Oncogene (2007) 26, 5489–5504 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW Sirtuins: critical regulators at the crossroads between cancer and aging LR Saunders and E Verdin Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, USA Sirtuins (SIRTs 17), or class III histone deacetylases (HDACs), are protein deacetylases/ADP ribosyltransferases that target a wide range of cellular proteins in the nucleus, cytoplasm, and mitochondria for post-translational modification by acetylation (SIRT1, -2, -3 and -5) or ADP ribosylation (SIRT4 and -6). The orthologs of sirtuins in lower organisms play a critical role in regulating lifespan. As cancer is a disease of aging, we discuss the growing implications of the sirtuins in protecting against cancer development. Sirtuins regulate the cellular responses to stress and ensure that damaged DNA is not propagated and that mutations do not accumulate. SIRT1 also promotes replicative senescence under conditions of chronic stress. By participating in the stress response to genomic insults, sirtuins are thought to protect against cancer, but they are also emerging as direct participants in the growth of some cancers. Here, we review the growing implications of sirtuins both in cancer prevention and as specific and novel cancer therapeutic targets. Oncogene (2007) 26, 5489–5504; doi:10.1038/sj.onc.1210616 Keywords: sirtuins; cancer; lifespan; lysine acetylation Introduction Cancer is a disease of aging, as the incidence of most cancers increases with age following an accumulation of mutations. Mutations arise from errors during DNA replication, environmental insults, and are induced by intracellular reactive oxygen species that increase during cellular stress. In many organisms, longer lifespan correlates with lower levels of reactive oxygen species (Finkel and Holbrook, 2000). Additionally, age-related changes may alter the internal microenvironment to favor tumor development (Anisimov et al., 1988). As the global human population ages, the ability to prevent and treat cancer becomes essential to allow people to live longer, healthier lives. Members of the silent information regulator 2 (Sir2) family, which are conserved from bacteria to humans, regulate lifespan in various organisms (Longo and Correspondence: Dr E Verdin, Gladstone Institute of Virology and Immunology, University of California, 1650 Owens St, San Francisco, CA 94158, USA. E-mail: [email protected] Kennedy, 2006). Seven human sirtuins (SIRT1–7), also referred to as class III histone deacetylases (HDACs), share a catalytic domain of B275 amino acids. Because of their established role in controlling lifespan in lower organisms, there is growing interest in determining whether the same effect can be seen in mammals and in humans in particular (Figure 1). The most effective and reproducible way to extend lifespan is with a calorie-restricted diet (Roth et al., 2001; Hursting et al., 2003). Calorie restriction (CR), a 2040% lowering of caloric intake, reduces spontaneous tumor formation in several mouse models in which a tumor suppressor is inactivated or an oncogene is overexpressed (Jones et al., 1997; Berrigan et al., 2002; Mai et al., 2003). Aging is associated with increased rates of stress-induced apoptosis, and CR lowers sensitivity to stress-induced apoptosis (Higami et al., 1997; Ando et al., 2002). Levels of SIRT1 are increased in response to CR, and the enzymatic activity of SIRT1 is required for resistance to apoptosis induced by CR (Cohen et al., 2004b; Nemoto et al., 2004). CR also induces SIRT3 expression, but appears to decrease SIRT4 (Shi et al., 2005; Haigis et al., 2006). Mice lacking the insulin receptor in adipose tissue live 18% longer on a regular diet, suggesting that a reduction in fat mass rather than lower caloric intake mediates longer lifespan (Bluher et al., 2003). Changes in energy balance and body mass composition lower cancer incidence in CR mice (Huffman et al., 2007). Because the prevalence of obesity in humans is expected to increase more than 40% by 2030, cancer incidence is also expected to rise (Vainio et al., 2002). As sirtuins promote longevity, one would expect that sirtuin activation or increased expression, should protect against cancer. Sirtuins could mediate this protective effect by limiting replicative lifespan, by protecting against DNA damage and oxidative stress or by guarding against accumulation of mutations and against genomic instability. In contrast, the loss of sirtuin expression, activity or regulation can bypass replicative senescence, allow cell division to proceed without the proper repair of DNA, and promote accumulation of mutations and genomic instability leading to tumor development. There is also growing evidence that tumor cells can become ‘addicted’ to sirtuin overexpression, which allows rapid proliferation and loss of checkpoints to promote continued propagation in the presence of accumulating mutations. Sirtuins and cancer LR Saunders and E Verdin 5490 Figure 1 Cellular localization, enzymatic activities and targets of the mammalian sirtuins. SIRT1 responds to changes in nutrient availability and cellular stress to promote cell survival by deacetylating histone and non-histone targets in the nucleus. SIRT2 is mainly localized in the cytoplasm and deacetylates a-tubulin, but during mitosis, it shuttles to the nucleus and deacetylates histone H4-K16 to promote chromatin condensation. SIRT2 may function as part of a mitotic checkpoint to ensure that cells do not pass through mitosis if a stress signal or DNA damage is present. SIRT3, 4 and 5 localize to the mitochondria. SIRT3 deacetylates and activates acetyl-CoA synthetase 2 (AceCS2) to enhance acetyl-coA production. SIRT4 ADP-ribosylates and represses glutamate dehydrogenase (GDH) to suppress insulin signaling. No targets or enzymatic activity have been identified for SIRT5. SIRT6 promotes DNA repair and guards against genomic instability in the nucleus. Its targets have not been identified. SIRT7 localizes to the nucleolus where it interacts with RNA pol-I to promote transcription of rRNA genes. SIRT1, sirtuins. These emerging data point to sirtuins as critical regulators at the crossroads between cancer and aging. Unraveling the cellular roles of the sirtuins in the control of cell growth and as guardians against genomic damage is likely to lead to the identification of novel cancer prevention and treatment strategies. Sirtuin enzymatic activities: deacetylation and mono-ADP-ribosylation The conserved catalytic domain shared by the sirtuins functions as a mono-ADP-ribosyltransferase and as a b-nicotinamide adenine dinucleotide (NAD þ )-dependent lysine deacetylase (Figure 2) (Frye, 1999; Imai et al., 2000; Landry et al., 2000; Smith et al., 2000). The deacetylase domain in the sirtuins is different from that of class I and II HDACs, which are Zn þ dependent enzymes (North and Verdin, 2004; Longo and Kennedy, 2006). Sirtuins use one NAD þ molecule and generate acetyl-ADP-ribose and nicotinamide during the deacetylation reaction (Landry et al., 2000). While SIRT1, 2, 3 and 5 have significant deacetylase activity towards a histone H4 peptide, SIRT4, 6 and 7 have low to undetectable deacetylase activity in vitro on tested substrates (Langley et al., 2002; North et al., 2003). SIRT1 deacetylates histones and Oncogene other nuclear targets, including p53, Ku70 and FOXO (Forkhead box, class O) in vivo (as discussed below). Of the sirtuins, only SIRT2 deacetylates tubulin, also a target of the class II deacetylase HDAC6 (Hubbert et al., 2002; Matsuyama et al., 2002; North et al., 