 [34,35]. Activation of this channel leads to an influx have potential to impact sirtuin enzymatic activity. of Na2+ and Ca2+ into the cytosol to induce cell death. Decreased Several studies have linked sirtuin activity with the organismal expression of SIRT2 and SIRT3 dampened TRPM2-mediated metabolic status and cellular NAD ratio. In many tissues, the

NAD+:NADH ratio is low during nutrient excess and high during studies and via multiple proposed mechanisms [47,64]. SIRT1 nutrient deprivation [43,44]. For example, in skeletal muscle activity is also dramatically enhanced upon phosphorylation by and white adipose, the NAD+:NADH ratio is elevated during cal- additional kinases in response to adrenergic signaling and orie restriction [45]. Due to their dependency on NAD, it is not stresses such as DNA damage, microtubule disruption and surprising that certain sirtuins are reported to have increased ac- heat or cold shock [65,66]. Phosphorylation at one particular tivity in response to high NAD+ levels [46]. For example, SIRT1 in site, Thr522, boosts SIRT1 activity by promoting its monomeric skeletal muscle and brain is activated by exercise, fasting and state rather than its oligomeric, aggregation-prone state [67]. calorie restriction [47–49]. In contrast, low NAD+ levels are In the case of SIRT4, activity likely does not parallel the cellular observed with obesity as well as old age, two factors that confer NAD+ level. SIRT4 plays a key role in inhibiting fat catabolism increased risk for many cancers and are also linked to decreased when mice are well fed, despite the low levels of NAD+ expected sirtuin activity [50,51]. Along these lines, growing evidence sug- under this condition [68]. SIRT4 mRNA and protein are more gests that loss of sirtuin function plays a role in obesity- and age- abundant in mouse tissues under fed versus fasted conditions associated cancers [8,52]. [69,70]. SIRT4 mRNA is also highly induced by DNA damage It is proposed that there are tissue-type and cellular-compart- and inhibition of mammalian target of rapamycin (mTOR), dis- mentalization variations in NAD+ and NADH levels that may lead cussed further below [71,72]. SIRT6 activity in vitro is induced to distinct alterations of sirtuin activity in different contexts [42]. by fatty acids [25]. It will be important for future studies to reveal NAD+ is generated by biosynthetic reactions in the mitochondria, further molecular mechanisms by which sirtuin levels and activity nucleus and cytosol [53]. Generally NAD+ is most abundant in are regulated, whether it is via post-translational modification, mitochondria, particularly in highly metabolically active tissues, transcriptional control, or even mRNA stability. such as cardiac myocytes, although the distribution varies across cell types [54]. Nuclear NAD+ can be depleted upon Connections between Sirtuins and Cancer DNA damage when this molecule is used as a substrate for the The associations between cancer metabolism and sirtuins often poly(ADP-ribose) polymerase (PARP) family of enzymes to acti- fall into one of two themes. First, loss of sirtuin activity may result vate DNA repair pathways [55]. Inhibiting PARPs elevates in increased susceptibility to cancer onset. Second, and some- NAD+ levels, presumably in the nucleus, and accordingly was what paradoxically, an already established tumor that expresses shown to boost activity of nuclear SIRT1 and not mitochondrial high levels of some sirtuins may possess survival advantages, SIRT3 [56]. Upon PARP activation, the mitochondrial perme- including resistance to chemotherapeutics. ability transition pore opens to allow flow of NAD+ from mito- On the one hand, loss of sirtuin activity has been shown to chondria to the cytosol and nucleus in order to allow further contribute to cancer onset in numerous studies. The link be- PARP function. Work by Yang et al. [38] suggests that upregula- tween sirtuin loss and tumor emergence is evidenced by several tion of NAD+ biosynthesis enables mitochondrial NAD+ to be models where SIRT1, SIRT2, SIRT3, SIRT4, or SIRT6 knockout maintained at physiological levels even though cytosolic and mice are more prone to cancer incidence [13,19]. Overexpres- nuclear NAD+ pools are depleted upon genotoxic stress. Promo- sion of SIRT1 in the gut epithelium was found to suppress tumor- tion of NAD+ biosynthesis in the mitochondria was further indi- igenesis in a mouse model of colon cancer [73], although other cated to be dependent on SIRT3 and SIRT4 [38]. It will be useful groups have drawn the opposite conclusion [74,75]. Further for future studies to validate these NAD+ measurements in live studies are needed to clarify this discrepancy. In humans, cells in order to avoid the reliance on subcellular fractionation. SIRT3 protein and mRNA levels are strongly decreased in breast Other Regulation of Sirtuins and ovarian cancer [76]. SIRT4 expression is decreased in lung, It is important to point out that sirtuin activity is not solely depen- breast, bladder and gastric cancer and specific leukemia sub- dent on NAD+ levels. Transcriptional, post-translational and types [71,72]. SIRT6 levels are reduced in colon carcinoma and allosteric regulation are all important physiological modulators pancreatic cancer [77]. The metabolic state maintained by sir- of sirtuin activity [57]. A major negative regulator of SIRT1 with tuins can be particularly incompatible with the onset of cancer, relevance in cancer is deleted in breast cancer-1 (DBC1), a as discussed further below. nuclear protein that functions as a tumor suppressor and is ho- On the other hand, however, in certain established tumors it is mozygously deleted in some breast cancers [58]. DBC1 inhibits possible that sirtuins have pro-tumorigenic roles by promoting SIRT1 by directly binding to its catalytic domain [58,59]. This survival under the stress conditions that dominate the cancer repressive interaction is induced upon DNA damage down- cell state. For example, high SIRT1 expression is observed in stream of ATM, a key mediator of the DNA damage response drug-resistant cancers [78]. In numerous studies, SIRT1 levels that acts as a kinase targeting multiple proteins including are elevated in human cancer relative to normal tissue. In fact, DBC1 [60,61]. Phosphorylation of DBC1 creates an additional maintaining SIRT1 expression appears so vital for cancer cells SIRT1-binding site. Strong repression of SIRT1 in this manner that there are no reported deletions of SIRT1 in cancer and stimulates apoptosis, a proper cellular response to excessive only extremely rare instances of SIRT1 mutation [13]. Thus, while genotoxic damage in many contexts. An AMP-activated protein SIRT1 may counter the onset of cancer, an established tumor kinase (AMPK)-dependent pathway disrupts the DBC1–SIRT1 can greatly benefit from ramping up SIRT1 expression to induce interaction via phosphorylation of SIRT1 [62,63]. AMPK is a pro-survival pathways [79]. Expression of another nuclear sirtuin, cellular sensor of a low-energy state that acts as a bioenergetic SIRT7, promotes survival of cancer cells and maintenance of rheostat by phosphorylating many metabolic proteins to restore a transformed state [80]. In breast, thyroid and liver cancer, energetic homeostasis [47]. Indeed, AMPK has been linked to SIRT7 is upregulated [13]. The mitochondrial sirtuin SIRT3 pro- activation of SIRT1 under low-energy conditions in multiple motes oral squamous cell carcinoma by preventing apoptosis

