Sorting out functions of sirtuins in cancer

Abstract

The sirtuins (SIRT 1-7) comprise a family of NAD⁺ dependent protein modifying enzymes with activities in lysine deacetylation, ADP-ribosylation, and/or deacylation. These enzymes are involved in the cell’s stress response systems and in regulating gene expression, DNA damage repair, metabolism and survival. Sirtuins play 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 sirtuin genes, particularly SIRT1, involved in maintaining genome integrity and DNA repair will promote tumorigenesis due to 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.

INTRODUCTION

Sirtuins are mammalian homologs of yeast silent information regulator 2 (Sir2) encoding a histone deacetylase.^{1, 2} The seven mammalian members of sirtuins (termed SIRT1-7) share a conserved catalytic core domain (Fig. 1). Despite this homology, sirtuins have divergent enzymatic activities with SIRT1-3 and SIRT7 primarily as lysine deacetylases, SIRT4 primarily as ADP-ribosyltransferase, SIRT5 as deacetylase and deacylase, and SIRT6 as ADP-ribosyltransferase and deacetylase.^1-5 In addition, primary localization of these proteins in the cell varies significantly, reflecting functional distinction of sirtuin members (See Fig. 1).

Fig. 1.

Comparison of mammalian sirtuins.

The amino acid length of each mature main form of sirtuins is indicated at the end of each protein, with the conserved catalytic domain illustrated in grey. Principal enzymatic activities and primary cellular localization are shown.

Growing interest in sirtuins largely stems from previous studies of yeast Sir2 showing that in lower organisms, increased Sir2 gene dosage is sufficient to extend lifespan,^6-8 and that Sir2 is at a nexus between caloric restriction (CR), resveratrol or other CR mimetics and longevity. ^9,10 More recently, these initial observations have been scrutinized and refined to show a lack of an effect of Sir2 in extending lifespan in lower species as well as in mammals.^11-13 Further adding to the complex nature of the role of sirtuins in extending lifespan, Kanfi et al described that transgenic SIRT6 male mice live approximately 10-15% longer than their wild-type littermates.¹⁴ The conflicting literature on the roles of Sir2 in longevity contributes in part to the confusion of functions of mammalian sirtuins, but may also foretell the complexity of these genes in mammalian cells.

Research in the past decade has revealed the perplexed and often controversial roles of sirtuins in promoting versus suppressing cancer. Cancer cells alter normal cellular machineries to promote unabated cell proliferation and maximize their lifespan, but as a consequence of their malignant growth, they cut short the lifespan of the host organism. Hanahan and Weinberg have elegantly outlined the fundamental mechanisms of cancer promotion as consisting of sustaining proliferative signaling, enabling replicative immortality, activating invasion and metastasis and inducing angiogenesis; meanwhile, as normal cells possess innate mechanisms to antagonize cancer promoting signals, the transformed cells must also overcome tumor suppression mechanisms, namely, by evading growth suppressor signals and resisting cell death.¹⁵ Crucially underlying these hallmarks of cancer is the genetic instability of cancer cells.¹⁵ Although cancer biologists tend to classify genes into either tumor promoting or tumor suppressing, only a limited number of genes unambiguously fall into one of these categories, for examples, MYC as an oncogene and retinoblastoma gene RB as a tumor suppressor gene.¹⁶ Other genes including sirtuins are less apparent, and the tumor promoting or inhibiting properties of the genes may depend on the stages of cancer development and contextual variables such as tissue of origin, the microenvironment and the exact experimental conditions.¹⁵ However, rapid progress has been made most recently in the research on mammalian sirtuins and cancer, which may improve our understanding of these elusive genes and will be highlighted in this review.