2003). Mono-ADP-ribosylation, during which ADP-ribose from NAD þ is transferred to an acetylated target protein, is conserved from bacteria to humans and is carried out by several families of proteins (Hassa et al., 2006). Mono-ADP-ribosylated cellular proteins include histones, high mobility group (HMG) family members, actin, a- and b-tubulin and glutamate dehydrogenase (GDH; Hassa et al., 2006). SIRT1 preferably transfers mono-ADP-ribose to histone H1 (Frye, 1999). SIRT6 is more efficient with bovine serum albumin (BSA), and SIRT2 can also ADP-ribosylate BSA in vitro (Frye, 1999; Liszt et al., 2005). SIRT6 robustly mono-ADPribosylates itself through an intramolecular reaction (Liszt et al., 2005). In vivo, SIRT4 ADP-ribosylates GDH, which inhibits its activity (Haigis et al., 2006). A conserved histidine residue in the catalytic core of the sirtuins is important for their deacetylase and monoADP-ribosyltransferase activities (Frye, 1999). Deacetylation and ADP-ribosylation may be linked and dependent on each other. ADP-ribose generated in the deacetylation reaction is added to a substrate following Sirtuins and cancer LR Saunders and E Verdin 5491 Figure 2 Enzymatic activities of sirtuins. The conserved catalytic domain shared by the sirtuins carries out deacetylation and ADPribosylation. In both reactions, sirtuins use b-NAD þ as a cofactor. During the deacetylation reaction (left panel), b-NAD þ is hydrolysed to generate nicotinamide, 20 -O-acetyl-ADP-ribose and the deacetylated target. During the ADP-ribosylation reaction (right panel), ADP-ribose is attached to an acetylated target protein and nicotinamide is released. NAD þ , nicotinamide adenine dinucleotide. its deacetylation. Alternatively, some sirtuins might have only mono-ADP-ribosyltransferase activity. Regulation of sirtuins by NAD þ /NADH ratios and inhibition by nicotinamide Since NAD þ is a critical cofactor for sirtuin enzymatic activity, changes in levels of NAD þ and NADH, or their ratio, induced by diet and metabolic status, may regulate the biological activity of sirtuins in vivo as discussed below. CD38, which localizes to the inner nuclear membrane, hydrolyses NAD þ to nicotinamide (Aksoy et al., 2006b). In CD38/ mice, NAD þ levels are increased 10- to 20-fold, and the SIRT1 substrate p53 is less acetylated (Aksoy et al., 2006a). Nicotinamide phosphoribosyltransferase (Nampt), which is induced after some forms of stress, converts nicotinamide to nicotinamide mononucleotide, which then reacts with ATP to regenerate NAD þ (Yang and Sauve, 2006). Overexpression of Nampt increases NAD þ levels and induces SIRT1 activity, with changes in gene expression paralleling those in cells overexpressing SIRT1 (Revollo et al., 2004). Nampt decreases as primary cells age and undergo replicative senescence, which lowers NAD þ levels and SIRT1 activity (van der Veer et al., 2007). Inhibition of Nampt activity induces premature senescence in early-passage primary cells, while overexpression of Nampt delays senescence and increases survival after oxidative stress in late-passage primary cells (van der Veer et al., 2007). These effects of Nampt are dependent on SIRT1, as overexpression of a catalytically inactive SIRT1 blocks lifespan extension and accelerates senescence (van der Veer et al., 2007). Another cellular enzyme that uses NAD þ is poly(ADPribose) polymerase (PARP), a nuclear protein involved in DNA repair that cleaves NAD þ into nicotinamide and ADP-ribose (Zhang, 2003). Poly(ADP-ribose) is added to histones and other nuclear proteins including p53 (Poirier and Savard, 1980; Mendoza-Alvarez and Alvarez-Gonzalez, 2001; Simbulan-Rosenthal et al., 2001). Activation of PARP after DNA damage quickly depletes cells of NAD þ with a concomitant increase in nicotinamide levels, which may suppress the activity of the sirtuins (Zhang, 2003). Future experiments aimed at elucidating Oncogene Sirtuins and cancer LR Saunders and E Verdin 5492 the consequences of and the interplay among mono-ADP ribosylation, poly-ADP ribosylation and deacetylation of the same target protein, like histones and p53, will reveal how sirtuins influence cell survival after DNA damage. Nicotinamide promotes cell survival and longevity by protecting cells against stress, injury and inflammatory responses (Li et al., 2006). As a precursor to NAD þ , nicotinamide inhibits sirtuins by blocking the regeneration of NAD þ through interception of an ADP-ribosylenzyme-acetyl peptide intermediate (Jackson et al., 2003; Porcu and Chiarugi, 2005). Nicotinamide extends the replicative lifespan of primary human fibroblasts (Lim et al., 2006), and inhibits SIRT1 with an IC50 of o50 mM and PARP-1 with an IC50B100 mM (Yamamoto and Okamoto, 1980; Bitterman et al., 2002). Since nicotinamide is found in mammalian cells at 50–150 mM, nicotinamide is a physiological inhibitor of both sirtuins and PARP (Yang and Sauve, 2006). Regulation of SIRT1 expression in primary cells and its overexpression in cancer SIRT1 is overexpressed in a number of cancers. This overexpression occurs in part at the transcriptional level following the loss of repressors of the SIRT1 promoter. Two p53 binding sites in the SIRT1 promoter normally repress SIRT1 expression. However, in the absence of nutrients, Foxo3a translocates to the nucleus, interacts with p53, inhibits its suppressive activity and leads to increased SIRT1 expression (Nemoto et al., 2004). p53/ mice show increased basal expression of SIRT1 in selective tissues, including adipose tissue, but SIRT1 levels were not further elevated upon nutrient withdrawal (Nemoto et al., 2004). SIRT1 levels may be higher in tumors that have lost p53. As discussed below, SIRT1 deacetylates both p53 and FOXO proteins, which feedback to regulate SIRT1 expression. Another tumor suppressor that regulates SIRT1 is E2F1, which binds the SIRT1 promoter and regulates basal expression and induction after DNA damage (Wang et al., 2006). E2F1-Rb complexes bind target promoters and recruit HDAC-containing complexes to repress transcription of genes that control cell-cycle progression (DeGregori and Johnson, 2006). E2F1 is acetylated by p300/CBP-associated factor (PCAF), which enhances its DNA-binding, transactivation activity, and stability, and transforms E2F1 into an activator of transcription (Martinez-Balbas et al., 2000). SIRT1 also deacetylates E2F1 and may regulate apoptosis induction in response to DNA damage through this factor (Wang et al., 2006). The fine balance and regulation of acetylases and deacetylases may determine the cellular response to DNA damage: DNA repair for low levels of DNA damage and apoptosis for extensive DNA damage. Loss of E2F1 may have a dual effect on SIRT expression: increased basal expression and impaired induction after DNA damage. Such dysregulation of SIRT1 expression might tip the cellular response Oncogene to DNA damage to favor apoptosis induction instead of DNA repair (Figure 3). SIRT1 is also regulated by the tumor suppressor gene hypermethylated in cancer 1 (HIC1). SIRT1 mRNA and protein levels are increased in HIC1/ mouse embryonic fibroblasts (MEFs) (Chen et al., 2005). Similar to the intimate feedback loop with E2F1, SIRT1 interacts with HIC1 and represses its own expression (Chen et al., 2005). SIRT1 also deacetylates HIC1 and promotes HIC1 sumoylation at the same lysine acetylated by the histone acetyltransferases (HATs) CBP/p300 (Stankovic-Valentin et al., 2007). HIC1 functions as a transcriptional repressor when deacetylated and sumoylated (Stankovic-Valentin et al., 2007). HIC1 promoter hypermethylation occurs during tumorigenesis and