Oncogene (2014) 33, 1609–1620 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

REVIEW Sorting out functions of in

M Roth and WY Chen

The sirtuins (SIRT 1–7) comprise a family of NAD þ -dependent protein-modifying with activities in lysine deacetylation, adenosinediphospho(ADP)-ribosylation, and/or deacylation. These enzymes are involved in the cell’s stress response systems and in regulating expression, DNA damage repair, metabolism and survival. Sirtuins have complex roles in both promoting and/or suppressing tumorigenesis. This review presents recent research progress concerning sirtuins and cancer. On one hand, functional loss of , particularly SIRT1, involved in maintaining genome integrity and DNA repair will promote tumorigenesis because of genomic instability upon their loss. On the other hand, cancer cells tend to require sirtuins for these same processes to allow them to survive, proliferate, repair the otherwise catastrophic genomic events and evolve. The bifurcated roles of SIRT1, and perhaps several other sirtuins, in cancer may be in part a result of the nature of the genes that are involved in the cell’s genome maintenance systems. The in-depth understanding of sirtuin functions may have significant implication in designing precise modulation of selective sirtuin members to aid cancer prevention or treatment under defined conditions.

Oncogene (2014) 33, 1609–1620; doi:10.1038/onc.2013.120; published online 22 April 2013 Keywords: acquired resistance; cancer; genetic instability; longevity; sirtuin; SIRT1

INTRODUCTION enabling replicative immortality, activating invasion and Sirtuins are mammalian homologs of yeast silent information metastasis and inducing angiogenesis; meanwhile, as normal regulator 2 (Sir2) encoding a .1,2 The seven cells possess innate mechanisms to antagonize cancer-promoting mammalian members of sirtuins (termed SIRT1–7) share a signals, the transformed cells must also overcome tumor conserved catalytic core domain (Figure 1). Despite this homology, suppression mechanisms, namely, by evading growth suppressor sirtuins have divergent enzymatic activities with SIRT1–3 and signals and resisting cell death. Crucially underlying these 15 SIRT7 primarily as lysine deacetylases, SIRT4 primarily as adeno- hallmarks of cancer is the genetic instability of cancer cells. sinediphospho (ADP)-ribosyltransferase, SIRT5 as deacetylase and Although cancer biologists tend to classify genes into either tumor deacylase, and SIRT6 as ADP-ribosyltransferase and deacetylase.1–5 promoting or tumor suppressing, only a limited number of genes In addition, primary localization of these proteins in the cell varies unambiguously fall into one of these categories, for examples, significantly, reflecting functional distinction of sirtuin members MYC as an oncogene and retinoblastoma gene RB as a tumor 16 (see Figure 1). suppressor gene. Other genes including sirtuins are less Growing interest in sirtuins largely stems from previous studies apparent, and the tumor-promoting or -inhibiting properties of yeast Sir2 showing that in lower organisms, increased Sir2 gene of the genes may depend on the stages of cancer development dosage is sufficient to extend lifespan,6–8 and that Sir2 is at a and contextual variables such as tissue of origin, the micro- 15 nexus between caloric restriction, resveratrol or other caloric environment and the exact experimental conditions. However, restriction mimetics and longevity.9,10 More recently, these initial rapid progress has been made most recently in the research on observations have been scrutinized and refined to show a lack of mammalian sirtuins and cancer, which may improve our an effect of Sir2 in extending lifespan in lower species as well as in understanding of these elusive genes and will be highlighted in mammals.11–13 Further adding to the complex nature of the role of this review. sirtuins in extending lifespan, Kanfi et al.14 described that transgenic SIRT6 male mice live B10–15% longer than their wild-type littermates. The conflicting literature on the roles of Sir2 SIRT1 in longevity contributes in part to the confusion of functions of Biochemical overview mammalian sirtuins, but may also foretell the complexity of these SIRT1 shares the greatest homology with yeast Sir2, which was genes in mammalian cells. initially characterized as an nicotinamide adenine dinucleotide Research in the past decade has revealed the perplexed and (NAD) þ -dependent histone deacetylase2 (so-called class III often controversial roles of sirtuins in promoting versus suppres- histone deacetylase that is structurally and biochemically distinct sing cancer. Cancer cells alter normal cellular machineries to from class I, II and IV histone deacetylases). SIRT1 deacetylates promote unabated cell proliferation and maximize their lifespan, histone H4 lysine 16 (H4K16) as well as histone H3 lysine 9 and 14 but as a consequence of their malignant growth, they cut short (H3K9 and H3K14, respectively).1 Additionally, SIRT1 deacetylates the lifespan of the host organism. Hanahan and Weinberg15 have histone H1 lysine 26 (H1K26) and is involved in the deposition of elegantly outlined the fundamental mechanisms of cancer histone variants.17 These modifications of histone tails are closely promotion as consisting of sustaining proliferative signaling, related to gene silencing and heterochromatin formation that may