of cancer cells [81]. This report is in line with a human genetics factor in complex with the coactivator p300/CBP [95]. This com- study showing that an extra germline copy of SIRT3 limits plex binds hypoxia-responsive elements in promoters of target apoptosis in glioma cells and predisposes patients to brain genes to activate a transcriptional program that boosts angio- tumors [82]. Thus, while sirtuins in many cases can suppress genesis, erythropoiesis and glycolytic metabolism [96]. cancer formation, sirtuins can also enhance the growth of Much like sirtuins, PHDs are perfectly poised to elicit meta- some tumors, depending on the cancer type, stage and accom- bolic alterations in response to changing nutrient availability or panying mutations. A more comprehensive understanding of stress. PHDs are a family of a-ketoglutarate-dependent dioxyge- sirtuin functions and relevant targets in cancer may shed light nases that hydroxylate proline residues of target proteins [97,98]. on the pro- or anti-tumorigenic roles of sirtuins in particular tumor There are three main mammalian PHDs (also called egg laying types. defective nine, or Egln, proteins in reference to their originally described function in egg laying in Caenorhabditis elegans) Altered Glucose Metabolism in Cancer and Regulation [99]. A little-studied fourth PHD family member is located in the by Sirtuins endoplasmic reticulum [100]. During catalysis, PHDs transfer In many cancers, metabolic reprogramming is characterized one atom of molecular oxygen to a proline residue of a substrate by increased glucose uptake [83]. Glucose is predominantly protein, resulting in prolyl-hydroxylation. The other oxygen atom used for biosynthetic purposes; intermediates of glycolysis are is transferred to a-ketoglutarate which is subsequently decar- directed toward pathways that build macromolecules, including boxylated to form carbon dioxide and succinate [97]. PHD cata- nucleotides, lipids and proteins. In addition to biosynthesis, lytic activity is sensitive to several key molecules that can be glucose contributes to ATP production, generation of signaling viewed as indicators of the cellular metabolic state: oxygen, molecules and antioxidants, and production of lactate, which ROS and specific TCA cycle intermediates [91]. In response to can acidify the tumor microenvironment to promote migration, changing levels of these inputs, PHDs have been shown to insti- genetic instability and cancer cell stemness [84–87]. Upregula- gate HIF-driven metabolic changes that restore homeostasis tion of glycolysis and lactic acid production even under normoxia and redox balance. when mitochondria are functional is termed the ‘Warburg effect’. Because the PHD catalytic mechanism requires molecular Elevated glycolysis can additionally fuel ATP production, even oxygen, a drop in intracellular oxygen levels can decrease PHD under hypoxic conditions in cancer cells. Rapidly growing or activity and consequently stabilize HIF [88]. Two PHD family metastatic tumors often have low oxygen due to inadequate members, PHD1 and PHD2, are quite sensitive to subtle blood supply. In the absence of sufficient oxygen, mitochondrial changes in cellular oxygen levels due to their weak affinity for