aging, leading to the upregulation of SIRT1 (Chen et al., 2003, 2004). The normal downregulation of SIRT1 protein seen during aging might be lost in cells without HIC1, making them resistant to replicative senescence after oxidative stress, and vulnerable to transformation if mutations are propagated. HIC þ / mice are tumor prone and show a p53- and SIRT1dependent block in apoptosis induction in response to DNA damage (Chen et al., 2003, 2005). Loss of HIC1 and concomitant increase in SIRT1 prolongs lifespan, but facilitates tumor development as less apoptosis is induced in response to DNA damage. SIRT1 expression is also regulated at the posttranscriptional level by HuR (Abdelmohsen et al., 2007). HuR is a ubiquitously expressed mRNA-binding Figure 3 Activation of SIRT1 after stress promotes survival through deacetylation of various substrates. Oxidative stress or DNA damage induces activation of HATs and histone deacetylases. The extent of activation of these factors and the balance of acetylation and deacetylation determines how a cell responds to the stress signal. SIRT1 expression is induced after stress by multiple transcription factors, such as p53, E2F1 and hypermethylated in cancer 1, and its enzymatic activity is dependent on nicotinamide adenine dinucleotide levels that may be regulated during stress as well. High SIRT1 activity (left panel) promotes deacetylation of Ku70, FOXO and p53, promotes DNA repair and cell-cycle arrest, and blocks apoptosis induction after various stresses. If the levels and activity of HATs predominate (right panel), acetylated forms of Ku70, Foxo and p53 induce apoptosis. HAT, histone acetyltransferases; SIRT, sirtuins. Sirtuins and cancer LR Saunders and E Verdin 5493 protein that binds the 30 UTR of SIRT1 to stabilize the SIRT1 transcript (Abdelmohsen et al., 2007). HuR decreases dramatically as cells age and reach senescence (Wang et al., 2001) and this leads to a destabilization of SIRT1 mRNA (Abdelmohsen et al., 2007). High levels of SIRT1 and HuR contribute to the enhanced survival of rapidly proliferating cells after oxidative damage, as opposed to senescent cells that are more sensitive to oxidative damage (Abdelmohsen et al., 2007). After oxidative damage, HuR is phosphorylated by Chk2, which promotes dissociation of HuR from SIRT1 mRNA, leading to lower levels of SIRT1 and an enhanced sensitivity to apoptosis induction (Abdelmohsen et al., 2007). Thus, the extent of DNA damage is relayed to SIRT1, in part, by activation of Chk2. High levels of DNA damage and increased activation of Chk2 decrease expression of SIRT1, tipping the balance towards acetylation of p53 and other factors that induce apoptosis. SIRT1 promotes replicative senescence during prolonged exposure to low-level stress Primary cells undergo a limited number of divisions before telomere shortening sends a stress signal that induces replicative senescence. Oncogene activation and other stress stimuli also induce the same replicative senescence program (Hanahan and Weinberg, 2000). During replicative senescence in primary human fibroblasts, SIRT1 levels decrease (Michishita et al., 2005; Abdelmohsen et al., 2007). A decrease in HuR during senescence destabilizes the SIRT1 mRNA (Abdelmohsen et al., 2007). SIRT1 enzymatic activity also decreases as NAD þ levels fall due to a decrease in Nampt activity (van der Veer et al., 2007). Several tumor suppressor genes, including p53, p19ARF and Rb, regulate senescence by inducing a permanent withdrawal from the cell-cycle. The transcription factor p53 regulates expression of genes, including p21, PUMA and Bax, to initiate cell-cycle arrest, senescence or apoptosis (Vousden and Lu, 2002). Loss of p53 function can compromise the ability of human cells to undergo replicative senescence (Itahana et al., 2001). The key role of p53 in regulating cell proliferation and stress response is highlighted by inactivation or loss-of-function of p53 in over 50% of human tumors (Hollstein et al., 1994). In response to stress signals, p53 is acetylated at multiple lysines, which enhances its activity and stability (Barlev et al., 2001). p53 is deacetylated on lysine 382 (K379 in mouse p53) both by SIRT1 and HDAC1 (Luo et al., 2001; Vaziri et al., 2001; Cheng et al., 2003). p53 recruitment of HATs and HDACs/SIRT1 to specific promoters may regulate replicative senescence via changes in gene expression (Han et al., 2006). Increased p53 acetylation is associated with senescence (Pearson et al., 2000). As would be expected if increased acetylation of p53 induces senescence, SIRT1 overexpression blocks oncogene-induced senescence through deacetylation of p53 (Langley et al., 2002). Contrary to the prediction that loss of SIRT1 would induce senescence through increased p53 acetylation, primary SIRT1/ MEFs are resistant to replicative senescence in response to prolonged cell replication in culture (Chua et al., 2005). However, induction of replicative senescence is not impaired after acute DNA damage or oncogene activation in SIRT1/ MEFs (Chua et al., 2005). Thus, SIRT1 limits replicative senescence during prolonged exposure to low levels of stress. In addition to lower levels of total p53 and less p53 acetylation, SIRT1/ MEFs show lower expression of the tumor suppressor gene p19ARF (Cheng et al., 2003; Chua et al., 2005). The increase in p19ARF and p53 expression normally observed during serial passage of MEFs is not seen in SIRT1/ MEFs, which could prevent induction of replicative senescence (Chua et al., 2005). The interplay between SIRT1 and p53 and their reciprocal regulation of expression may be critical for replicative senescence. p19ARF does not appear to be acetylated, and it is unclear how SIRT1 regulates p19ARF expression, but it appears to play a critical role in how SIRT1 regulates replicative senescence (Chua et al., 2005). A role for SIRT1 in limiting replicative lifespan is also suggested by knockdown of SIRT1 with siRNAs in human diploid fibroblasts, which leads to enhanced cell proliferation (Abdelmohsen et al., 2007). Thus, SIRT1 seems to limit replicative lifespan and induce replicative senescence in response to chronic nonlethal forms of stress. An in-depth analysis of specific stresses will further define SIRT1’s role in limiting replicative senescence. Overexpression of SIRT1 alone does not affect replicative lifespan (Michishita et al., 2005), but Nampt overexpression, which increases SIRT1 activity through enhanced production of NAD þ , promotes p53 deacetylation and extends replicative lifespan of human smooth muscle cells (van der Veer et al., 2007). These results suggest that increased NAD þ levels regulate SIRT1 and at least one other factor to promote longevity, or that increased SIRT1 levels are not sufficient and SIRT1 must be activated to extend replicative lifespan. Alternatively, as NAD þ binds tetrameric p53, alters its conformation and inhibits DNA binding at some promoters (McLure et al., 2004), increased NAD þ may inhibit p53, perhaps at specific promoters, to block replicative senescence. Thus, the proper equilibrium of SIRT1 expression levels and activation limits replicative lifespan, along with the interaction and balance of HATs and tumor suppressor genes, such as p53 and p19ARF. As discussed above, cells can respond to DNA damage either by inducing cell-cycle arrest and DNA repair or by inducing apoptosis, depending on the extent of the damage and the ability to repair the damage before cell division. The balance of NAD þ and NADH control activation of SIRT1, which may signal the extent of DNA damage and influence the cellular