Department of Cancer Biology, Beckman Research Institute, Duarte, CA, USA. Correspondence: Dr WY Chen, Department of Cancer Biology, Beckman Research Institute, City of Hope, 1500 East Duarte Road, Duarte, CA 91010, USA. E-mail: [email protected] Received 4 January 2013; revised 11 February 2013; accepted 18 February 2013; published online 22 April 2013 Functions of sirtuins in cancer M Roth and WY Chen 1610 min/ þ Enzymatic Primary formation in adenomatous polyposis coli (APC) mice. They Sirtuin protein and catalytic domain Activities Localization demonstrated that SIRT1 deacetylates b-catenin and reduces its 254 495 nuclear presence and transactivation potential. Similarly, SIRT1 747 Deacetylase Nucleus Oberdoerffer et al.26 showed that SIRT1 conditional 40 294 þ / À SIRT2 352 Deacetylase Cytosol overexpression reduces tumor burden in mice. In a 11 137 373 recent report, Herranz et al. undertook a 3-year study of SIRT1 SIRT3 399 Deacetylase Mitochondria transgenic mice with SIRT1 overexpression under its own 47 308 , and showed that increased SIRT1 expression by 314 SIRT4 ART Mitochondria threefold improves healthy mouse aging and reduces 51 301 Deacetylase& SIRT5 310 Mitochondria spontaneous carcinomas and sarcomas, as well as carcinogen- Deacylase induced liver cancer incidence. Taken together, in vivo mouse 45 257 Deacetylase& SIRT6 355 Nucleus models suggest that SIRT1 may improve genetic stability of ART normal cells and suppress tumor formation. 100 314 SIRT7 410 Deacetylase Nucleolus In addition to genetic stability, SIRT1 may regulate other pathways that contribute to its tumor suppression function. SIRT1 Figure 1. Comparison of mammalian sirtuins. The amino-acid length has been shown to inhibit the NF-kB pathway28 that promotes of each mature main form of sirtuins is indicated at the end of each 29 protein, with the conserved catalytic domain illustrated in grey. inflammation, survival and cancer metastasis. NF-kB comprises Principal enzymatic activities and primary cellular localization are heterodimers with p65 and p50 subunits. SIRT1 deacetylates the shown. p65/Rel-A subunit of NF-kB and blocks its transactivation of downstream anti-apoptotic target genes, cIAP-2 and BCL-xL.28 underlie certain biological processes.18,19 Notably, global genomic Further in line with tumor suppressor function, SIRT1 acts hypoacetylation at H4K16 is a hallmark of human cancer cells, downstream of BReast CAncer 1 (BRCA1) to negatively regulate both cell lines and clinical samples.20 the anti-apoptotic gene, Survivin, by deacetylating H3K9 on its The biological roles of SIRT1, however, are mostly revealed promoter and thereby repressing its transcription. Thus, BRCA1 ablation, via reduced SIRT1, results in elevated Survivin levels and through its deacetylation of a growing number of non-histone 30 substrates that are involved in a wide variety of cellular functions, enhances tumor growth. particularly in metabolic, oxidative/genotoxic and oncogenic stress responses. These substrates can be broadly categorized as: (1) transcriptional factors p53, forkhead transcription factors Dysregulation of SIRT1 and enzymatic activity in cancer (FOXO)1, FOXO3a, nuclear factor (NF)-kB, c-MYC, N-MYC, E2F1 and hypoxia-inducible transcription factors (HIF)-1a/HIF-2a, for reg- Although SIRT1 has tumor suppression function in the mouse ulating cell cycle progression and promoting survival under studies described above, so far there is no reported functional various conditions; (2) DNA repair machinery elements Ku70, SIRT1 genetic mutation or deletion, or SIRT1 promoter hyper- RAD51, NBS1, APE1, XPA/C and WRN, for improving DNA damage methylation in human cancer. The Sanger’s Catalogue of Somatic repair; (3) nuclear receptor, circadian clock and related factors LXR, Mutations in Cancer database registers only 14 uncharacterized FXR, ERa, AR, PPARg, PGC1a, CLOCK and PER2, for regulating point mutations among 4357 unique samples as of December metabolism; (4) histone-modifying enzymes SUV39H1, p300, TIP60 2012, perhaps as background mutations. and PCAF, for regulating gene expression; (5) cell-signaling In contrast to rare SIRT1 genetic alterations, numerous studies molecules STAT3, b-catenin and SMAD7, as detailed in previous have revealed that human cancer samples display elevated levels reviews.21,22 of SIRT1 relative to their nontransformed counterparts. These studies span different cancer types including liver,31 breast,32 gastric,33 prostate34,35 and hematopoietic36–41 origin. In colorectal SIRT1, genetic stability and tumor suppression cancer, Kabra et al.42 observed reduction of SIRT1 expression, but Several studies using mouse models provide evidence that Nosho et al.43 showed that SIRT1 overexpression correlates with SIRT1 may improve genetic stability and suppress tumor microsatellite instability as well as a CpG island methylator formation (Table 1). SIRT1 is a critical gene for mouse early phenotype. Similarly, in mice, overexpression of SIRT1 is development. Homozygous deletion of SIRT1 (DExon5–6 or reported in leukemia, lymphoma, sarcoma, lung adenocarcinoma DExon5–7) results in perinatal and postnatal lethality in C57BL/6 and prostate cancer.34,39,44,45 and 129 strains.23,24 More severe and early embryonic lethality Alteration of SIRT1 gene expression in cancer is mediated by phenotype occurs in another strain with mixed genetic multiple mechanisms affecting transcription, and post- background containing FVB upon SIRT1 deletion (DExon5–6).25 translational modifications. At the transcriptional level, tumor In C57BL/6-129 background, wild-type and SIRT1 À / À mouse suppressor HIC1 directly represses SIRT1 transcription, thereby embryonic stem cells exhibit similar levels of chromosomal leading to and activation of another tumor suppressor abnormality, but SIRT1 À / À embryonic stem cells are more p53 (Chen et al.,44 and Figure 2). Loss of HIC1 results in prone to genomic instability under oxidative stress.26 In mixed deacetylation of p53 and survival of aged and damaged cells, FVB background, SIRT1 À / À early embryos and mouse embryonic which may predispose them to .46,47 p53 itself has been fibroblasts display markedly increased spontaneous gross shown to directly repress SIRT1 transcription as well.48 Conversely, chromosomal abnormalities accompanied with altered global SIRT1 is upregulated by oncogenic transformation in several histone modifications.25 The genetic instability in SIRT1 À / À settings. Yuan et al.39 have recently shown that transformation of embryonic cells is attributed to defective DNA damage repair hematopoietic stem/progenitor cells by oncogenic tyrosine kinase characterized by reduced gH2AX foci formation and reduced BCR-ABL activates SIRT1 transcription in part through STAT5 recruitment of DNA-damage repair factors to the foci.25,26 In (signal transducer and activator of transcription 5). STAT5 is accord with reduced genetic stability upon SIRT1 loss, SIRT1 downstream of numerous cytokine and growth factor receptor- heterozygous deletion accelerates tumor formation in p53 þ / À signaling cascades and is constitutively active in many cancer knock-out mice.25 types.49 STAT5 also acts downstream of BCR-ABL protein in Using a conditional SIRT1 overexpression allele that is chronic myelogenous leukemia (CML).50,51 STAT5 directly binds specifically expressed in the cells lining the intestine, Firestein the SIRT1 promoter and enhances SIRT1 expression in CML cells39 et al.27 showed that SIRT1 overexpression reduces intestinal tumor (see Figure 2). SIRT1 levels are increased stepwise from normal

Oncogene (2014) 1609 – 1620 & 2014 Macmillan Publishers Limited Functions of sirtuins in cancer M Roth and WY Chen 1611 Table 1. Mouse cancer phenotypes with sirtuin gene knockout or overexpression