SIRT1

Biochemical Overview

SIRT1 shares the greatest homology with yeast Sir2 that was initially characterized as an NAD⁺ dependent histone deacetylase² (so called Class III HDAC that is structurally and biochemically distinct from Class I, II and IV HDACs). SIRT1 deacetylates histone H4 lysine 16 (H4K16) as well as histone H3 lysine 9 and 14 (H3K9 and H3K14, respectively).¹ Additionally, SIRT1 deacetylates histone H1 lysine 26 (H1K26) and is involved in the deposition of histone variants.¹⁷ These modifications of histone tails are closely related to gene silencing and heterochromatin formation that may underlie certain biological processes.^{18, 19} Notably, global genomic hypoacetylation at H4K16 is a hallmark of human cancer cells, both cell lines and clinical samples.²⁰

The biological roles of SIRT1, however, are mostly revealed through its deacetylation of a growing number of non-histone substrates that are involved in a wide variety of cellular functions, particularly in metabolic, oxidative/genotoxic and oncogenic stress responses. These substrates can be broadly categorized as: (1) transcriptional factors p53, FOXO1, FOXO3a, NF-κB, c-MYC, N-MYC, E2F1, and HIF-1α/HIF-2α, for regulating cell cycle progression and promoting survival under various conditions; (2) DNA repair machinery elements Ku70, RAD51, NBS1, APE1, XPA/C and WRN, for improving DNA damage repair; (3) Nuclear receptor, circadian clock and related factors LXR, FXR, ERα, AR, PPARγ, PGC1α, CLOCK, and PER2, for regulating metabolism; (4) histone-modifying enzymes SUV39H1, p300, TIP60 and PCAF, for regulating gene expression; (5) cell signaling molecules STAT3, β-catenin and SMAD7, as detailed in previous reviews.^{21, 22}

SIRT1, genetic stability and tumor suppression

Several studies using mouse models provide evidence that SIRT1 may improve genetic stability and suppress tumor formation (Table 1). SIRT1 is a critical gene for mouse early development. Homozygous deletion of SIRT1 (ΔExon5-6 or Δxon5-7) results in peri- and post-natal lethality in C57BL/6 and 129 strains.^{23, 24} More severe and early embryonic lethality phenotype occurs in another strain with mixed genetic background containing FVB upon SIRT1 deletion (ΔExon5-6).²⁵ In C57BL/6 -129 background, wild type and SIRT1^−/− mouse embryonic stem cells (ESCs) exhibit similar levels of chromosomal abnormality, but SIRT1^−/− ESCs are more prone to genomic instability under oxidative stress.²⁶ In mixed FVB background, SIRT1^−/− early embryos and mouse embryonic fibroblasts display markedly increased spontaneous gross chromosomal abnormalities accompanied with altered global histone modifications.²⁵ The genetic instability in SIRT1^−/− embryonic cells is attributed to defective DNA damage repair characterized by reduced γH2AX foci formation and reduced recruitment of DNA damage repair factors to the foci.^{25, 26} In accord with reduced genetic stability upon SIRT1 loss, SIRT1 heterozygous deletion accelerates tumor formation in p53^+/− knockout mice.²⁵

Table 1.

Mouse cancer phenotypes with sirtuin gene knockout or over-expression

Sirtuin	Genotype	Phenotype	Organ sites of tumors	Role of Sirtuins	Reference
SIRT1	Constitutive deletion of SIRT1	SIRT1 knockout inhibited BCR-ABL transformation and CML development	Blood	cancer promoting	Yuan et al (39)
	SIRT1Tg Pten+/−	SIRT1 transgenic mice had increased incidence of thyroid cancer with lung metastases. SIRT1 expression also increased prostate carcinomas in situ.	Thyroid and prostate	cancer promoting	Herranz et al (75)
	SIRT1+/− p53+/−	Double heterozygous knockout mice had increased mammary tumor incidence	Mammary glands	cancer suppressing	Wang RH et al (25)
	Villin-Cre SIRT1ΔSTOP APCmin/+	Intestinal SIRT1 expression reduced intestinal tumors	Intestine	cancer suppressing	Firestein et al (27)
	Mx-Cre SIRT1ΔSTOP p53+/−	SIRT1 over-expression reduced overall tumor incidence, particularly thymic lymphoma, in p53+/− background	Thymus	cancer suppressing	Oberdoerffer et al (26)
	SIRT1Tg	Transgenic SIRT1 mice had reduced incidence of spontaneous carcinomas and sarcomas, but not lymphoma. SIRT1Tg mice had lower incidence of carcinogen-induced liver carcinomas	Liver, and other epithelial and mesenchymal tissues	cancer suppressing	Herranz et al (11)
SIRT2	Constitutive deletion of SIRT2.	SIRT2−/− female mice developed mammary tumors, while males predominately developed liver cancers and intestinal tumors	Liver and intestine in males and mammary glands in females	cancer suppressing	Kim et al (142)
SIRT3	Constitutive deletion of SIRT3	SIRT3−/− female mice developed mammary tumors with 35% penetrance at about 24 months of age.	Mammary glands	cancer suppressing	Kim et al (164)
SIRT6	Constitutive SIRT6 deletion	Premature aging and death at post- natal week 3	-	-	Mostoslavsky et al (192)
	Villin-Cre SIRT6fl/fl APC+/−	SIRT6−/− mice developed larger intestinal tumors	Intestine	cancer suppressing	Sebastian et al (200)