ATP production is limited [88]. To circumvent an energetic oxygen, with the Km for oxygen being only slightly higher than deficit, glycolysis can be upregulated to generate ATP via sub- the normal oxygen concentration in the cell. This suggests strate-level phosphorylation. The amount of ATP produced by PHD1 and PHD2 normally operate at sub-optimal conditions glycolysis is only a fraction of that generated by the electron and any drop in oxygen can potentially make PHDs much less transport chain; however, greatly induced glycolysis could sup- active [96]. Oxygen sensing by PHD1 and PHD2 situates these port bioenergetic homeostasis when oxygen is limiting [89]. enzymes as integral components in the HIF-driven transcrip- HIF, PHDs and Metabolic Stress Sensing tional response to low intracellular oxygen. PHD activity is One mechanism by which sirtuins have been shown to control frequently repressed in tumors that have become hypoxic due glycolysis is via regulation of the hypoxia-inducible factor (HIF) to excessive oxygen consumption or insufficient blood supply signaling pathway. HIF is a master transcriptional activator of [101]. glycolysis with strong links to cancer and functions as an a/b het- The use of a-ketoglutarate as a co-substrate makes PHDs erodimer [90]. Of the three HIFa isoforms, HIF1a and 2a are the sensitive to TCA cycle imbalances that are observed in some most well-studied [91]. Increased levels of HIF1a and HIF2a are cancers. Thus, PHD activity and HIF stability can be regulated observed in many cancers and correlate with worse prognosis even under normoxic conditions. At high concentrations, succi- [92]. While HIF1a and HIF2a have overlapping target genes, a nate and fumarate competitively inhibit the PHD-binding site that number of genes are exclusively modulated by just one HIF iso- is normally occupied by the structurally similar molecule a-keto- form. In healthy cells, HIF is activated under hypoxia to promote glutarate [102,103]. In some tumors, deficiencies in the TCA glycolytic metabolism along with other pathways that mediate cycle enzymes succinate dehydrogenase and fumarate hydra- cell survival under low oxygen. However, under some conditions tase lead to a build-up of succinate and fumarate, respectively of cellular oxidative stress and in many cancers, aberrantly acti- [104]. The overabundance of these metabolites inhibits PHD vated HIF facilitates metabolic reprogramming and upregulation function and is linked to HIF-driven metabolic reprogramming of glycolysis even when oxygen levels are sufficient [93]. Physi- in cancer. ologically under normoxia, HIF transcriptional activity is limited PHD enzymatic activity is intrinsically sensitive to redox status to a low, basal level. Cytosolic HIFa is hydroxylated by oxy- due to the requirement for reduced iron in the catalytic site. The gen-dependent prolyl hydroxylase domain (PHD) enzymes and PHD catalytic domain contains a conserved triad of two histi- subsequently ubiquitinated by the von Hippel-Lindau (VHL) E3 dines and one aspartate that coordinate iron [105]. To enable ubiqutin ligase, targeting HIFa for degradation [94]. oxygen binding at this catalytic site, iron must be maintained Under hypoxia, PHD activity is inhibited by low oxygen avail- in the reduced (Fe2+) state, a function achieved, in part, by ability and HIFa is stabilized. HIFa translocates to the nucleus the cellular antioxidants ascorbate (vitamin C) or glutathione and forms a heterodimer with HIFb (also called aryl hydrocarbon [106,107]. Under conditions of high intracellular ROS, these anti- nuclear receptor, ARNT) resulting in a functional transcription oxidant molecules may be depleted leading to oxidation of the

ABHigh SIRT activity Loss of SIRT activity

SIRT2 SIRT3 ROS ROS Ac

α OH HIF1 PHD ROS PHD Ac p-VHL Ub Ac Ub α Ub HIF1α hydroxylated HIF1 HIF1α stabilized and degraded and translocated Cytoplasm Cytoplasm Nucleus Nucleus Ac SIRT1 p300 HIF1β

Ac HIF1-mediated HIF1-mediated SIRT6 HIF1α Ac p300 glycolytic program Ac glycolytic program repressed activated SIRT6 Ac HIF1α HIF1β

H3 HRE H3 HRE Current Biology

Figure 2. Sirtuin-mediated repression of the basal HIF response and glycolytic metabolism during normoxia. (A) Sirtuins obstruct HIF-mediated reprogramming of cell metabolism under normoxia. HIF activity is restricted under normoxia due to proteasomal degradation of HIF1a, which is triggered by PHD family members and the p-VHL ubiquitin ligase. Degradation prevents movement of HIF1a to the nucleus. In mitochondria, SIRT3 limits ROS levels, thus helping maintain PHD function so that HIF1a can be degraded. In the cytosol, SIRT2 represses basal HIF1a by direct deacetylation. Any HIF1a molecules that do enter the nucleus are deacetylated and repressed by SIRT1. SIRT6 also inhibits HIF1a in the nucleus via direct binding to HIF1a at hypoxia-responsive elements (HRE) on gene promoters, preventing formation of a functional transcription factor complex. SIRT6 further represses HIF1- regulated genes by deacetylating histone H3K9 at glycolytic gene promoters. (B) Loss of sirtuin activity, for example during the transition to cancer or in genetic mouse knockout models, leads to activation of HIF1-mediated glycolytic metabolism even under normoxia. In the absence of SIRT3, ROS levels increase and inhibit PHD-catalyzed hydroxylation of HIF1a. Also, in the absence of specific sirtuins, HIF1a is hyperacetylated and more readily moves to the nucleus and forms a functional transcription factor in complex with HIF1b and p300. HIF, hypoxia-inducible factor; PHD, prolyl hydroxylase domain; VHL, von Hippel-Lindau E3 ubiquitin ligase; OH, prolyl hydroxylation; Ac, lysine acetylation; Ub, ubiquitination; ROS, reactive oxygen species. catalytic iron and inhibition of PHD activity. In this way, the HIF target gene [112]. It is possible that upregulation of SIRT1 expres- response can also be turned on downstream of increased sion under hypoxia serves to build a pool of SIRT1 that can rapidly ROS, a common scenario in cancer [108,109]. dampen the HIF signal as soon as adequate oxygen is achieved. Modulation of HIF by Sirtuins A similar rationale has been suggested to explain why some PHD Several studies have shown that the stress- and nutrient-sensing family members are HIF target genes [105]. pathways that coordinate HIF activity also intersect with sirtuins Also in the nucleus, SIRT6 represses HIF transcriptional activ- at numerous nodes. For example, multiple sirtuins oppose HIF- ity to limit glycolysis in cancer (Figure 2A) [113]. First, SIRT6 driven metabolic rewiring (Figure 2). Elaborate control mecha- deacetylates histone H3K9 on the promoter of HIF target nisms enforced by SIRT1, SIRT2, SIRT3, SIRT6 and SIRT7 genes, aiding in gene silencing. SIRT6 also directly interacts counter HIF activity to keep glucose metabolism in check [13]. with and inhibits HIF1a on hypoxia-responsive elements of In the nucleus, there is complex and considerable interplay be- glycolytic genes. Loss of SIRT6 in mouse embryonic fibroblasts tween SIRT1 and HIF, in line with the role of SIRT1 as a promoter (MEFs) boosts expression of key glycolytic enzymes, including of oxidative metabolism and mitochondrial function (Figure 2A). pyruvate dehydrogenase kinase 1 (PDK1). Elevated PDK1 was Under normoxia, SIRT1 inhibits the basal HIF response by pro- additionally shown to be vital for the transformation phenotype moting stability of the VHL transcript to drive degradation of of SIRT6-deficient MEFs [77]. The authors of this study further HIFa [50]. SIRT1 further inactivates HIF1a in the nucleus by show that glycolysis is increased upon SIRT6 conditional removing an acetyl group that is key to the interaction between knockout in an APCmin/+ mouse model of colon cancer and is HIF1a and p300 [110]. Under hypoxia, the gradual drop in linked to increased tumor incidence [77]. NAD+ decreases SIRT1 function and contributes to HIF activation Nuclear SIRT7 in cell culture has been shown to inhibit the HIF [110], which the authors of this study propose synergizes with response by decreasing HIF1a and HIF2a protein levels [114]. intratumoral PHD inhibition to maximize HIF-driven glycolysis. The mechanism remains to be elucidated, but appears to be Dioum et al. [111] show that, despite declining NAD+, residually independent of SIRT7 catalytic activity as well as PHD- and pro- active SIRT1 can deacetylate and activate HIF2a under hypoxia, teasome-mediated degradation pathways. Of note, SIRT2 de- helping drive a switch toward isoform-specific target genes (dis- acetylates and represses HIF1a in the cytosol [115]. However, cussed further below). Interestingly, SIRT1 is a HIF1 and HIF2 this interaction has been less extensively studied.