Sirtuin Genotype Phenotype Organ sites of tumors Role of sirtuins Reference

SIRT1 Constitutive SIRT1 knockout inhibited BCR-ABL Blood Cancer promoting Yuan et al.39 deletion of SIRT1 transformation and CML development SIRT1tg Pten þ / À SIRT1 transgenic mice had increased Thyroid and prostate Cancer promoting Herranz et al.75 incidence of thyroid cancer with lung metastases. SIRT1 expression also increased prostate carcinomas in situ SIRT1 þ / À p53 þ / À Double heterozygous knock-out mice had Mammary glands Cancer Wang RH et al.25 increased mammary tumor incidence suppressing Villin-Cre SIRT1DSTOP Intestinal SIRT1 expression reduced intestinal Intestine Cancer Firestein et al.27 APCmin/ þ tumors suppressing Mx-Cre SIRT1DSTOP SIRT1 overexpression reduced overall tumor Thymus Cancer Oberdoerffer p53 þ / À incidence, particularly thymic lymphoma, in suppressing et al.26 p53 þ / À background SIRT1tg Transgenic SIRT1 mice had reduced Liver, and other Cancer Herranz et al.11 incidence of spontaneous carcinomas and epithelial and suppressing sarcomas, but not lymphoma. SIRT1tg mice mesenchymal tissues had lower incidence of carcinogen-induced liver carcinomas SIRT2 Constitutive SIRT2 À / À -female mice developed mammary Liver and intestine in Cancer Kim et al.142 deletion of SIRT2 tumors, whereas -males predominately males and mammary suppressing developed liver cancers and intestinal tumors glands in females SIRT3 Constitutive SIRT3 À / À -female mice developed mammary Mammary glands Cancer Kim et al.164 deletion of SIRT3 tumors with 35% penetrance at about 24 suppressing months of age SIRT6 Constitutive SIRT6 Premature aging and death at postnatal — — Mostoslavsky deletion week 3 et al.192 Villin-Cre SIRT6fl/fl SIRT6 À / À mice developed larger intestinal Intestine Cancer Sebastian APCmin/ þ tumors suppressing et al.200 Abbreviations: APC, adenomatous polyposis coli; CML, chronic myelogenous leukemia; SIRT, sirtuins; SIRT1tg, SIRT1 transgenic. No tumors have been reported in constitutive SIRT4, SIRT5 and SIRT7 knock-out mice.

hematopoietic cells to chronic-phase CML to accelerated and blast dependent kinase cyclin B/CDK1 controls cell proliferation.66 The crisis CML.39 In the same setting, Yuan et al.39 also showed that dual specificity tyrosine phosphorylation-regulated kinases SIRT1 can be activated by KRAS transformation. Recently, several DYRK1A and DYRK3 phosphorylate SIRT1 and increase SIRT1 groups have demonstrated that MYC (c-MYC and N-MYC) proto- enzymatic activity to promote cell survival.67 SIRT1 oncogenes directly activates SIRT1 transcription.52,54 This occurs phosphorylation by c-Jun N-terminal kinase 2 stabilizes the despite incomplete agreement of its effect on MYC protein protein,68 whereas SIRT1 phosphorylation by JNK1 facilitates stability: three groups showing that SIRT1 and MYC form a positive ubiqitination-mediated degradation.69 Under genotoxic stress, feedback loop in which SIRT1 deacetylates MYC and enhances the proapoptotic nuclear desumoylase SENP1 removes SIRT1 MYC protein stability,52,54,55 whereas another group showing that sumoylation and reduces its deacetylase activity.70 SIRT1 destabilizes c-MYC protein.53 In addition, the cell cycle and Finally, SIRT1 enzymatic activity could be regulated during apoptosis regulator E2F1 induces SIRT1 transcription when cancer tumorigenesis. Deleted in breast cancer 1 (DBC1) is a tumor cells are under genotoxic stress.56 suppressor that is lost in a portion of breast cancer patients. DBC1 At the message level, SIRT1 expression is increased by suppresses SIRT1 activity by direct binding to the SIRT1 catalytic stabilizing its messenger RNA (mRNA) in cancer cells. core domain.71,72 In contrast, active regulator of SIRT1 (AROS) are small, RNA polymerase II-transcribed genes that generally form increases SIRT1 activity through direct interaction with N-terminus clusters and operate by binding to the 30 untranslated regions of of SIRT1 protein.73 Furthermore, increasing SIRT1 expression is genes and thereby negatively regulating mRNA stability and/or concomitant with activation of the mammalian NAD þ salvage translation.57 Several families of microRNAs have been shown to biosynthesis nicotinamide phosphoribosyltransferase in negatively regulate SIRT1, particularly miR-34a.58 The several types of cancer cells to provide sufficient NAD þ for SIRT1 chromosomal region where miR-34a resides is frequently lost in functions, which is important for cancer cell survival and stress human cancers, while reintroduction of miR-34a into cancer cells response.52,74 triggers apoptosis.59,60 Other miR families including miR-200a that target SIRT1 have been characterized as possessing tumor suppressor function.32,57,61 Conversely, SIRT1 mRNA is stabilized SIRT1 promotes cancer genomic instability and cancer evolution by RNA-binding protein HuR62, which has a role in promoting Several recent studies provide crucial insight into the roles of tumor cell proliferation and survival.63–65 SIRT1 dysregulation in cancer. Yuan et al.39 demonstrated that SIRT1 protein stability is regulated by phosphorylation, sumoy- SIRT1 homozygous knockout significantly inhibits BCR-ABL lation and ubiquitination. SIRT1 phosphorylation by cell cycle- transformation of mouse bone marrow stem/progenitor cells

& 2014 Macmillan Publishers Limited Oncogene (2014) 1609 – 1620 Functions of sirtuins in cancer M Roth and WY Chen 1612 SIRT1 BCR-ABL Survival Normal Cells DNA repair with fixed Tumor suppression STAT5 with fidelity DNA HIC1 p53 Myc E2F1

SIRT1 Evolve to STAT5A STAT5B high grade malignancy Survival -2235 -1838 -1116 -1039 -178 -168 -80 -65 Cancer Cells DNA repair with increased with infidelity chemo mutations Relapse and miRNAs 34a, drug resistance 200a, etc. HuR Figure 3. A model for bifurcated SIRT1 roles in cancer through genome maintenance. SIRT1 promotes genome maintenance in both normal and cancer cells under genotoxic stress and DNA UAG AAAA damage. Activation of DNA repair with high fidelity in normal cells improves genome stability and suppresses tumor formation. AUG Activation of DNA repair with low fidelity in cancer cells prevents catastrophic genomic events and renders cancer cell survival, but allows cancer cells to accumulate nonfatal lesions and more mutations to evolve toward high-grade malignancy and drug resistance under chemotherapy.