No tumors have been reported in constitutive SIRT4, SIRT5 and SIRT7 knockout mice.

Using a conditional SIRT1 over-expression allele that is specifically expressed in the cells lining the intestine, Firestein et al. showed that SIRT1 over-expression reduces intestinal tumor formation in APC^+/min mice.²⁷ They demonstrated that SIRT1 deacetylates β-catenin and reduces its nuclear presence and trans-activation potential. Similarly, Oberdoerffer et al showed that SIRT1 conditional over-expression reduces tumor burden in p53^+/− mice.²⁶ In a recent report, Herranz et al undertook a three-year study of SIRT1 transgenic mice with SIRT1 over-expression under its own promoter, and showed that increased SIRT1 expression by 3 fold improves healthy mouse aging and reduces spontaneous carcinomas and sarcomas, as well as carcinogen-induced liver cancer incidence.¹¹ Taken together, in vivo mouse models suggest that SIRT1 may improve genetic stability of normal cells and suppress tumor formation.

In addition to genetic stability, SIRT1 may regulate other pathways that contribute to its tumor suppression function. SIRT1 has been shown to inhibit the NF-κB pathway²⁸ that promotes inflammation, survival and cancer metastasis.²⁹ NF-κB is comprised of heterodimers with p65 and p50 subunits. SIRT1 deacetylates the p65/Rel-A subunit of NF-κB and blocks its transactivation of downstream anti-apoptotic target genes, cIAP-2 and BCL-xL.²⁸ Further in line with tumor suppressor function, SIRT1 acts downstream of BRCA1 to negatively regulate the anti-apoptotic gene, Survivin, by deacetylating H3K9 on its promoter and thereby repressing its transcription. Thus, BRCA1 ablation, via reduced SIRT1, results in elevated Survivin levels and enhances tumor growth.³⁰

Dysregulation of SIRT1 gene expression and enzymatic activity in cancer

Although SIRT1 has tumor suppression function in the mouse studies described above, so far there is no reported functional SIRT1 genetic mutation or deletion, or SIRT1 promoter hypermethylation in human cancer. The Sanger’s Catalogue of Somatic Mutations in Cancer (COSMIC) database registers only 14 uncharacterized point mutations among 4357 unique samples as of December 2012, perhaps as background mutations.

In contrast to rare SIRT1 genetic alterations, numerous studies have revealed that human cancer samples display elevated levels of SIRT1 relative to their non-transformed counterparts. These studies span different cancer types including liver,³¹ breast,³² gastric,³³ prostate^{34, 35} and hematopoietic^36-41 origin. In colorectal cancer, Kabra et al observed reduction of SIRT1 expression⁴², but Nosho et al showed that SIRT1 over-expression correlates with microsatellite instability as well as a CpG Island methylator phenotype.⁴³ Similarly, in mice, over-expression of SIRT1 is reported in leukemia, lymphoma, sarcoma, lung adenocarcinoma and prostate cancer.^{34, 39, 44, 45}