response. Proper feedback mechanisms turn off the response once the damage is repaired. Extended survival responses signaling through SIRT1 during chronic stress could lead to tumor development through inactivation of p53. Therefore, SIRT1 may induce senescence, promote longevity and block tumor Oncogene Sirtuins and cancer LR Saunders and E Verdin 5494 development when cells are under chronic stress. While senescence protects against tumor development earlier in life, senescent cells may contribute to aging and agerelated diseases, such as cancer, by compromising tissue renewal and repair (Campisi, 2005). Interestingly, transgenic mice expressing a truncated form of p53, which has high activity, are resistant to tumor formation, yet show premature signs of aging and have a shorter lifespan (Tyner et al., 2002). In addition to p53, the related proteins p63 and p73 mediate cell growth and tumorigenesis by regulating target genes similar to those of p53, and p73 is deacetylated by SIRT1 (Dai et al., 2007). Mice overexpressing an isoform of p63, DNp63a, under the keratin 14 promoter for specific expression in the basal cells of the skin, also age prematurely (Sommer et al., 2006). In both transgenic mice with truncated p53 and those expressing DNp63a, SIRT1 levels are lower (Sommer et al., 2006). As the DNp63a isoform is overexpressed in the transgenic mice with high p53 activity and DNp63a interacts with SIRT1, elevated levels of DNp63a might directly regulate SIRT1 stability or turnover (Sommer et al., 2006). These mouse models suggest that lower levels of SIRT1 lead to premature aging, but could also protect against tumorigenesis. Conditional deletion of both p53 and SIRT1 will be needed to determine whether SIRT1 exerts its activity primarily via p53 deacetylation. SIRT1 also promotes replicative senescence by deacetylating histone H1 and forming heterochromatin (Figure 4). Heterochromatin, which is associated with gene silencing, contains several distinct alterations in acetylation and methylation, including hypoacetylated H4-K16 (Jeppesen and Turner, 1993), which can be carried out by SIRT1 (Vaquero et al., 2004). SIRT1 also recruits and deacetylates linker histone H1 on K26 to establish heterochromatin (Vaquero et al., 2004). Aging changes the distribution of H1 subtypes (Lennox and Cohen, 1983), and changes in chromatin mediate the silencing of specific genes during senescence (Narita et al., 2003). H1 protein is absent from senescent cells, and its loss induces formation of senescence-associated heterochromatic foci (SAHFs; Narita et al., 2003; Funayama et al., 2006). This loss of H1 is thought to occur post-translationally (Funayama et al., 2006), indicating that as levels of SIRT1 decrease during cellular aging, the resulting hyperacetylation of H1 might target it for degradation. Future experiments will determine if the loss of histone H1 during replicative senescence is indeed dependent on lower levels of SIRT1. Deacetylation of SIRT1 targets in response to cellular stress promotes survival SIRT1 localizes to the nucleus where it deacetylates various targets and regulates the cellular response to stress (Figure 3). SIRT1 promotes cellular survival by initiating cell-cycle arrest and DNA repair through deacetylation of p53, FOXO and Ku70. While SIRT1 clearly deacetylates p53, the consequence of this Figure 4 SIRT1 expression and activity decrease during aging. High levels of SIRT1 protein in young cells in combination with high levels of nicotinamide adenine dinucleotide lead to deacetylation of p53 and histone proteins to promote longevity (left panel). SIRT1 levels are lower in aged cells, and higher levels of nicotinamide inhibit SIRT1 activity (right panel). The resulting hyperacetylation of p53 can induce replicative senescence. Deacetylation of histone H1 leads to its degradation that promotes formation of senescenceassociated heterochromatic foci (SAHFs). Failure to downregulate SIRT1 during aging may promote cell survival after oxidative damage, leading to the accumulation of mutations, and an increased risk of cancer development. SIRT, sirtuins. Oncogene Sirtuins and cancer LR Saunders and E Verdin 5495 modification on p53 after DNA damage is controversial. In response to DNA damage, SIRT1/ MEFs show hyperacetylation of p53 at multiple lysines, not just the SIRT1 target K379, but this is not accompanied by a further increase in expression of p53 target genes such as p21, Bax or mdm2, nor by an increase in apoptosis (Cheng et al., 2003; Kamel et al., 2006). A SIRT1specific inhibitor, EX-527, increases p53 acetylation without any consequence on cell survival after DNA damage, unlike TSA, which inhibits HDAC1 and results in decreased survival following DNA damage (Solomon et al., 2006). Thus, more than just SIRT1-mediated deacetylation of p53 may be required to promote survival after DNA damage. MEFs from mice engineered to express p53 with seven C terminal lysines mutated to arginines (p537KR), to mimic unacetylated p53, respond to DNA damage similarly to MEFs from wild-type mice, but show enhanced stability of p537KR that allows for enhanced induction of target genes in thymocytes after DNA damage (Krummel et al., 2005). Immortalized p537KR MEFs undergo senescence earlier, suggesting that regulation of p53 acetylation may fine-tune p53 activity over the lifespan of an organism (Krummel et al., 2005). ES cells from mice engineered to express p53 with six C terminal lysines mutated to arginines (p536KR) show less transactivation of targets after DNA damage than ES cells that are p53 þ / (Feng et al., 2005). MEFs from these mice show no difference in p53 stability or target transactivation after DNA damage (Feng et al., 2005). Thymocytes from p536KR mice are slightly more resistant to apoptosis after irradiation (IR), with impaired upregulation of PUMA and DR5, but equivalent induction of mdm2, Bax and Pidd, and no alteration in p53 stability (Feng et al., 2005). Since these C terminal lysines are also modified by ubiquitination, neddylation, methylation and sumoylation, these studies suggest a role for acetylation in conjunction with other post-translational modifications in controlling p53 stability, localization and activity. A specific role for SIRT1 deacetylation of p53 in thymocytes and other tissues should be examined through tissue-specific alterations in SIRT1 levels. Additionally, conditional double-knockout mice lacking both SIRT1 and HDAC1 will address whether these deacetylases are redundant in targeting of p53 or if both are needed in response to certain types of stress and signaling pathways. FOXO transcription factors, including FOXO1, FOXO3a, FOXO4 and FOXO6, respond to DNA damage and oxidative stress and regulate expression of cell-cycle, DNA repair and apoptosis genes (FurukawaHibi et al., 2005). The importance of FOXO proteins in cancer is underscored as the FOXO proteins are important regulators of cell growth and are found as novel fusion proteins after chromosomal translocations in several types of cancer. The FOXOs are regulated by post-translational modifications including phosphorylation and acetylation. After oxidative stress, FOXO proteins are phosphorylated and relocalize to the nucleus, where they associate with HATs to form active transcriptional complexes, but the FOXOs themselves are acetylated on several lysine residues by multiple HATs, which inhibits their transactivation activity (Fukuoka et al., 2003; Daitoku et al., 2004; Motta et al., 2004; van der Horst et al., 2004). SIRT1 deacetylation of FOXO suppresses transactivation of proapoptotic proteins