AROS DBC1 repair including both non-homologous end joining (NHEJ) and (HR) repair in CML cells76 and osteosarcoma cells.26 It is shown that SIRT1 is recruited to sites 254 495 of DNA damage, presumably to remodel local structure N-catalytic core -C to facilitate repair in cancer cells.77,78 Therefore, it is likely that Figure 2. Regulation of SIRT1 gene expression and activities in SIRT1 may have a role in genome maintenance in both normal cancer cells. SIRT1 transcription is activated by Myc, E2F1 or BCR-ABL and cancer cells. However, this may lead to complex outcomes in in part through STAT5, but repressed by HIC1 and p53. The sites for cancer cells. On one hand, the maintenance by SIRT1 may ensure transcriptional factor binding are shown. SIRT1 mRNA is stabilized cancer cell survival and proliferation (see more in next section) by by HuR but degraded by miR34a and miR200a. SIRT1 activity is avoiding deleterious impact of DNA damage on key oncogenes, enhanced by AROS but inhibited by DBC1. given that transformation is accompanied with increased production of reactive oxygen species (ROS) and oxidative DNA damage.79,80 On the other hand, increased DNA repair by SIRT1 and development of CML-like myeloid leukemia in a mouse model could lead to more mutations in cancer cells and further genomic in the BALB/c strain. In this model, bone marrow cells were instability for cancer evolution. This is supported by a recent harvested from adult wild-type and SIRT1 À / À mice and report by Wang et al.76, showing that SIRT1 promotes de novo transduced by BCR-ABL in vitro, followed by transplantation to acquisition of genetic mutations for drug resistance in CML and lethally irradiated syngenic recipients. The effect of SIRT1 prostate cancer cells upon therapeutic (by tyrosine kinase knockout on leukemogenesis is thus not influenced by SIRT1 inhibitors) and/or genotoxic (by camptothecin) stress. SIRT1 loss on mice, but is a result of cell autonomous impact on promotion of mutation acquisition is associated with its ability leukemia cells. Pharmacological inhibition of SIRT1 also inhibits to increase error-prone DNA damage repair in cancer cells, in leukemia development to a similar extent as SIRT1 knockout. This particular, through deacetylating the key NHEJ repair factor Ku70. study provides the first genetic evidence for a causal role of Wang et al.76 proposed that the unusual effect of SIRT1 in dysregulated SIRT1 expression in efficient oncogenic trans- mutation promotion may be attributed to the compromised DNA formation and leukemia progression. repair fidelity in cancer cells,81,82 and thus new genetic mutations In another study, Herranz et al.75 crossed SIRT1 transgenic mice may arise as a consequence of misrepair under stress. to PTEN þ / À mice, and found that SIRT1 overexpression increased Genomic instability is one of the most critical enabling incidence of thyroid carcinomas and their lung metastasis, characteristics of cancer. As cancer progresses toward higher- suggesting that increased SIRT1 gene expression has a direct grade malignancy, more mutations are acquired, but mechanisms role in promoting tumor progression. These authors found that for such increasing mutation acquisition are not well under- SIRT1 enhanced Myc stability, although they did not directly link stood.15 The new findings by Wang et al.76 may suggest a deacetylation of Myc to protein stability. Furthermore, Herranz previously underestimated pathway for cancer genomic instability et al.75 showed that PTEN þ / À SIRT1 transgenic mice also and evolution, that is, to accumulate mutations through enhanced developed prostate carcinomas in situ, whereas PTEN þ / À or infidelity DNA repair, which has important biological significance SIRT1 transgenic cohort mice did not, indicating that SIRT1 for cancer drug resistance. SIRT1 appears to be at the center overexpression may also have a role in promoting tumor initiation. of such an evolution pathway;83 however, more detailed This study provides additional genetic evidence that SIRT1 mechanisms still need to be worked out in the future. activation can facilitate tumorigenesis. The above studies ostensibly contrast other mouse genetic studies suggesting roles of SIRT1 in tumor suppression, as SIRT1 promotes cancer hallmark capability described above. The precise mechanisms for this difference are Cancer evolves not only with accumulation of genetic alterations, not clear, but recent studies raise the possibility that SIRT1 might but also with profound epigenetic changes. Cancer cells display differentially regulate genomic stability in normal versus cancer global genomic DNA hypomethylation with concurrent hyper- cells (Figure 3). In normal cells, loss of SIRT1 causes defective DNA methylation at tumor suppressor gene loci. In addition, cancer repair leading to genomic instability. Intriguingly, in cancer cells, cells display gross aberrant histone modification patterns relative SIRT1 knockdown also reduces the efficiency of DNA damage to normal cells.84 In addition to SIRT1 directly deacetylating