Alteration of SIRT1 gene expression in cancer is mediated by multiple mechanisms affecting transcription, translation and post-translational modifications. At the transcriptional level, tumor suppressor HIC1 directly represses SIRT1 transcription thereby leading to acetylation and activation of another tumor suppressor p53 (ref. 44, and Fig. 2). Loss of HIC1 results in deacetylation of p53 and survival of aged and damaged cells, which may predispose them to cancers.^{46, 47} p53 itself has been shown to directly repress SIRT1 transcription as well.⁴⁸ Conversely, SIRT1 is up-regulated by oncogenic transformation in several settings. Yuan et al have recently shown that transformation of hematopoietic stem/progenitor cells by oncogenic tyrosine kinase BCR-ABL activates SIRT1 transcription in part through STAT5 (signal transducer and activator of transcription 5).³⁹ STAT5 is downstream of numerous cytokine and growth factor receptor signaling cascades and is constitutively active in many cancer types.⁴⁹ STAT5 also acts downstream of BCR-ABL protein in chronic myelogenous leukemia (CML).^{50, 51} STAT5 directly binds the SIRT1 promoter and enhances SIRT1 expression in CML cells³⁹ (See Fig. 2). SIRT1 levels are increased stepwise from normal hematopoietic cells to chronic phase CML to accelerated and blast crisis CML.³⁹ In the same setting, Yuan et al also showed that SIRT1 can be activated by KRAS transformation. Recently, several groups have demonstrated that MYC (c-MYC and N-MYC) proto-oncogenes directly activates SIRT1 transcription.^52-54 This occurs despite incomplete agreement of its effect on MYC protein stability: three groups showing that SIRT1 and MYC form a positive feedback loop in which SIRT1 deacetylates MYC and enhances MYC protein stability; ^{52, 54, 55} whereas another group showing that SIRT1 destabilizes c-MYC protein.⁵³ In addition, the cell cycle and apoptosis regulator E2F1 induces SIRT1 transcription when cancer cells are under genotoxic stress.⁵⁶

Fig 2.

Regulation of SIRT1 gene expression and activities in cancer cells

SIRT1 transcription is activated by Myc, E2F1, or BCR-ABL in part through STAT5, but repressed by HIC1 and p53. The sites for transcriptional factor binding are shown. SIRT1 mRNA is stabilized by HuR but degraded by miR34a and miR200a. SIRT1 activity is enhanced by AROS but inhibited by DBC1.

At the message level, SIRT1 expression is increased by stabilizing its mRNA in cancer cells. MicroRNAs (miRs) are small, RNA polymerase II-transcribed genes that generally form clusters and operate by binding to the 3′ untranslated regions (UTR) of genes and thereby negatively regulating mRNA stability and/or translation.⁵⁷ Several families of miRs have been shown to negatively regulate SIRT1, particularly miR-34a.⁵⁸ The chromosomal region where miR-34a resides is frequently lost in human cancers, while re-introduction of miR-34a into cancer cells 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 by RNA binding protein HuR⁶² that plays a role in promoting tumor cell proliferation and survival.^63-65

SIRT1 protein stability is regulated by phosphorylation, sumoylation and ubiquitination. SIRT1 phosphorylation by cycle-dependent kinase Cyclin B/CDK1 controls cell proliferation.⁶⁶ The dual specificity tyrosine phosphorylation-regulated kinases DYRK1A and DYRK3 phosphorylate SIRT1 and increase SIRT1 enzymatic activity to promote cell survival.⁶⁷ SIRT1 phosphorylation by c-Jun N-ternimal kinase 2 (JNK2) stabilizes the protein,⁶⁸ whereas SIRT1 phosphorylation by JNK1 facilitates ubiqitination-mediated degradation.⁶⁹ Under genotoxic stress, the proapoptotic nuclear desumoylase SENP1 removes SIRT1 sumoylation and reduces its deacetylase activity.⁷⁰

Finally, SIRT1 enzymatic activity could be regulated during tumorigenesis. Deleted in breast cancer 1 (DBC1) is a tumor suppressor that is lost in a portion of breast cancer patients. DBC1 suppresses SIRT1 activity by direct binding to the SIRT1 catalytic core domain.^{71, 72} In contrast, active regulator of SIRT1 (AROS) increases SIRT1 activity through direct interaction with N-terminus of SIRT1 protein.⁷³ Furthermore, increasing SIRT1 expression is concomitant with activation of the mammalian NAD⁺ salvage biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT) in several types of cancer cells to provide sufficient NAD⁺ for SIRT1 functions, which is important for cancer cell survival and stress response.^{52, 74}