In the mitochondria, SIRT3 promotes mitochondrial meta- note, SIRT6 represses gluconeogenic gene expression in con- bolism and limits HIF-driven glycolysis by two mechanisms: de- cert with p53, the histone acetyltransferase GCN5, the transcrip- acetylation and coordination of ROS signaling (Figure 2A). SIRT3 tion factor FOXO1 and the transcriptional coactivator peroxi- boosts mitochondrial metabolism by deacetylating and acti- some proliferator-activated receptor g coactivator 1a (PGC-1a) vating enzymes involved in the TCA cycle and fatty acid oxida- [124,125]. Zhang et al. [125] observed that the tumor suppressor tion, including succinate dehydrogenase (SDH), long chain p53 boosts SIRT6 expression in order to limit gluconeogenesis, a acyl-CoA dehydrogenase (LCAD), glutamate dehydrogenase finding observed in both liver and colon cancer cell lines. The au- (GDH) and isocitrate dehydrogenase 2 (IDH2) [15]. In tandem, thors propose that suppressing gluconeogenesis via SIRT6 may SIRT3 limits glycolytic metabolism by coordinating a multi- be an additional anti-neoplastic function of p53. Future studies pronged strategy to decrease ROS (discussed further below) are needed to explore whether regulation of gluconeogenesis and reduce HIF function. By repressing ROS, SIRT3 promotes by other sirtuins is meaningful for cancer metabolism. PHD activity and HIF degradation. SIRT3 loss dramatically boosts ROS, which is proposed to deactivate PHD family mem- Alternative Fuel Sources in Cancer and Their bers and consequently stabilize HIF1a (Figure 2B) [76,116]. Coordination by Sirtuins Indeed, in SIRT3-deficient MEFs, increased ROS promotes a Beyond rewired glucose utilization, many cancers additionally or HIF-mediated transition to the Warburg effect [76]. alternatively display addiction to fatty acids or amino acids, such Thus, loss of sirtuin function has been shown in many cases as glutamine. An intensely studied use of these alternative fuels to shift the cell toward glycolytic metabolism in a process that is anaplerosis, the process of refilling the TCA cycle. Anaplerotic is amenable to transformation (Figure 2B). In cancer cell lines pathways provide alternative entry sites to generate TCA cycle and sirtuin knockout mouse models, low expression of SIRT1, intermediates, which are often used for anabolic and bioener- SIRT3 and SIRT6 correlates with increased levels of HIF1 target getic purposes in cancer [126]. In many healthy tissues, PDH is genes, including the glucose transporter GLUT1 as well as the major enzyme that channels glucose-derived pyruvate into enzymes involved in glycolysis and lactate production, including the TCA cycle [127]. PDH converts pyruvate to acetyl-CoA, hexokinase (HK), PDK1, phosphoglycerate kinase 1 (PGK1) and and then acetyl-CoA condenses with oxaloacetate to form cit- lactate dehydrogenase (LDHA) [50,76,113]. rate. However, PDH activity is often limited in cancer; addition- Additional Glycolytic Regulation by Sirtuins ally, roadblocks at other steps in the TCA cycle or shunting of Aside from HIF regulation, sirtuins can modulate glycolysis in metabolites toward biosynthetic pathways can limit production other ways. SIRT1 has been shown to regulate glycolytic gene of oxaloacetate, which is needed to fuel subsequent rounds of expression downstream of the transcriptional activator MYC, the TCA cycle [126]. In cancer, the TCA cycle can be refueled although there are conflicting reports concerning the direction by alternative pathways including glutaminolysis, reverse TCA of this modulation [117–120]. Loss of SIRT3 in cancer cells cycling and fatty acid oxidation. additionally boosts glycolytic metabolism via pyruvate dehydro- Regulation of Glutamine Anaplerosis by Sirtuins genase (PDH), the major enzyme that channels glucose-derived A major driver of glutamine metabolism in cancer is the MYC pyruvate into the TCA cycle. SIRT3 deacetylates and activates family of transcriptional activators, including MYC (c-MYC), the PDH catalytic subunit E1a, which directs pyruvate to the L-MYC and N-MYC. This family is known to ubiquitously amplify TCA cycle [121,122]. In the absence of SIRT3, pyruvate entry expression of most genes undergoing transcription, and MYC into the TCA cycle is blocked. This is thought to enable glycolytic target sequences have been identified in 30% of all genes intermediates to be redirected toward biosynthetic pathways [128]. In tumors, aberrant upregulation of specific gene sets by and lactate production. Overexpression of a constitutively de- MYC is linked to growth advantages in cancer cells. Specifically, acetylated mimetic of PDH in MCF7 breast cancer cells resulted MYC boosts glutamine metabolism by increasing the expression in a less transformed phenotype in soft agar assays, while of glutamine transporters as well as enzymes involved in direct- a constitutively acetylated mimetic — which mimics loss of ing glutamine toward the TCA cycle, including glutaminase (GLS) SIRT3 — had a more highly transformed phenotype [122]. [129]. Many sirtuins have been linked to the regulation of Sirtuin-mediated Regulation of Gluconeogenesis in MYC, but SIRT6 has most strongly been shown to coordinate Cancer glutamine metabolism via MYC [77]. By deacetylating H3K56 Gluconeogenesis is a lesser-studied arm of glucose metabolism residues at MYC target gene promoters, SIRT6 suppresses in cancer. A number of sirtuins coordinate gluconeogenesis, and MYC transcription activity specifically toward genes involved in the links to cancer are just beginning to be explored [15]. Phys- glutamine as well as glucose metabolism. Accordingly, SIRT6- iologically, glucose production occurs in the liver and to a small deficient MEFs show increased glutamine uptake, an advantage extent the kidneys in order to maintain blood glucose between that may promote the increased growth of SIRT6-deficient MEF meals. However, recent studies demonstrate that cancer cells xenograft models compared with wild type [77]. SIRT7 also sup- may operate at least portions of the gluconeogenic pathway. presses MYC, while SIRT1 boosts MYC activity, and future Leithner et al. [123] found that lung cancer cell lines and tissue studies may reveal the relevance of these interactions for gluta- samples overexpress PCK2, the mitochondrial isoform of phos- mine metabolism in cancer [128]. phoenolpyruvate carboxykinase, a gluconeogenic gene. Meta- SIRT4 coordinates glutamine anaplerosis in an alternative bolic tracing demonstrated that lung cancer cells convert lactate manner. SIRT4 shuts down an access point that directs gluta- to pyruvate, then to oxaloacetate via pyruvate carboxylase, and mine into the TCA cycle (Figure 3A). In cancer cells, the then to phosphoenolpyruvate via PCK2. This pathway generates ability of SIRT4 to restrict the supply of this alternative fuel limits glycolytic intermediates in cancer cells deprived of glucose. Of tumorigenesis [71]. SIRT4 impedes glutamine anaplerosis by