Oncogene (2014) 1609 – 1620 & 2014 Macmillan Publishers Limited Functions of sirtuins in cancer M Roth and WY Chen 1613 histone substrates to affect the epigenetic state, SIRT1 can drugs. Another group reported that a novel SIRT1 inhibitor, deacetylate DNA methyltransferase 1 and can either enhance or Amurensin G, reduces expression of multidrug resistance 1 and hinder its methyltransferase activity,85 thus indirectly affecting sensitizes otherwise resistant cells to cytotoxic drugs.106 global or local DNA methylation patterns. SIRT1 is a component of the polycomb repressor complex (PRC), which is involved in silencing genes during normal development. In cancer cells, PRC is Cancer hallmark 2: sustaining proliferation signaling. As discussed also involved in silencing tumor suppressor genes.86,87 above, SIRT1 is activated by MYC and forms a positive feedback The catalytic subunit of the PRC II complex is EZH2, which is loop to stabilize MYC protein in most studies. Stabilized MYC responsible for trimethylation of H3K2788 and itself a noted proto- further enables cell proliferative signaling and stimulates cell oncogene frequently mutated in human cancers.89 SIRT1 directly growth. This likely explains why Burkitt’s lymphoma, which is complexes with EZH2 and other components of the initiated by c-MYC transformation, is most sensitive to the SIRT1 inhibitor cambinol used as a single agent.104 SIRT1 is also shown PRC complex and is an integral part of the PRC’s silencing 107 functions.90 Kuzmichev et al.45 identified a SIRT1-containing to regulate WNT signaling, and WNT activation is associated with transcriptional activation of c-MYC in high-grade malignancy PRC complex, termed PRC4, which is specifically found in 108 transformed cells and embryonic stem cells. Inhibition of SIRT1 of colorectal cancer in the serrated route. has been shown to reactivate silenced tumor suppressor genes.91 In addition, SIRT1 interacts and deacetylates the SUV39H1 Cancer hallmark 3: evading growth suppressors. As described methyltransferase, promoting histone H3 methylation and above, SIRT1 deacetylates and inactivates p53, a negative cell 92 fostering heterochromatin formation and repressing ribosomal cycle regulator, to bypass growth arrest. Another key tumor RNA transcription to protect cells from energy deprivation- suppressor family that is deacetylated by SIRT1 is the FOXO.109–112 93 dependent apoptosis. The dysregulated epigenome by SIRT1 FOXO transcription factors are involved in cell cycle control113 as in cancer cells may act in concert with genetic alterations to well as antioxidant and DNA-damage repair pathways.114 Motta facilitate cancer evolution and reach the biological endpoint et al.110 showed that SIRT1 deacetylation of FOXO3a suppresses 15 hallmarks. This section will discuss the cancer hallmarks that are FOXO3-mediated cell apoptosis induction, in parallel to p53 affected by SIRT1. inhibition. Wang et al.115 showed that deacetylation of FOXO3 by SIRT1 or SIRT2 results in its ubiquitination and subsequent Cancer hallmark 1: resisting cell death. SIRT1 regulates multiple degradation, thereby facilitating cell cycle progression and cell death pathways. SIRT1 deacetylates p53 at lysine 382, contributing to the promotion of cancer. reducing its transcriptional activity and leading to a blockade of p53-dependent apoptosis in response to DNA damage signals.94,95 Similar patterns are observed for p73, which is part of the p53 Cancer hallmark 4: inducing angiogenesis. It is known that SIRT1 family.96 Leukemia stem cells are typically refractory to controls endothelial angiogenic functions through deacetylating chemotherapy. Recently, Li et al.40 have demonstrated that FOXO1 and notch1 intracellular domain during vascular 116,117 inhibiting SIRT1 in CD34 þ CML stem/progenitor cells results in growth. SIRT1 enhances tumor angiogenesis through nega- enhanced p53 acetylation, transcriptional activity and apoptosis of tively modulating d-like ligand 4 (DLL4)/notch signaling in Lewis 118 these cells, and sensitizes them to the BCR-ABL inhibitor, imatinib, lung carcinoma xenograft-derived vascular endothelial cells. both in vitro and in vivo. This effect is dependent on p53, as cells SIRT1 also activates endothelial nitric oxide synthase by with p53 knockdown fail to respond to SIRT1 inhibition. This study deacetylation to enhance nitric oxide production and improve 119 reveals a novel drug resistance mechanism mediated by SIRT1 in vascular function. leukemia stem cells. The intrinsic apoptotic pathway is tightly orchestrated by Cancer hallmark 5: activating invasion and metastasis. Epithelial- the BCL-2 family, which contains pro- and anti-apoptotic 97 to-mesenchymal transition (EMT) is an essential process to promote members, of which Bax is the prototypical pro-apoptotic gene. cancer invasion and metastasis. It has been shown that SIRT1 is Cancer cells can resist apoptosis by sequestering Bax from activated during EMT-like transformation of mammary epithelial mitochondria. This is accomplished through the non-canonical 98 cells, in part due to epigenetic silencing of miR-200a, which action of Ku70. SIRT1 regulates Bax sequestration through negatively regulates SIRT1.32 SIRT1 is also a positive regulator of deacetylation of Ku70, thereby increasing Ku70 interaction with 120 99 EMT and metastasis of prostate cancer. Overexpression of SIRT1 Bax and blocking Bax translocation to mitochondria. By induces EMT through ZEB1 in prostate cancer modulating Ku70, SIRT1 inhibition induces apoptosis in leukemia cells; SIRT1 knockdown restores cell-cell adhesion and reverses EMT cells lacking p53 in vitro and suppresses growth of tumor of prostate cancer cells in vitro and in vivo.120 xenograft in vivo.39 The transcriptional regulator BCL6 is also subjected to deacetylation by SIRT1.100 BCL6 is expressed in the germinal Cancer hallmark 6: deregulating cellular energetics and tumor center within lymph nodes and represses genes that are involved microenvironment. The HIF-1 and HIF-2 are activated in cancer in cell cycle regulation, apoptosis and differentiation.101 cells because of chronically low oxygen tension in the tumor bulk. Translocations involving BCL6 are a frequent event in B-cell HIF-1 activates numerous genes that promote angiogenesis, lymphomas.102,103 Deacetylation by SIRT1 promotes BCL6 survival and glucose uptake, all necessary for tumor growth. transcriptional repression activity and resists cell apoptosis.100 HIF-1 is emerging as a major player in metastasis, chemo- and Heltweg et al.104 demonstrate that a chemical inhibitor of SIRT1, radiotherapy resistance.121 HIF-1a is the regulatory subunit of cambinol, has anti-lymphoma properties in vitro and in vivo in part HIF-1 and is subjected to post-translational modification.122,123 Lim by preventing the deacetylation and activation of BCL6. et al.124 showed that SIRT1 can deacetylate HIF-1a and repress its As described above, SIRT1 can promote cancer cell death biological function in vitro and in tumor xenografts. However, such resistance by facilitating acquisition of genetic mutations.76 In effect of SIRT1 on HIF-1a has been recently disputed by Laemmle addition, SIRT1 promotes chemotherapy resistance by enhancing et al.,125 showing that SIRT1 stabilizes HIF-1a under hypoxia and efflux of drugs. Chu et al.105 report that SIRT1 is elevated in drug- SIRT1 inhibition impairs hypoxic response of hepatocellular resistant cell lines as well as in patient samples. SIRT1 increases the carcinoma cells. The study by Laemmle et al.,125 is in line with a expression of multidrug resistance 1 in cancer cells, whereas previous report demonstrating that SIRT1 helps cells survive inhibition of SIRT1 sensitizes these cancer cells to anticancer against hypoxic environment by activating HIF-2a.126