Bim and Fas ligand by FOXO, while promoting expression of p27kip and GADD45a to induce cell-cycle arrest (Brunet et al., 2004; van der Horst et al., 2004; Kobayashi et al., 2005). While overexpression of SIRT1 inhibits apoptosis induced by FOXO3a after cellular stress and SIRT1/ MEFs are more sensitive to oxidative damage, the ability of FOXO3a to induce cell-cycle arrest is enhanced by overexpression of SIRT1 and diminished in SIRT1/ cells (Brunet et al., 2004; Kobayashi et al., 2005). SIRT1 and FOXO1 are recruited to the manganese superoxide dismutase promoter, and the deacetylase activity of SIRT1 is required for transactivation of this antioxidant gene, indicating that SIRT1 also promotes survival by inducing the repair of oxidative damage (Daitoku et al., 2004; van der Horst et al., 2004). Thus, SIRT1 appears to promote cell-cycle arrest and DNA repair downstream of FOXO proteins, promoting survival rather than apoptosis. The ability of SIRT1 to promote survival after oxidative stress agrees with the model that SIRT1 promotes a longer lifespan, as increased resistance to oxidative stress correlates with longevity (Finkel and Holbrook, 2000). While the FOXO homolog in Caenorhabditis elegans plays a key role in lifespan regulation, demonstration of a similar role in mammalian lifespan will require development of conditional transgenic mouse models. As FOXO and p53 share many transcriptional targets, the consequences of stress signals in cells containing wild-type FOXO and p53, versus in tumor cells containing a mutation in either or both p53 or FOXO, are predicted to vary. The ability of SIRT1 to promote cellcycle arrest and DNA repair after DNA damage might require both p53 and FOXO deacetylation. The fact that p53 and FOXO interact following oxidative stress, highlight the possibility that SIRT1 mediates survival following cellular stress through both p53 and FOXO (Brunet et al., 2004). In primary cells with wild-type p53 and FOXO proteins, SIRT1 will promote cell-cycle arrest versus apoptosis after DNA damage. However, in tumor cells lacking wild-type p53 or FOXO, SIRT1 may have a different effect, and careful studies with various mutants of p53, FOXO and SIRT1 are needed to determine the interplay among these factors. CBP/p300 acetylation of FOXO proteins shifts the response towards apoptosis (van der Heide and Smidt, 2005). Again, overexpression of SIRT1 in primary cells may influence the downstream signaling pathways initiated after DNA damage and determining whether cells with DNA damage proliferate without arresting for repair, potentially leading to tumorigenesis. Tumor cells might require high levels of SIRT1 to protect against apoptosis, therefore allowing for the continued proliferation of tumor cells with genomic instability. Another SIRT1 target that promotes survival in cells that have suffered DNA damage is Ku70. Ku70 is Oncogene Sirtuins and cancer LR Saunders and E Verdin 5496 mainly localized to the nucleus where it is involved in DNA damage repair, but a small fraction is localized in the cytoplasm where Ku70 regulates apoptosis through sequestration of the proapoptotic protein Bax (Sawada et al., 2003). DNA damage promotes Ku70 acetylation on multiple lysines in its C terminus, disrupting the association with Bax, which on release transits to the mitochondria and initiates apoptosis (Cohen et al., 2004a). SIRT1, along with a classI/II HDAC, maintains Ku70 in a deacetylated state (Cohen et al., 2004a, b). SIRT1, while mainly localized in the nucleus, can shuttle to the cytoplasm (Tanno et al., 2006), but the site of SIRT1-mediated deacetylation of Ku70 is not yet clear. Only nuclear SIRT1 appears to be important for cell survival in response to DNA damage, as mutants that are constitutively cytoplasmic do not protect against oxidative stress (Tanno et al., 2006). Future experiments will determine if acetylation of Ku70 regulates its DNA repair functions in the nucleus. For SIRT1 to promote cell survival in cells with DNA damage, the increased acetylation of Ku70 after DNA damage must be promptly reversed by deacetylation of Ku70. The factors that fine-tune Ku70 acetylation and deacetylation are not known. High levels of SIRT1 in primary cells may tip the balance towards survival after DNA damage. As cells age and SIRT1 levels are diminished, DNA damage should result in increased levels of apoptosis. In pre-malignant cells overexpressing SIRT1, Ku70 may not be properly activated in response to DNA damage that is too extensive to be repaired, allowing for the accumulation of mutations without the induction of apoptosis. Treatment of cancers with a combination of HDAC and sirtuin inhibitors might promote apoptosis in combination with standard chemotherapy agents that damage DNA. The balance between acetylation and deacetylation of specific factors is regulated at an additional level, since SIRT1 itself deacetylates several HATs and regulates their enzymatic activities (Kalkhoven et al., 2002; Thompson et al., 2004). SIRT1 directly interacts with PCAF and p300 to promote their deacetylation and enzymatic inactivation (Fulco et al., 2003; Motta et al., 2004; Cohen et al., 2004a). The importance of HATs in cancer is highlighted by their frequent mutation, deletion and translocation in several types of cancer (Iyer et al., 2004). Recruitment of SIRT1 to its targets brings it in close proximity to HATs and allows SIRT1mediated deacetylation of p300, CBP and PCAF. The balance of HAT and HDAC/SIRT1 levels and their dysregulation in cancer alter the expression, stability and localization of target proteins. The critical role of SIRT1 during development may be coopted for tumor cell survival SIRT1 clearly plays an important role in development: knockout mice show multiple defects, and most die before or just after birth (Cheng et al., 2003; Ford et al., 2005). SIRT1 and p53 double-knockout mice have the same phenotype as SIRT1/ mice indicating that p53 Oncogene hyperacetylation is not responsible for the developmental defects downstream of SIRT1 (Kamel et al., 2006). Generation of conditional mice lacking both SIRT1 and FOXO will reveal the contribution of FOXO proteins to SIRT1’s role in development. SIRT1 normally limits proliferation and possibly tumor development as small interfering RNA (siRNA)mediated SIRT1 depletion in primary human fibroblasts enhances their proliferation (Abdelmohsen et al., 2007). In the context of cells containing wild-type p53 and intact cell-cycle checkpoints, SIRT1 limits tumor formation by inducing cellular senescence. However, loss of p53 and other tumor suppressor genes increases SIRT1 expression and can lead to transformation and formation of tumor cells that are addicted to SIRT1 overexpression. Various tumor cell lines cease growth and undergo apoptosis after knockdown of SIRT1 expression via siRNA (Ford et al., 2005; Abdelmohsen et al., 2007). Therefore, SIRT1 appears to be a key survival factor for some tumor cells (Ford et al., 2005; Ota et al., 2006). SIRT1 is overexpressed in acute myeloid leukemia (AML) blasts from patients as well as AML cell lines (Bradbury et al., 2005). The overexpression found in multiple types of nonmelanoma skin cancers, including early stages, suggests that SIRT1 may be playing a critical role in promoting proliferation in skin cancer (Hida et al., 2006). The ability of cancer cells to undergo senescence correlates with their response to treatment. Interestingly, SIRT1 mRNA and protein levels are higher in drug-resistant cancer cell lines than in the original cell lines and in patient tumors after chemotherapy than before