SIRT1 promotes cancer genomic instability and cancer evolution

Several recent studies provide crucial insight into the roles of SIRT1 dysregulation in cancer. Yuan et al demonstrated that SIRT1 homozygous knockout significantly inhibits BCR-ABL transformation of mouse bone marrow stem/progenitor cells and development of CML-like myeloid leukemia in a mouse model in the BALB/c strain.³⁹ In this model, bone marrow cells were harvested from adult wild type and SIRT1^−/− mice and transduced by BCR-ABL in vitro followed by transplantation to lethally irradiated syngenic recipients. The effect of SIRT1 knockout on leukemogenesis is thus not influenced by SIRT1 loss on mice, but is a result of cell autonomous impact on leukemia cells. Pharmacological inhibition of SIRT1 also inhibits leukemia development to a similar extent as SIRT1 knockout. This study provides the first genetic evidence for a causal role of dysregulated SIRT1 expression in efficient oncogenic transformation and leukemia progression.

In another study, Herranz et al crossed SIRT1 transgenic (SIRT1^tg) mice to PTEN^+/− mice, and found that SIRT1 over-expression increased incidence of thyroid carcinomas and their lung metastasis,⁷⁵ suggesting that increased SIRT1 gene expression has a direct role in promoting tumor progression. These authors found that SIRT1 enhanced Myc stability although they did not directly link deacetylation of Myc to protein stability. Furthermore, Herranz et al showed that PTEN^+/− SIRT1^tg mice also developed prostate carcinomas in situ, whereas PTEN^+/− or SIRT1^tg cohort mice did not,⁷⁵ indicating that SIRT1 over-expression may also have a role in promoting tumor initiation. This study provides additional genetic evidence that SIRT1 activation can facilitate tumorigenesis.

The above studies ostensibly contrast other mouse genetic studies suggesting roles of SIRT1 in tumor suppression as described above. The precise mechanisms for this difference are not clear, but recent studies raise the possibility that SIRT1 might differentially regulate genomic stability in normal versus cancer cells (Fig. 3). In normal cells, loss of SIRT1 causes defective DNA repair leading to genomic instability. Intriguingly, in cancer cells, SIRT1 knockdown also reduces the efficiency of DNA damage repair including both non-homologous end joining (NHEJ) and homologous recombination (HR) repair in CML cells⁷⁶ and osteosarcoma cells.²⁶ It is shown that SIRT1 is recruited to sites of DNA damage, presumably to remodel local chromatin structure to facilitate repair in cancer cells.^{77, 78} Therefore, it is likely that SIRT1 may play a role in genome maintenance in both normal and cancer cells. However, this may lead to complex outcomes in cancer cells. On one hand, the maintenance by SIRT1 may ensure cancer cell survival and proliferation (see more in next section) by avoiding deleterious impact of DNA damage on key oncogenes, given that transformation is accompanied with increased production of reactive oxygen species and oxidative DNA damage.^{79, 80} On the other hand, increased DNA repair by SIRT1 could lead to more mutations in cancer cells and further genomic instability for cancer evolution. This is supported by a recent report by Wang et al showing that SIRT1 promotes de novo acquisition of genetic mutations for drug resistance in CML and prostate cancer cells upon therapeutic (by tyrosine kinase inhibitors) and/or genotoxic (by camptothecin) stress.⁷⁶ SIRT1 promotion of mutation acquisition is associated with its ability to increase error-prone DNA damage repair in cancer cells, in particular, through deacetylating the key NHEJ repair factor Ku70. Wang et al⁷⁶ proposed that the unusual effect of SIRT1 in mutation promotion may be attributed to the compromised DNA repair fidelity in cancer cells,^{81, 82} and thus new genetic mutations may arise as a consequence of misrepair under stress.

Fig. 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 damage. Activation of DNA repair with high fidelity in normal cells improves genome stability and suppresses tumor formation. 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 non-fatal lesions and more mutations to evolve towards high grade malignancy and drug resistance under chemotherapy.

Genomic instability is one of the most critical enabling characteristics of cancer. As cancer progresses towards higher grade malignancy, more mutations are acquired; but, mechanisms for such increasing mutation acquisition are not well understood.¹⁵ The new findings by Wang et al may suggest a previously underestimated pathway for cancer genomic instability and evolution, that is to accumulate mutations through enhanced infidelity DNA repair, which has important biological significance for cancer drug resistance.⁷⁶ SIRT1 appears to be at the center of such an evolution pathway;⁸³ however, more detailed mechanisms still need to be worked out in the future.