& 2014 Macmillan Publishers Limited Oncogene (2014) 1609 – 1620 Functions of sirtuins in cancer M Roth and WY Chen 1614 Summary of SIRT1 and cancer accumulation and promotes apoptosis of cancer cells.138,139 152 All the data so far suggest that SIRT1 has both pro- and anticancer Recently, Liu et al. showed that SIRT2 is activated by N-MYC roles. We propose that SIRT1 may act as a genome caretaker in in neuroblastoma cells and by c-MYC in pancreatic cancer cells. normal cells, and therefore suppress tumorigenesis. However, Activated SIRT2 stabilizes N-MYC and c-MYC protein and promotes upon oncogenic events, tumor cells co-opt SIRT1-regulated cell proliferation through downregulating ubiquitin-protein ligase 152 cellular pathways to promote unabated proliferation, progression, NEDD4. Liu et al. also found that SIRT2 increases Aurora A 142 resisting death signals and genetic/epigenetic evolution. expression in these cells, in contrast to the finding by Kim et al. Such dual roles in tumorigenesis are not unprecedented. For In this regard, it is worthy to note that Aurora A has bifurcated examples, telomerase reverse transcriptase (TERT) can both hinder roles in cancer with either gene overexpression or knockout 153,154 and promote tumor formation;16 Transforming growth factor-b promoting tumorigenesis. More studies are needed to can be antiproliferative, but is redirected toward promoting EMT further delineate the precise roles of SIRT2 in cancer. and high-grade malignancy at the later stages of tumorigenesis;127 aberrant DNA hypermethylation by DNA methyltransferase 1 promotes tumorigenesis by silencing tumor suppressor genes, but SIRT3 DNA methyltransferase 1 deletion or expression of a hypomorphic Biochemical overview 128,129 allele in mice results in tumorigenesis. As such, the precise SIRT3 possesses NAD þ -dependent deacetylase as well as ADP- role of SIRT1 in tumorigenesis may be dependent on cellular and ribosyltransferase activity. SIRT3 is the major mitochondrial deace- molecular contexts. tylase with a broad range of substrates.155 Although principally localized to mitochondria, SIRT3 can be detected in the nucleus as well.156 There are two forms of SIRT3, and processing of the SIRT2 N-terminus results in a smaller protein that localizes to Biochemical overview mitochondria.157,158 Within the nucleus, SIRT3 deacetylates H4K16 SIRT2 shares certain biochemical parallels with SIRT1. SIRT2 has and H3K9 to repress transcription.156 In mitochondria, SIRT3 thus far been characterized primarily as an NAD þ -dependent regulates metabolism, energy homeostasis, thermogenesis and protein deacetylase. Although being mostly cytoplasmic, SIRT2 mitochondrial biogenesis. For a more thorough review on SIRT3 can translocate to the nucleus where it deacetylates H4K16 during and metabolic regulation, we refer the readers to other mitosis.130,131 SIRT2 has also been shown to deacetylate H3K56.132 reviews.159,160 SIRT3 increases fatty acid b-oxidation by Much fewer non-histone substrates have thus far been identified deacetylating, and therefore enhancing the activity of long-chain for SIRT2. a-tubulin is the first known SIRT2 substrate,133 but the acyl CoA dehydrogenase.161 The catabolism of fatty acids by b- exact biological role of tubulin acetylation/deacetylation remains oxidation yields acetyl CoA, which gets further metabolized by the elusive. Sharing substrates with SIRT1, SIRT2 also deacetylates Kreb’s cycle. SIRT3 regulates several components of the Kreb’s cycle FOXO1,134,135 FOXO3115,136 and p53137–139 for regulating auto- and electron transport chain to promote adenosine triphosphate phagy, apoptosis and differentiation. Most recently, SIRT2 is production.160,162 This is punctuated by the phenotype seen in SIRT3 shown to regulate programmed necrosis by targeting receptor- knock-out mice that show reduced cellular adenosine triphosphate interacting proteins.140 Mitotic regulation is another biological levels as well as increased ROS.162 Uncontrolled ROS in the cell function frequently ascribed to SIRT2, as detailed in a previous results in DNA and protein damage, aging and cancer.163 review.141 Kim et al.142 described that SIRT2 regulates mitosis by deacetylating components of the anaphase-promoting complex/ SIRT3 and cancer cyclosome ubiquitin ligase machinery to enhance its activity, SIRT3 acts like a tumor suppressor as evidenced by increased directing degradation of aurora kinases as cells exit mitosis. In the 164 dietary setting, the intra-adipose tissue hypoxia and mammary tumor formation in SIRT3 knock-out mice. One major activation of HIF-1a results in transcriptional repression of SIRT2, tumor suppression mechanism of SIRT3 is through modulation of ROS. As noted above, SIRT3 À / À cells show increased ROS due to leading to increased acetylation of PGC-1a, and affecting 162,164,165 b-oxidation and mitochondrial gene expression.143 faulty electron transport. Another means by which SIRT3 regulates ROS is through the activation of antioxidant enzymes, such as superoxide dismutase that is a major mitochondrial SIRT2 and cancer detoxifying enzyme.166,167 Further, Someya et al.168 revealed that Analogous to the bifurcated results with SIRT1 in cancer, SIRT2 has SIRT3 lowers ROS by deacetylation and activation of mitochondrial both roles in tumor suppression and promotion. SIRT2 is reported isocitrate dehydrogenase 2, which in turn increases NADPH levels to be a tumor suppressor in gliomas144,145 and melanomas,146 and the abundance of active glutathione, a major cellular with chromosomal loss at the SIRT2 locus and point mutations antioxidant. Deacetylation and activation of FOXO3a by SIRT3 also within the catalytic domain that abrogates its enzymatic activity. promotes its activation of antioxidant enzymes.169 Yet another SIRT2 expression is reduced in certain cancers,142,145,147 and gene mechanism whereby SIRT3 reduces ROS, and therefore silencing by histone deacetylation is associated with SIRT2 tumorigenesis is in its capacity to destabilize HIF-1a.165,170 Because downregulation in glioma cells.148 However, forced SIRT2 of SIRT3’s effect in regulating oxidative phosphorylation and HIF-1, expression only moderately reduces proliferation of glioma SIRT3 À / À cells may be further prone to cancer by shifting its cells.149 The direct support of SIRT2’s tumor suppressor role is metabolism toward aerobic glycolysis, a phenomenon termed the provided by Kim et al.,142 showing that SIRT2 knock-out mice Warburg effect, which is thematic of most cancer cells.171,172 The develop tumors in various organs due to abnormal chromosomal studies with SIRT3 knock-out mice lend credence to the notion that segregation and aneuploidy caused by increased expression of SIRT3 acts to protect against tumorigenesis. However, it is unclear mitotic regulators including aurora kinases upon SIRT2 knockout. why SIRT3 knockout only increases mouse mammary tumors, given On the other hand, it is observed that SIRT2 may promote that these pathways are not mammary gland-specific. oncogenic phenotypes. SIRT2 is increased in acute myeloid In human cancers, Kim et al.164 showed that SIRT3 protein and leukemia cells compared with normal bone marrow cells, and mRNA are reduced in breast cancer. Consistently, Bell et al.165 SIRT2 inhibition causes apoptosis of acute myeloid leukemia cells showed that knockdown of SIRT3 in human cancer cells increases in vitro.150 Hou et al.151 showed that SIRT2 expression positively tumors size and reduces latency when injected into mice. correlates with cortactin in high-grade prostate cancer samples Conversely, forced expression of SIRT3 blocks proliferation and and predicts poor prognosis. Knockdown of SIRT2 induces p53 reduces tumor xenografts. Finley et al.170 found that about 20% of