therapy (Chu et al., 2005). siRNAmediated knockdown of SIRT1 expression partially reverses the drug-resistant phenotype, while SIRT1 overexpression or activation of SIRT1 with resveratrol increases expression of the multidrug resistance protein, MDR1 (Chu et al., 2005). Differences in the expression level of SIRT1 in primary versus transformed cells needs to be expanded to other cancer types, to identify cancers which might benefit from treatment with SIRT1-specific inhibitors. SIRT1-specific inhibitors may be useful chemotherapeutics to target tumors whose survival depends on SIRT1. Overexpression of SIRT1 in tumors is likely to affect both histone and non-histone acetylated targets. An example of a tumor dependent on SIRT1-mediated deacetylation of a non-histone target is B-cell lymphoma; SIRT1 deacetylates and deactivates the critical oncogene B-cell lymphoma 6 protein (BCL6). BCL6, a protooncogene, functions as a transcriptional repressor and recruits HDACs and other corepressors to unique promoters (Dhordain et al., 1998). BCL6 is specifically expressed in mature B cells and is required for the formation of germinal centers through repression of genes involved in differentiation and apoptosis (Niu, 2002). Chromosomal translocations involving BCL6 are found in non-Hodgkin’s lymphoma and BCL6 is constitutively expressed in some B-cell lymphomas (Niu, 2002). BCL6 is regulated by several post-translational modifications, including phosphorylation, which Sirtuins and cancer LR Saunders and E Verdin targets BCL6 for proteasome-mediated degradation (Niu et al., 1998), and acetylation by p300, which inactivates BCL6 repression of targets through disruption of the interaction with HDACs (Bereshchenko et al., 2002). Like nuclear factor-kB (NF-kB), p53 and Ku70, BCL6 is deacetylated by both HDACs and SIRT1 (Bereshchenko et al., 2002; Heltweg et al., 2006). BCL6 repressor activity is controlled by competing HATs and HDACs/SIRT1, and acetylation impairs the oncogeneic properties and transforming capabilities of BCL6 (Bereshchenko et al., 2002). Cambinol, a SIRT1 inhibitor, inactivates BCL6 in Burkitt’s lymphoma cells by promoting its acetylation, and leads to apoptosis induction (Heltweg et al., 2006). In mouse xenograft models, cambinol alone was effective specifically against tumors expressing BCL6 (Heltweg et al., 2006). DNA damage does not promote BCL6 acetylation (Bereshchenko et al., 2002), but inhibition of SIRT1 with cambinol sensitizes cells to DNA-damage-induced apoptosis independently of p53 (Heltweg et al., 2006). Cambinol also induced p53, FOXO3a and Ku70 acetylation (Heltweg et al., 2006), indicating that multiple targets of SIRT1 may control the response to DNA damage. Tumor cells addicted to SIRT1 may be sensitized to apoptosis induction by combined use of SIRT1 and HDAC inhibitors to sensitize them to DNAdamaging chemotherapeutic agents. In addition to its effects on specific non-histone targets, SIRT1 may promote cancer development by deacetylating histones. SIRT1 preferentially deacetylates H3-K9, H3-K14 and H4-K16 in vitro and in vivo (Imai et al., 2000; Vaquero et al., 2004). Importantly, loss of H4-K16 acetylation is a hallmark of human tumors (Fraga et al., 2005), and while this could be due to a loss of HAT association or function, it could also reflect overexpression of SIRT1 in tumors. Overexpression of SIRT1, other deacetylases, and/or chromatin remodeling factors may silence key tumor suppressor genes as an early event in the transformation process (Jones and Baylin, 2007). Loss of these tumor suppressor genes can relieve constraints on replicative senescence and allow tumor cells to proliferate without normal checkpoints in place. Epigenetic changes at the level of histone acetylation are mitotically inherited, allowing for improper silencing to be passed on during cell division. Long-term silencing of genes in embryonic stem cells is controlled by polycomb group proteins (PcG; Schuettengruber et al., 2007). PcG proteins function as large complexes that regulate growth, development and differentiation by acting on chromatin to alter gene expression, and PcGs are overexpressed in several cancers (Gil et al., 2005). The recent ‘cancer stem cell’ hypothesis implicates genes controlling self-renewal in pluripotent stem cells with the aberrant survival of tumor cells that have regained the ability to self-renew (Reya et al., 2001). In stem cells, SIRT1 directly associates with Suz12, a member of PcG complexes and like PcG proteins, SIRT1 is overexpressed in breast and colon cancers (Kuzmichev et al., 2005). In potential glioblastoma stem cells expressing the marker CD133, SIRT1 was overexpressed B5-fold compared to CD133negative cells from tumor samples (Liu et al., 2006), indicating a potential role for SIRT1 in driving the selfrenewal and resistance to apoptosis characteristic for these cancer stem cells. 5497 Activation of SIRT1 may prevent age-related cancers Prostate cancer incidence increases with age more rapidly than any other type of cancer, indicating that factors that increase risk of development of prostate cancer might also be associated with controlling lifespan (Waterbor and Bueschen, 1995). Development of prostate cancer depends on the activity of the androgen receptor (AR), which functions as an oncogene, and while androgen ablative therapy works initially, most patients relapse (Shand and Gelmann, 2006; Singh et al., 2006). Interestingly, the only predictor of tumor recurrence in patients with low-grade prostate cancer is the presence of changes in global histone acetylation and methylation levels (Seligson et al., 2005). Hormones, including dihydrotestosterone, activate the AR by promoting its acetylation, which enhances its transactivation activity through increased association with coactivators (Fu et al., 2000, 2003). SIRT1 deacetylates AR, and SIRT1 overexpression can block hormonal activation of AR (Fu et al., 2006). Furthermore, overexpression of SIRT1 inhibits the in vitro growth and proliferation of prostate cancer cells that express the AR (Fu et al., 2006). This suggests that resveratrol and other SIRT1 activators might be good chemopreventative and chemotherapeutic agents for prostate cancer. The interaction of SIRT1 with several of its targets, including the AR, may be regulated by four and a half LIM-domain protein 2 (FHL2) (Yang et al., 2005). LIM domain proteins function as adapters and modifiers mediating protein interactions, and FHL2 interacts with CBP/p300 and SIRT1 to influence their interaction with AR (Dawid et al., 1998; Labalette et al., 2004; Yang et al., 2005). In the normal prostate, FHL2 is localized to the cytoplasm, but in response to RhoA activation in prostate cancer, FHL2 relocalizes to the nucleus, and the amount of nuclear FHL2 correlates with more aggressive prostate cancer (Muller et al., 2002; Kahl et al., 2006). FHL2 may cooperate with androgens to inhibit FOXO1 activity by promoting the interaction of FOXO1 with the AR (Yang et al., 2005). FHL2 interacts with SIRT1 to promote interaction with FOXO and attenuate FOXO-dependent expression of proapoptotic genes in prostate cancer cells (Yang et al., 2005). As FHL2 can bridge CBP/p300 and SIRT1 to target proteins, future studies will need to dissect the precise role of FHL2 in prostate cancer in influencing acetylation and deacetylation of targets. It is not clear if FHL2 is simply bridging HATs and SIRT1 to FOXO and AR or playing an active role in determining access of acetylation and deacetylation factors to targets. As FHL2 is cytoplasmic in normal prostate cells but nuclear in