SIRT1 promotes cancer hallmark capability

Cancer evolves not only with accumulation of genetic alterations, but also with profound epigenetic changes. Cancer cells display global genomic DNA hypomethylation with concurrent hypermethylation at tumor suppressor gene loci. Additionally, cancer cells display gross aberrant histone modification patterns relative to normal cells.⁸⁴ In addition to SIRT1 directly deacetylating histone substrates to affect the epigenetic state, SIRT1 can deacetylate DNA methyltransferase 1 (DNMT1) and can either enhance or hinder its methyltransferase activity,⁸⁵ thus indirectly affecting global or local DNA methylation patterns. SIRT1 is a component of the polycomb repressor complex (PRC) that is involved in silencing genes during normal development. In cancer cells, PRC is also involved in silencing tumor suppressor genes.^{86, 87} The catalytic subunit of the PRC II complex is EZH2 that is responsible for trimethylation of H3K27 ⁸⁸ and itself a noted proto-oncogene frequently mutated in human cancers.⁸⁹ SIRT1 directly complexes with EZH2 and other components of the PRC complex and is an integral part of the PRC’s silencing functions.⁹⁰ Kuzmichev et al identified a SIRT1-containing PRC complex, termed PRC4, that is specifically found in transformed cells and ESCs.⁴⁵ Inhibition of SIRT1 has been shown to reactivate silenced tumor suppressor genes.⁹¹ In addition, SIRT1 interacts and deacetylates the SUV39H1 methyltransferase, promoting histone H3 methylation and fostering heterochromatin formation⁹² and repressing rRNA transcription to protect cells from energy deprivation-dependent apoptosis.⁹³ The dysregulated epigenome by SIRT1 in cancer cells may act in concert with genetic alterations to facilitate cancer evolution and reach the biological endpoint hallmarks.¹⁵ This section will discuss the cancer hallmarks that are affected by SIRT1.

Cancer hallmark 1: resisting cell death. SIRT1 regulates multiple cell death pathways. SIRT1 deacetylates p53 at lysine 382, 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 family.⁹⁶ Leukemia stem cells are typically refractory to chemotherapy. Recently, Li et al. have demonstrated that inhibiting SIRT1 in CD34⁺ CML stem/progenitor cells results in enhanced p53 acetylation, transcriptional activity and apoptosis of these cells, and sensitizes them to the BCR-ABL inhibitor, imatinib, both in vitro and in vivo. This effect is dependent on p53 as cells with p53 knockdown fail to respond to SIRT1 inhibition.⁴⁰ This study reveals a novel drug resistance mechanism mediated by SIRT1 in leukemia stem cells.

The intrinsic apoptotic pathway is tightly orchestrated by the BCL-2 family that contains pro- and anti-apoptotic members, of which Bax is the prototypical pro-apoptotic gene.⁹⁷ Cancer cells can resist apoptosis by sequestering Bax from mitochondria. This is accomplished through the non-canonical action of Ku70.⁹⁸ SIRT1 regulates Bax sequestration through deacetylation of Ku70, thereby increasing Ku70 interaction with Bax and blocking Bax translocation to mitochondria.⁹⁹ By modulating Ku70, SIRT1 inhibition induces apoptosis in leukemia cells lacking p53 in vitro and suppresses growth of tumor xenograft in vivo.³⁹

The transcriptional regulator BCL6 is also subjected to deacetylation by SIRT1.¹⁰⁰ BCL6 is expressed in the germinal center within lymph nodes and represses genes that are involved in cell cycle regulation, apoptosis and differentiation.¹⁰¹ Translocations involving BCL6 are a frequent event in B-cell lymphomas.^{102, 103} Deacetylation by SIRT1 promotes BCL6 transcriptional repression activity and resists cell apoptosis.¹⁰⁰ Heltweg et al. demonstrate that a chemical inhibitor of SIRT1, Cambinol, has anti-lymphoma properties in vitro and in vivo in part by preventing the deacetylation and activation of BCL6.¹⁰⁴