Oncogene (2014) 1609 – 1620 & 2014 Macmillan Publishers Limited Functions of sirtuins in cancer M Roth and WY Chen 1615 all human cancer samples and 40% of breast and ovarian cancer SIRT6 samples contain deletions of SIRT3. Biochemical overview Recently, a novel tumor suppressor function of SIRT3 was þ 173 SIRT6 is a chromatin-bound NAD -dependent deacetylase and uncovered in its ability to deacetylate the proto-oncogene, Skp2. ADP-ribosyltransferase.189 Its major histone substrates are H3K9 Skp2 is overexpressed in a wide range of cancers, and its expression and H3K56.190,191 Michisita et al.190 showed that SIRT6 associates levels foretell a poor clinical prognosis. Skp2 functions biochemically with and deacetylates H3K9 to maintain proper as an E3 ubiquitin ligase and is responsible for targeting numerous telomeric chromatin, and disruption of functions likely tumor suppressors such as p21 and p27 for proteasome-mediated 174 accounts for the premature aging phenotype observed in degradation. Deacetylation of Skp2 by SIRT3 leads to its nuclear SIRT6 À / À mice.192 SIRT6 has an integral role in DNA repair import where Skp2 is excluded from targeting E-cadherin. In contrast, pathways including BER,192 HR193,194 and NHEJ.195,196 SIRT6 can acetylated Skp2 gets exported to the cytoplasm where it 173 ADP-ribosylate PARP1, leading to its activation and promotion of ubiquitinates E-cadherin for proteasome-mediated degradation. double-strand break repair.196 Intriguingly, overexpression of Reduced E-cadherin is observed in many cancers and is a feature of 175 SIRT6, but not SIRT1, increases NHEJ and HR repair efficiency in EMT and cancer metastases. Together, these studies indicate that nonmalignant cells.196 Kawahara et al.197 demonstrated that SIRT6 SIRT3 suppresses tumorigenesis. is recruited with NF-kB to deacetylate H3K9 and silence its Yet, as with SIRT1 and SIRT2, there are opposing studies that 176 target genes. SIRT6 is also a corepressor of HIF1 and represses propose a tumor-promoting role for SIRT3. Alhazzazi et al. expression of the genes involved in energy metabolism by showed that SIRT3 levels are elevated in oral squamous carcinoma deacetylating H3K9.198 cell lines and patient samples, and that knockdown of SIRT3 in OSCC lines reduces cell viability and proliferation as well as tumors in xenografts. Ectopic SIRT3 expression reverses p53-induced cell SIRT6 and cancer cycle arrest and senescence, which would provide a mechanism 177 Forced expression of SIRT6 induces apoptosis of cancer cells for a tumor-promoting role of SIRT3. Like SIRT1, SIRT3 can 199 deacetylate Ku70 and thereby facilitate its interaction with Bax to but not in nontransformed cells. SIRT6-mediated apoptosis mitigate Bax-mediated apoptosis.178 These studies, albeit few and of cancer cells requires its ADP-ribosyltransferase, but not deacetylase, activity, as well as the intact ataxia telangiectasia contradictory, provide some evidence that SIRT3 may have a cell- 199 protective role under stress and may give cancer cells a growth mutated (ATM) and p53 pathways. Mice with constitutive SIRT6 advantage. knockout display genetic instability and die of a premature aging syndrome B25 days postnatally.192 Using a conditional gene knock-out strategy, Sebastian et al.200 very recently demonstrated SIRT4 a role of SIRT6 in tumor suppression. These authors revealed that SIRT4 is a mitochondrial ADP-ribosyltransferase without recog- loss of SIRT6 is sufficient to transform immortalized mouse embryonic fibroblasts. Conditional knockout of SIRT6 in the nized deacetylase activity. Its primary function thus far uncovered min/ þ is in regulation of metabolic function. In vitro, SIRT4 can intestine of APC mice results in increased tumor incidence, ADP-ribosylate histones.179 SIRT4 ADP-ribosylates glutamate in part by derepression of Myc activity and activation of aerobic dehydrogenase and inhibits its catalytic activity. Glutamate glycolysis. They further showed that SIRT6 is frequently deleted dehydrogenase catalyzes the conversion of glutamate to and its expression is significantly reduced in human cancer a-ketoglutarate.179,180 Yang et al.181 reported that SIRT3 and samples. Together with SIRT6’s roles in genome maintenance, the SIRT4 are required for cell viability in response to genotoxic stress data so far suggest that SIRT6 may act as a tumor suppressor. along with nicotinamide phosphoribosyltransferase to maintain NAD þ levels within mitochondria. To date, there are no systematic studies evaluating the role of SIRT7 SIRT4 in cancer, although Bradbury et al.41 revealed that SIRT4 Biochemical overview levels are consistently down in acute myeloid leukemia samples. SIRT7 is primarily a deacetylase and is localized to the nucleolus as Speculatively, as SIRT4 regulates a-ketoglutarate production 3 from glutamate, we wonder what role SIRT4 may have on well as the nucleus. Within nucleoli, SIRT7 regulates ribosomal DNA gene expression in part by activating RNA polymerase I.201 a-ketoglutarate-dependent enzymes that have been shown to be 202 involved in tumorigenesis, such as the TET enzymes and jmjC SIRT7 deacetylates H3K18, specifically, to repress transcription. 182,183 SIRT7 is also shown to deacetylate p53 in vitro and in vivo, histone demethylases. Metabolic dysfunction leading to 203 abnormal epigenetic regulation and ultimately tumorigenesis has and SIRT7 ablation increases p53-mediated apoptosis. a precedent, as is seen with isocitrate dehydrogenase mutations in several types of cancers.184,185 SIRT7 and cancer Several groups showed that SIRT7 is elevated in human cancers SIRT5 of breast,204 thyroid205 and liver206 origin. Ford et al.201 Mitochondrial SIRT5 is perhaps the most unusual of sirtuins, as it demonstrated that knockdown of SIRT7 inhibits cell growth and possess NAD þ -dependent deacetylase as well as deacylase induces apoptosis. Barber et al.202 showed that knockdown of (demalonylase and desuccinylase) activities.4 The best studied SIRT7 in cancer cell lines reduces cell growth both in vitro and substrate of SIRT5 is carbamoyl phosphate synthetase 1 for the in vivo, and demonstrated a novel oncogenic mechanism of SIRT7 rate-limiting step in urea synthesis. Several groups have demon- by deacetylating H3K18. Interestingly, many different cancers strated that SIRT5 can deacetylate, demalonylate and desucciny- display global hypoacetylation of H3K18, which is associated with late carbamoyl phosphate synthetase 1.4,186,187 Deacetylation of a poor prognosis in prostate cancers.207,208 Viral oncogenes can carbamoyl phosphate synthetase 1 activates its enzymatic activity, stimulate hypoacetylation of H3K18.209,210 Consistently, Barber which promotes the production of urea and clearance of excessive et al.202 showed that knockdown of SIRT7 abolishes adenoviral ammonia from the cell. The biological significance of E1A-mediated proliferation and transformation of fibroblasts. demolonylation and desuccinylation of carbamoyl phosphate These studies point to a growth advantage that SIRT7 renders synthetase 1 by SIRT5 remains unknown.188 To date, there are no cancer cells. Further in vivo studies should be undertaken to studies evaluating the role of SIRT5 in tumorigenesis. evaluate roles of SIRT7 in cancer in the future.