A B SIRT4 HIF1α mTORC1 CREB2 CREB2 expression

mTORC, SIRT4 and SIRT1 SLC1A5 DNA damage linked? GLS1 Glucose Glucose Low pH p300 IDH1 HIF2α HIF2β Pyruvate DNA damage Pyruvate Lactate x SIRT4

Isocitrate Glutamine SLC1A5 Glutamine TCA TCA IDH1 cycle GDH cycle GLS1 α-ketoglutarate Glutamate Glutamine α-ketoglutarate Glutamate

Current Biology

Figure 3. Sirtuin-mediated regulation of TCA cycle anaplerosis. (A) SIRT4 leads to repression of glutamine anaplerosis. SIRT4 expression is strongly induced in response to various cellular stresses, including dysfunctional mTORC1 and DNA damage. The connections between mTORC1, DNA damage and SIRT4 are unclear. In cancer cells, induction of SIRT4 limits entry of glutamine into the TCA cycle, in part by ADP-ribosylating and inhibiting GDH. (B) SIRT1 promotes glutamine anaplerosis and reductive carboxylation during chronic acidosis. Chronic acidosis can occur in cancer cells due to a metabolic switch to increased glycolysis and lactate production. Shunting pyruvate toward lactate can deplete the TCA cycle, driving the need for anaplerosis by other fuels. SIRT1 is activated by chronic acidosis. Under this condition, SIRT1 deacetylates HIF1a. Deacetylation inhibits HIF1a but activates HIF2a, leading to expression of a speciﬁc subset of target genes (in red) that promote glutamine anaplerosis and reductive carboxylation. SLC1A5 is induced to increase glutamine import into mitochondria. GLS1 is induced to convert glutamine to glutamate. IDH1 is induced to redirect a-ketoglutarate towards isocitrate via a reductive carboxylation. GDH, glutamate dehydrogenase; GLS1, glutaminase isoform 1; IDH1, isocitrate dehydrogenase 1.