prostate cancer, small molecules that disrupt Oncogene Sirtuins and cancer LR Saunders and E Verdin 5498 the interaction of SIRT1 and its substrates may promote apoptosis specifically in tumor cells. Furthermore, a potential role of FHL2 in regulating SIRT1 in other cancer types needs to be addressed. Specifically, FHL2 and SIRT1 may play a role in colon cancer as FHL2 influences the acetylation of b-catenin by CBP/p300 and a nuclear sirtuin could play an opposing role. Chronic inflammation-induced tumorigenesis may be prevented by SIRT1 deacetylation of NF-jB Chronic inflammation is a major risk factor for several cancers and NF-kB activation plays a key role in inflammation-associated cancers. Activation of NF-kB in cancer is associated with resistance to apoptosis and is a key determinant in chemotherapy response. NF-kB is maintained in an inactive form in the cytoplasm, and translocates to the cytoplasm in response to stress, infection and inflammation. Once in the nucleus, NF-kB induces transcription of cell-cycle, proliferation, migration and apoptosis genes (Piva et al., 2006). Phosphorylation of NF-kB on Ser276 recruits HAT-containing complexes to target promoters (Zhong et al., 2002), while acetylation of NF-kB on multiple lysines affects both its DNA binding and transactivation activity (Chen et al., 2002; Kiernan et al., 2003). Several class I HDACs as well as SIRT1 can deacetylate NF-kB (Ashburner et al., 2001; Chen et al., 2002; Zhong et al., 2002; Yeung et al., 2004). A direct interaction between endogenous SIRT1 and NF-kB not only promotes the deacetylation of NF-kB on lysine 310, but also of HATs on target promoters and local histone proteins to actively repress target gene expression (Yeung et al., 2004). As chronic inflammation and activation of NF-kB may contribute to several types of cancers, treatment with resveratrol may be a potential chemopreventative or chemotherapeutic agent. Resveratrol inhibits NF-kB-dependent transcription, but it is not known if this is directly and solely through activation of SIRT1 or also dependent on resveratrol’s effects on other regulators of NF-kB (Holmes-McNary and Baldwin, 2000; Manna et al., 2000). Regulation of a mitotic checkpoint by SIRT2 During mitosis, phosphorylation stabilizes SIRT2, which shuttles to the nucleus and colocalizes with chromatin (Dryden et al., 2003; Inoue et al., 2006). In the nucleus, SIRT2 deacetylates H4-K16, and during the G2/M transition, global levels of H4-K16 acetylation decrease, which may aid in chromatin condensation (Vaquero et al., 2006). As overexpression of SIRT2 delays mitotic exit, SIRT2 might function as a mitotic checkpoint protein by preventing chromatin condensation in response to mitotic stress (Dryden et al., 2003; Inoue et al., 2006). A loss of SIRT2 expression is seen in gliomas, which might compromise the mitotic checkpoint, contributing to genomic instability and tumorigenesis (Dryden et al., 2003; Hiratsuka et al., 2003; Oncogene Inoue et al., 2006). The proposed role of SIRT2 in regulating a mitotic checkpoint through global deacetylation of H4K-16 suggests that SIRT2 could guard against cells undergoing division when DNA damage is present. SIRT2 also colocalizes with microtubules in the cytoplasm and deacetylates a-tubulin (North et al., 2003). The consequences of tubulin acetylation are not understood, but may affect tubulin stability (Piperno et al., 1987). As several microtubule poisons are used as chemotherapeutic agents, SIRT2 expression may contribute to the response to these drugs (Rowinsky and Calvo, 2006). Tumors with high levels of SIRT2 may affect their response to chemotherapy drugs that target tubulin stability. Mitochondrial sirtuins: SIRT3, SIRT4 and SIRT5 Three sirtuins, SIRT3, SIRT4 and SIRT5 localize to the mitochondria. A proteomics approach recently suggested that 20% of mitochondrial proteins are acetylated, especially those involved in lifespan and metabolism (Kim et al., 2006). As mitochondria are the major site for production of reactive oxygen species that causes oxidative stress, this organelle is key in lifespan and aging. Mitochondria function in energy metabolism, and acetylation of key metabolic proteins may regulate changes in energy production in response to changes in the environment. Acetyl-CoA and NAD þ are key indicators of cellular energy status, and acetyl-CoA is a substrate for HATs, while NAD þ is a cofactor for sirtuins. While the cellular role and deacetylation targets are just starting to be identified, the mitochondrial sirtuins will likely play a role in the altered energy metabolism and response to oxidative stress in tumor cells. The first targets of SIRT3 and SIRT4 have recently been identified, and will be discussed in detail below. SIRT5 has deacetylase activity, but no targets have yet been identified (North et al., 2003; Michishita et al., 2005). SIRT3 shows high expression in brown adipose tissue, where its expression is further increased upon CR and exposure to cold temperatures (Shi et al., 2005). SIRT3 functions as a protein deacetylase (Onyango et al., 2002; Schwer et al., 2002), and specifically targets K642 of the mitochondrial protein acetyl-CoA synthetase 2. Deacetylation of acetyl-CoA synthetase 2 activates the enzyme and promotes the production of acetyl-CoA (Hallows et al., 2006; Schwer et al., 2006). Several genetic studies have linked SIRT3 to aging, including in humans (Rose et al., 2003; Glatt et al., 2007). SIRT4 does not possess deacetylase activity, but functions as an ADP-ribosyltransferase (Michishita et al., 2005; Haigis et al., 2006). Its only known target is GDH, a mitochondrial enzyme that converts glutamate to a-ketoglutarate (Haigis et al., 2006). ADPribosylation represses the activity of GDH (Haigis et al., 2006). In contrast to SIRT1 and SIRT3 that are upregulated during CR, SIRT4 activity may be downregulated by CR (Haigis et al., 2006). SIRT4 knockout Sirtuins and cancer LR Saunders and E Verdin 5499 mice are viable, and pancreatic islets isolated from these mice secrete higher levels of insulin, revealing a role for SIRT4 in downregulating insulin secretion through repression of GDH activity (Haigis et al., 2006). While additional targets of SIRT4 remain to be identified, the repression of GDH activity by SIRT4 may protect against diabetes and aging. The loss of SIRT4 could contribute to diabetes, a major risk factor for cancer (Strickler et al., 2001). Future experiments aimed at evaluating cancer incidence in aging SIRT4 knockout mice, especially those on a western diet that could increase diabetes risk, may identify a role for SIRT4 in protecting against cancer development. Guarding against aging and genomic instability by SIRT6 SIRT6 is a nuclear protein associated with heterochromatin that functions in base excision repair (BER). BER serves to protect the genome from single-strand breaks that arise spontaneously from alkylation, oxidation and deamination events, or that are induced by chemical mutagens (Michishita et al., 2005; Mostoslavsky et al., 2006). SIRT6 is widely expressed with the highest levels found in the brain and skeletal muscle and has no detectable deacetylase activity, but displays robust ADP-ribosyltransferase activity in vitro and can autoADP-ribosylate (North et al., 2003; Liszt et al., 2005; Michishita et al., 2005). No ADP-ribosylated targets of SIRT6 have been identified, but histone proteins and DNA repair proteins are possible targets. It will also be interesting to determine if SIRT2 and SIRT6 work in concert in the nucleus, with SIRT6 signaling to SIRT2 when DNA damage is repaired