& 2014 Macmillan Publishers Limited Oncogene (2014) 1609 – 1620 Functions of sirtuins in cancer M Roth and WY Chen 1616 CONCLUDING REMARKS 17 Vaquero A, Scher M, Lee D, Erdjument-Bromage H, Tempst P, Reinberg D. Studies of sirtuins are rapidly growing in the field of cancer and Human SirT1 interacts with histone H1 and promotes formation of facultative other diseases. Sirtuins are generally involved in stress response, heterochromatin. Mol Cell 2004; 16: 93–105. DNA damage repair and metabolism. SIRT1, SIRT2 and SIRT3 18 Vaquero A. The conserved role of sirtuins in chromatin regulation. Int J Dev Biol appear to have both roles in tumor inhibition and promotion. 2009; 53: 303–322. Despite the controversy, it appears that SIRT1 has a consistent role 19 Guarente L. Sir2 links chromatin silencing, metabolism, and aging. Genes Dev 2000; 14: 1021–1026. in mediating cancer cell survival, in particular for drug resistance, 20 Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G et al. and that SIRT6 has more pronounced roles in tumor suppression. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a Currently, both sirtuin activators and inhibitors have been common hallmark of human cancer. Nat Genet 2005; 37: 391–400. developed or under development, and they may be suited for 21 Houtkooper RH, Pirinen E, Auwerx J. Sirtuins as regulators of metabolism and 211 distinct therapeutic purposes. For cancer treatment, inhibitors healthspan. Nat Rev Mol Cell Biol 2012; 13: 225–238. like tenovin-6212 and cambinol,104 which targets both SIRT1 and 22 Saunders LR, Verdin E. Sirtuins: critical regulators at the crossroads between SIRT2, have shown antitumor effects in vivo. However, sirtuin cancer and aging. Oncogene 2007; 26: 5489–5504. modulators are in general not specific and potent enough. Given 23 McBurney MW, Yang X, Jardine K, Hixon M, Boekelheide K, Webb JR et al. The the complex roles of each sirtuin, future effort is needed to mammalian SIR2alpha protein has a role in embryogenesis and gametogenesis. develop more selective sirtuin modulators for human use to treat Mol Cell Biol 2003; 23: 38–54. cancer or other diseases. We expect to see continued rapid growth 24 Cheng HL, Mostoslavsky R, Saito S, Manis JP, Gu Y, Patel P et al. Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice. Proc in sirtuin research in coming years, which will help us better Natl Acad Sci USA 2003; 100: 10794–10799. understand functions of this family of proteins for the good of 25 Wang RH, Sengupta K, Li C, Kim HS, Cao L, Xiao C et al. Impaired DNA damage human health. response, genome instability, and tumorigenesis in SIRT1 mutant mice. Cancer Cell 2008; 14: 312–323. 26 Oberdoerffer P, Michan S, McVay M, Mostoslavsky R, Vann J, Park SK et al. SIRT1 CONFLICT OF INTEREST redistribution on chromatin promotes genomic stability but alters gene The authors declare no conflict of interest. expression during aging. Cell 2008; 135: 907–918. 27 Firestein R, Blander G, Michan S, Oberdoerffer P, Ogino S, Campbell J et al. The SIRT1 deacetylase suppresses intestinal tumorigenesis and colon cancer growth. ACKNOWLEDGEMENTS PLoS One 2008; 3: e2020. We thank the research support from the National Cancer Institute of the 28 Yeung F, Hoberg JE, Ramsey CS, Keller MD, Jones DR, Frye RA et al. Modulation of National Institutes of Health under award number R01 CA143421, and the State of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. California Tobacco Related Disease Research Program (TRDRP) award 20XT-0121 EMBO J 2004; 23: p 2369–2380. to WYC. 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