ADP-ribosylating and inhibiting GDH [28]. To direct glutamine to- to redox stress, impaired respiration or hypoxia [131–133]. For- ward the TCA cycle, glutamine can be converted to glutamate by ward TCA cycling requires pyruvate-derived acetyl-CoA to the glutaminase family of enzymes. Glutamate is then converted condense with oxaloacetate and form citrate. Under harsh to the TCA cycle intermediate a-ketoglutarate by mitochondrial conditions, such as cells undergoing proliferation in hypoxia, py- GDH [130] or alternatively by transaminases. In cancer cells, ruvate is directed almost entirely toward lactate [134]: acetyl- DNA damage dramatically induces SIRT4 expression by an CoA may be sufficiently depleted such that forward TCA cycling unknown mechanism, and glutamine anaplerosis is inhibited is limited. In this case, an alternative pathway is needed for cit- [71]. Consequently, TCA cycle intermediates are depleted rate production. Citrate is especially essential for cancer cells and, via mechanisms yet to be elucidated, the cell cycle stalls. because it is a key building block for fatty acid synthesis [134]. This SIRT4-mediated metabolic pause allows time for DNA In cancer cells, glutamine-derived a-ketoglutarate can undergo repair before the cell proceeds through the cell cycle. In the reductive carboxylation catalyzed by IDH1 or IDH2 to generate absence of SIRT4, glutamine anaplerosis remains activated citrate [135,136]. even during DNA damage. As a consequence, DNA damage per- A recent study shows that SIRT1 promotes reductive car- sists and cellular proliferation and transformative properties are boxylation (Figure 3B) [137]. In diverse cancer cell lines, pro- increased, possibly due to newly occurring DNA mutations. longed acidosis (pH 6.5), which mimics extensive lactate pro- In related studies, Csibi et al. [72] found that mTOR complex 1 duction, upregulates genes important for reverse TCA cycle (mTORC1), a serine/threonine kinase that drives cellular nutrient flux in a SIRT1-dependent manner. Mechanistically, under low uptake and proliferation, inhibits SIRT4-mediated repression of pH SIRT1 deacetylates HIF1a and 2a. Deacetylation inhibits anaplerosis (Figure 3A). mTORC1 represses SIRT4 expression HIF1a but activates HIF2a. By activating HIF2a, SIRT1 triggers by inhibiting CREB2, a transcription factor that induces SIRT4 the expression of key target genes, including the glutamine [72]. In MEFs with hyperactivated mTORC1, SIRT4 expression transporter SLC1A5, the mitochondrial glutaminase isoform 1 was decreased, thus activating glutamine metabolism and ana- (GLS1) and IDH1. Thus, the SIRT1/HIF2a axis promotes a meta- plerosis. High mTORC1 signaling commonly occurs in cancer bolic shift to reductive glutamine metabolism in order to maintain and is proposed to benefit cancer cell growth and survival by levels of TCA cycle intermediates under the harsh conditions increasing glutaminolysis via SIRT4 repression. In future studies, experienced by cancer cells. it will be interesting to examine whether mTOR signaling con- Sirtuins and Subcellular Metabolic Crosstalk verges on DNA damage responses via SIRT4 (Figure 3). A fertile area for future sirtuin research is the possible link between Sirtuins and Reverse TCA Cycling cancer and transcriptional control of mitochondrial biogenesis Although the TCA cycle had long been thought to operate in one and oxidative metabolism. PGC-1a is a master transcriptional co- direction, it is now known that at least a portion of the TCA cycle activator of mitochondrial metabolism and oxidative phosphory- operates in reverse in some cancer cells, particularly in response lation [138]. It is known that increased mitochondrial biogenesis