and mitosis can proceed. While normal human fibroblasts overexpressing SIRT6 do not have an extended replicative lifespan, SIRT6/ MEFs and SIRT6/ mouse embryonic stem cells (mESCs) show a reduced proliferative rate (Michishita et al., 2005; Mostoslavsky et al., 2006). SIRT6/ MEFs and mESCs have an increased sensitivity to irradiation but normal UV sensitivity (Mostoslavsky et al., 2006). SIRT6/ cells show multiple chromosomal defects including fragmentation, detached centrosomes, gaps and translocations due to a defect in BER (Mostoslavsky et al., 2006). Reconstitution of SIRT6/ MEFs with wild-type and catalytically inactive SIRT6 showed that an intact deacetylase domain of SIRT6 is required for BER and maintenance of genomic stability (Mostoslavsky et al., 2006). Proper regulation of genomic stability protects against tumor formation and aging (Lombard et al., 2005) and SIRT6/ mice show premature aging-like symptoms and die several weeks after birth (Mostoslavsky et al., 2006). Conditional knockdown of SIRT6 will be needed to determine if SIRT6 also guards against tumor formation by protecting the genome from DNA damage. SIRT6 may be a tumor suppressor gene whose loss contributes to the accumulation of mutations and increased genomic stability. Future screening of human tumors for loss of SIRT6 will determine if SIRT6 functions as a tumor suppressor gene. SIRT7 regulation of rRNA genes in proliferating tissues SIRT7 localizes to nucleoli, associates with condensed chromosomes during mitosis and is widely expressed in proliferating mouse tissues, such as liver, testes and spleen (Michishita et al., 2005; Ford et al., 2006). Low levels of SIRT7 are found in nonproliferating tissues, such as heart, brain and skeletal muscle (Michishita et al., 2005). SIRT7 interacts with RNA Polymerase I and promotes active transcription of rRNA genes, an activity that requires a wild-type catalytic domain (Ford et al., 2006). No deacetylase or ADP-ribosyltransferase activity has been demonstrated for SIRT7, and while SIRT7 has been reported to copurify with a high molecular weight complex in human cells, no specific interacting partners or targets have been identified (North et al., 2003; Ford et al., 2005, 2006). SIRT7 was identified in a subtractive library screen as being overexpressed in human thyroid papillary carcinoma compared to normal thyroid cells (De Nigris et al., 2002; Frye, 2002). As primary breast cells (HMECs) reach senescence, expression of SIRT3 and SIRT7 increases (Ashraf et al., 2006). Additionally, increased SIRT7 expression was seen in breast cancers as compared to nonmalignant breast tissue, with a significantly higher expression of SIRT7 in nodepositive than in node-negative breast cancers (Ashraf et al., 2006). Knockdown of SIRT7 expression in human U20S cells induces apoptosis, indicating that SIRT7 is required for cell survival (Ford et al., 2006). SIRT7 functions to regulate rRNA transcription and therefore is likely essential for cell survival and proliferation in both primary and transformed cells. It is not clear if elevated SIRT7 levels are a cause or consequence of cells bypassing senescence and becoming malignant, as increased proliferation rates of tumors require increased RNA pol-I transcription of rRNA. This potential role of SIRT7 in driving proliferation of malignant growth is in line with its higher expression in proliferating versus nonproliferating tissues (Ford et al., 2006). Tumor cells may be addicted to SIRT7 to maintain rapid proliferation, but the use of SIRT7 inhibitors as potential cancer therapeutic agents might be impeded by an essential role of SIRT7 in primary cells. Therapeutic potential of sirtuin regulation As the list of sirtuin targets rapidly expands and the cellular function of each sirtuin becomes clearer, the critical role that sirtuins play at the crossroads between longevity and cancer will be further elucidated. Current evidence suggests that several sirtuins protect against cancer. Primary cells express high levels of SIRT1, and SIRT1 levels normally decrease during cellular aging to promote replicative senescence and protect against tumorigenesis (Chua et al., 2005). As discussed above, replicative senescence is a form of tumor suppression and the critical role that SIRT1 plays in limiting replicative lifespan may indicate that SIRT1 functions as a tumor suppressor. Oncogene Sirtuins and cancer LR Saunders and E Verdin 5500 If SIRT1 indeed functions as a tumor suppressor, the SIRT1 activator resveratrol may function as a beneficial chemopreventative agent. Resveratrol is a natural product induced in plants after a variety of stresses and shows potential cancer chemoprotective properties (Athar et al., 2007). Activation of SIRT1 may be a useful chemopreventative agent in age-related cancers, such as prostate cancer in which deacetylation of the AR inactivates its ability to transform prostate cells (Fu et al., 2006). Another example in which SIRT1 activators, such as resveratrol, might be useful as a chemopreventative agent would be in promoting deacetylation and inactivation of NF-kB to prevent chronic inflammation, a contributing factor to tumor development (Yeung et al., 2004). In addition to its ability to function as a SIRT1 activator, resveratrol also downregulates the anti-apoptotic gene survivin and blocks activation of the pro-survival activity of NF-kB, PI3K/ AKT and mitogen-activated protein kinase pathways (Fulda and Debatin, 2006). Future experiments will clarify whether SIRT1 activation by resveratrol is upstream of all of these pathways, or if there are multiple mechanisms by which resveratrol works to protect against cancer. While SIRT1 clearly plays a critical role in development, lifespan and protection against cancer development, several forms of cancer have elevated levels of SIRT1 and may be dependent on SIRT1 for proliferation and survival. Loss of tumor-suppressor genes that regulate SIRT1 expression may lead to the overexpression of SIRT1 (Nemoto et al., 2004; Chen et al., 2005; Wang et al., 2006). Therefore, SIRT1-specific inhibitors may be useful chemotherapeutic agents for cancers ‘addicted’ to SIRT1 expression. Specifically, treatment of B-cell lymphomas with a SIRT1 inhibitor would increase acetylation of BCL6 and inactivate it as an oncogene (Heltweg et al., 2006). In addition to inactivation of an oncogene, SIRT1 inhibition may also reactivate expression of proapoptotic genes and other tumor suppressor genes through increased histone acetylation. Tumor cells show global changes in their chromatin, which can in part be due to the dysregulation of sirtuins (Jones and Baylin, 2007). Overexpression of SIRT1, SIRT2, SIRT3 and SIRT7 are found in tumors (De Nigris et al., 2002; Hiratsuka et al., 2003; Bradbury et al., 2005; Ashraf et al., 2006; Hida et al., 2006), indicating that sirtuin inhibitors may be useful chemotherapeutic agents against cancers that overexpress these sirtuins. The increased acetylation of targets, such as p53, Foxo, Ku70 and BCL6 induced by inhibition of sirtuins, in conjunction with chemotherapy agents that damage DNA, might induce tumor cell apoptosis. Future work in this area will more clearly define the precise role of each sirtuin at the crossroads between cancer and longevity and will hopefully identify new chemopreventative and chemotherapeutic uses of sirtuin activators and inhibitors. Acknowledgements We thank Stephen Ordway and Gary Howard for editorial assistance and John Carroll, Alisha Wilson, and Chris Goodfellow for graphics assistance. 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