Figure 4. Roles of sirtuins in ROS defense. SIRT1 Sirtuins coordinate a multi-faceted regimen to NADPH 3-Phospho- 2-Phospho- FOXO FOXO synthesis glycerate glycerate Ac Ac limit ROS. In the nucleus, SIRT1 deacetylates a x number of transcriptional regulators to boost PGAM FOXO FOXO HSF1 gene expression programs that increase antioxi- Ac SIRT2 dant defenses, limit ROS production and drive apoptosis in the event of uncontrollable ROS. In PGAM p53 NRF2 the cytosol, SIRT2 deacetylates FOXO to drive its nuclear import and activity. SIRT2 also deacety- 3-Phospho- 2-Phospho- Glucose Pyruvate lates and activates the glycolytic enzyme PGAM. glycerate glycerate FOXO3a PGC1α Activated PGAM boosts conversion of 3-phosphoglycerate to 2-phoshoglycerate. PGAM pro- ETC I II III IV V motes production of the antioxidant molecule NADPH, whereas a buildup of 3-phosphoglyc- SDHA erate would otherwise inhibit NADPH synthesis. In FAD FADH 2 mitochondria, SIRT3 boosts oxidative capacity SIRT3 Succinate FOXO3a Fumarate and limits ROS production via the deacetylation α-kg TCA and activation of subunits in all five electron SOD2 IDH2 cycle SIRT3 ROS production transport chain (ETC) complexes, including SDH- Isocitrate NADPH synthesis A which dually serves as a TCA cycle enzyme that •- H O O + 2 2 2 NADPH NADP ROS clearance generates FADH2. SIRT3 interacts with FOXO3a to FOXO3a elevate expression of ROS defense pathways in Oxidized Reduced Oxidative capacity glutathione both the nucleus and mitochondria. SIRT3 also glutathione Apoptosis with excessive ROS drives antioxidant strategies to clear ROS. De- H O H O 2 2 2 acetylation and activation of IDH2 generates the Current Biology antioxidant molecule NADPH. Finally, SIRT3 deacetylates and activates SOD2, an enzyme that clears superoxide. PGAM, phosphoglycerate mutase; SDH-A, succinate dehydrogenase A; IDH2, isocitrate dehydrogenase 2; SOD2, superoxide dismutase; a-kg, a-ketoglutarate; FAD, flavin adenine dinucleotide; HSF1, heat shock factor protein 1; NRF2, nuclear erythroid factor 2-related factor 2; PGC-1a, peroxisome proliferator-activated receptor g coactivator 1a. and respiration driven by PGC-1a promote tumor cell invasion major driver of cell growth that is hyperactivated in many cancers and metastasis [139]. Further, SIRT1 and SIRT3 can promote [109]. ROS activates PI3K by inhibiting the major negative regu- mitochondrial metabolism by turning on PGC-1a-mediated tran- lator of this pathway, PTEN [151]. Mechanistically, cytosolic ROS scriptional programs [140]; however, the direct connections be- oxidizes the catalytic cysteine residue of PTEN and promotes tween sirtuins, mitochondrial biogenesis and cancer metabolism formation of a disulfide bond to block PTEN function and enable are unclear. Under fasting conditions, SIRT1 deacetylates and unrestrained PI3K activity [152]. ROS additionally boosts activity activates PGC-1a [141]. Additionally, SIRT3 enhances expres- of AKT, a mediator of the PI3K pathway, by blocking the activity sion of PGC-1a and is essential for turning on PGC-1a-induced of a phosphatase that otherwise inhibits AKT [153]. mitochondrial biogenesis [142,143]. Thereby, these sirtuins While in some contexts ROS boosts cellular transformation promote efficient energy production during nutrient deprivation. and cancer cell growth, high levels of ROS have been shown in By favoring mitochondrial metabolism over glycolytic meta- other contexts to act as a pro-apoptotic signal instructing cancer bolism, it is reasonable to hypothesize on the one hand that sir- cells to die [154,155]. Accelerated metabolism in cancer often tuins hinder the metabolic changes needed for cellular transfor- generates high ROS levels. Because ROS can reach toxic con- mation. However, on the other hand functional mitochondria centrations, adaptive mechanisms must be put in place by can- are still vital for an established cancer cell population to grow cer cells to restore ROS homeostasis and allow survival of tumor and metastasize [144], and therefore sirtuin-induced upregula- cells [156,157]. Therefore, many cancer cells upregulate antiox- tion of mitochondrial metabolism may be one reason why sirtuin idant pathways that endow tumors with additional stress protec- expression can benefit many existing cancers. tion. In this way, antioxidant programs may actually promote the progression of established tumors [158–161]. Sirtuin-driven Mitochondrial Programs for ROS Coordination of ROS by Nuclear and Cytosolic Sirtuins Homeostasis Several sirtuins have major roles in preventing excessive, The role of ROS in cancer is complex. High levels of ROS have damaging levels of ROS (Figure 4). In the nucleus, SIRT1 pro- been linked to cancer incidence in numerous studies [145– motes ROS defense via deacetylation of key transcriptional 147]. ROS production can be increased by inefficiencies or stall- regulators of stress resistance, including p53, FOXO proteins, ing in the electron transport chain. ROS have both adverse and PGC-1a, heat shock factor protein 1 (HSF1) and nuclear beneficial consequences on cancer cells, which may in part be erythroid factor 2-related factor 2 (NRF2) [19,162]. These targets determined by the stage of tumor progression or specific can- increase antioxidant defenses, limit ROS production and drive cer-associated mutations [109]. First, at excessive levels ROS apoptosis in the event of uncontrollable ROS. For example, in can damage cellular machinery, including proteins, lipids, DNA response to oxidative stress or nutrient deprivation, SIRT1 de- and RNA. By causing genetic damage, ROS may have muta- acetylates FOXO [163,164]. This promotes nuclear retention of genic and pro-tumorigenic capacities. ROS also serve as impor- FOXO where it can turn on oxidative stress resistance genes, tant signaling molecules that can drive growth and cell division in such as those encoding mitochondrial superoxide dismutase 2 cancer [148–150]. For example, ROS have been shown to stim- (SOD2), catalase and the pro-apoptotic factor Bim. SIRT2 can ulate the phosphoinositide 3-kinase (PI3K) signaling pathway, a also deacetylate cytosolic FOXO, causing it to move to and

remain in the nucleus. By upregulating Bim, SIRT2 was shown to thione. Finally, SIRT3 has been shown to boost transcription of promote cell death when cells are under severe stress [165] —an antioxidant enzymes by interacting with mitochondrial FOXO3a appropriate response that can benefit survival of the surrounding and boosting its affinity for promoters of antioxidant genes in tissue. When PGC-1a is deacetylated by SIRT1, this directs the nucleus and promoters of genes encoding oxidative phos- PGC-1a toward a specific gene set that promotes increased phorylation subunits in mitochondria [171,180,181]. Through mitochondrial biogenesis and oxidative capacity to limit ROS these multiple mechanisms, SIRT3 boosts cellular oxidative ca- production [166]. pacity, decreases ETC stalling and promotes antioxidant de- In the cytosol, SIRT2 also combats ROS by promoting the fenses. generation of the antioxidant molecule NADPH (Figure 4). In Recent studies suggest SIRT3 is not the only mitochondrial response to H2O2 treatment in cell culture, SIRT2 deacetylates sirtuin that coordinates ROS; SIRT5 also limits ROS by at least and activates phosphoglycerate mutase (PGAM) [167]. PGAM two mechanisms. SIRT5 desuccinylates and activates SOD1, is a glycolytic enzyme that converts 3-phopshoglycerate to the largely cytosolic isoform of SOD that is also present at low 2-phosphoglycerate. In the absence of SIRT2, PGAM is less amounts in mitochondria [182]. SIRT5 additionally boosts tran- active and levels of 3-phosphoglycerate increase. Not only is scription of NRF2, consequently promoting gene expression glycolysis impeded, but also high amounts of 3-phosphoglyc- programs important for maintaining redox homeostasis [183]. erate inhibit 6-phosphogluconate dehydrogenase (6PGD), an Through regulation of ROS, mitochondrial sirtuins play a critical enzyme in the pentose phosphate pathway that produces yet complex role in cancer progression [184]. By decreasing NADPH. Thus, SIRT2 is required to activate PGAM and help ROS, SIRT3 has the capacity to limit tumorigenesis. In healthy maintain NADPH synthesis for use in clearing ROS. cells, SIRT3 decreases ROS, maintains PHD activity and re- Regulation of ROS by Mitochondrial Sirtuins presses HIF, as described above [76,116]. Consequently SIRT3 Mitochondrial SIRT3 is a major player in cellular antioxidant represses the transition to the Warburg effect, thus restricting a strategies. SIRT3 coordinates a multi-faceted post-translational metabolic pathway that is quite often vital to neoplastic transfor- program to reduce ROS (Figure 4). First, SIRT3 limits ROS produc- mation [185]. SIRT3 may further impede cancer onset by limiting tion by promoting efficient electron flow through the ETC. SIRT3 DNA damage caused by ROS [171]. Additionally, in pancreatic deacetylates and activates specific subunits in all five ETC com- cancer cell lines, SIRT3 suppression of ROS was shown to limit plexes. SIRT3 activates complex I [168] and SDH-A in complex II proliferation via coordination of iron metabolism [186]. By limiting [169,170], components of the ETC where electrons are initially ROS, SIRT3 represses redox-sensitive iron-responsive proteins, donated. SDH-A dually functions as a TCA cycle enzyme that thus downregulating an iron-related gene set that includes the oxidizes succinate to fumarate while converting flavin adenine transferrin receptor. In the absence of SIRT3, increased levels dinucleotide (FAD) to FADH2. Electrons from FADH2 are directly of transferrin receptor correlated with a growth advantage for fed into the SDH-B subunit of complex II and then on through pancreatic cancer cells, at least in part due to an abundance of the ETC. By activating complex I and II, SIRT3 enables NADH intracellular iron, an essential cofactor in DNA synthesis. and FADH2 to more readily contribute electrons to the ETC. While limiting ROS levels helps sirtuins act as tumor suppres- SIRT3 activates complex III [171,172] and IV [43,51] to further sors in certain cases, established tumors may benefit from promote efficient electron flow and generation of a proton maintaining sirtuin function to avoid ROS-induced apoptosis. gradient. Activation of complex V (ATP synthase) boosts ATP For example, high SIRT3 expression was observed in oral squa- production [172,173]. By activating all ETC components, SIRT3 mous cell carcinoma cell lines and human samples [81], and in increases mitochondrial oxidative capacity, prevents ETC stalling cardiomyocytes, overexpression of SIRT3 conferred resistance and limits ROS production. Of note, mitochondrial SIRT5 sup- to genotoxic and oxidative stress-inducing agents, including presses SDH activity, possibly via desuccinylation, but the rele- camptothecin and H2O2 [187]. Thus, it is tempting to speculate vance to cancer is not known [174]. In cancer cells grown in galac- that, in an already established tumor, high sirtuin expression tose — a condition that increases the dependency on oxidative may promote cancer cell viability by limiting ROS, a pro- phosphorylation — SIRT3 has been shown to boost the ETC in apoptotic signal that would otherwise instruct cancer cells to an additional way, via deacetylation of cyclophilin D, leading to die. Future studies are needed to reveal the range of cancer con- release of HK2 from mitochondria [175]. HK2 in mitochondria is texts in which ROS are either beneficial or harmful and in which linked to increased reliance on glycolytic metabolism, but released sirtuins have tumor-suppressive or tumor-promoting functions. HK2 stimulates oxidative phosphorylation. In addition to limiting ROS production, SIRT3 also promotes Conclusions ROS clearance. SIRT3 deacetylates SOD2, a key mitochondrial The role of sirtuins in cancer echoes that of other stress sensors enzyme in antioxidant defense which initiates ROS detoxification such as AMPK [188], whereby distinct cancer contexts ultimately by converting superoxide to H2O2 [176,177]. Mice heterozygous determine whether these enzymes confer pro- or anti-cancer for SOD2 have more DNA damage and 100% increased cancer properties. On the one hand, sirtuin activation promotes good incidence than wild-type controls [178]. To complete ROS clear- health by restricting metabolic pathways linked to neoplastic ance, H2O2 is reduced to water by the antioxidant glutathione. transformation, and sirtuin loss predisposes animal models to SIRT3 indirectly boosts levels of glutathione. By deacetylating cancer. On the other hand, sustained sirtuin activity in the stress- and activating the TCA cycle enzyme IDH2, SIRT3 promotes ful environment of a tumor may endow cancer cell survival conversion of isocitrate to a-ketoglutarate in a reaction that mechanisms or resistance to chemotherapeutics. Overall, our simultaneously produces NADPH [179]. NADPH is a reducing knowledge of the seven sirtuins in distinct cancer contexts agent with antioxidant functions that generates reduced gluta- is revealing metabolic susceptibilities that can potentially be

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