As described above, SIRT1 can promote cancer cell death resistance by facilitating acquisition of genetic mutations.⁷⁶ In addition, SIRT1 promotes chemotherapy resistance by enhancing efflux of drugs. Chu et al report that SIRT1 is elevated in drug resistant cell lines as well as in patient samples.¹⁰⁵ SIRT1 increases the expression of multi-drug resistance 1 (MDR1) in cancer cells, while inhibition of SIRT1 sensitizes these cancer cells to anti-cancer drugs. Another group reported that a novel SIRT1 inhibitor, Amurensin G, reduces expression of MDR1 and sensitizes otherwise resistant cells to cytotoxic drugs.¹⁰⁶

Cancer hallmark 2: sustaining proliferation signaling. As discussed above, SIRT1 is activated by MYC and forms a positive feedback loop to stabilize MYC protein in most studies. Stabilized MYC further enables cell proliferative signaling and stimulates cell growth. This likely explains why Burkitt’s lymphoma, that is initiated by c-MYC transformation, is most sensitive to the SIRT1 inhibitor Cambinol used as a single agent.¹⁰⁴ SIRT1 is also shown to regulate WNT signaling,¹⁰⁷ and WNT activation is associated with transcriptional activation of c-MYC in high grade malignancy of colorectal cancer in the serrated route.¹⁰⁸

Cancer hallmark 3: evading growth suppressors. As described above, SIRT1 deacetylates and inactivates p53, a negative cell cycle regulator, to bypass growth arrest. Another key tumor suppressor family that is deacetylated by SIRT1 is the Forkhead transcription factors (FOXO).^109-112 FOXO transcription factors are involved in cell cycle control¹¹³ as well as anti-oxidant and DNA damage repair pathways.¹¹⁴ Motta et al showed that SIRT1 deacetylation of FOXO3a suppresses FOXO3-mediated cell apoptosis induction, in parallel to p53 inhibition.¹¹⁰ Wang et al showed that deacetylation of FOXO3 by SIRT1 or SIRT2 results in its ubiquitination and subsequent degradation,¹¹⁵ thereby facilitating cell cycle progression and contributing to the promotion of cancer.

Cancer hallmark 4: inducing angiogenesis. It is known that SIRT1 controls endothelial angiogenic functions through deacetylating FOXO1 and Notch1 intracellular domain (NICD) during vascular growth.^{116, 117} SIRT1 enhances tumor angiogenesis through negatively modulating Delta-like ligand 4 (DLL4)/Notch signaling in Lewis lung carcinoma xenograft-derived vascular endothelial cells.¹¹⁸ SIRT1 also activates endothelial nitric oxide synthase by deacetylation to enhance nitric oxide production and improve vascular function.¹¹⁹

Cancer hallmark 5: Activating invasion and metastasis. Epithelial-to-mesenchymal transition (EMT) is an essential process to promote cancer invasion and metastasis. It has been shown that SIRT1 is activated during EMT-like transformation of mammary epithelial cells, in part due to epigenetic silencing of miR-200a, which negatively regulates SIRT1.³² SIRT1 is also a positive regulator of EMT and metastasis of prostate cancer.¹²⁰ Overexpression of SIRT1 induces EMT through transcription factor ZEB1 in prostate cancer cells; SIRT1 knockdown restores cell-cell adhesion and reverses EMT of prostate cancer cells in vitro and in vivo.¹²⁰

Cancer hallmark 6: deregulating cellular energetics and tumor microenvironment. The hypoxiainducible transcription factors HIF-1 and HIF-2 are activated in cancer cells due to chronically low oxygen tension in the tumor bulk. HIF-1 activates numerous genes that promote angiogenesis, survival and glucose uptake, all necessary for tumor growth. HIF-1 is emerging as a major player in metastasis, chemo and radiotherapy resistance.¹²¹ HIF-1α is the regulatory subunit of HIF-1 and is subjected to post-translational modification.^{122, 123} Lim et al. showed that SIRT1 can deacetylate HIF-1α and repress its biological function in vitro and in tumor xenografts.¹²⁴ However, such effect of SIRT1 on HIF-1α has been recently disputed by Laemmle et al showing that SIRT1 stabilizes HIF-1α under hypoxia and SIRT1 inhibition impairs hypoxic response of hepatocellular carcinoma cells.¹²⁵ Laemmle’s study is in line with a previous report demonstrating that SIRT1 helps cells survive against hypoxic environment by activating HIF-2α.¹²⁶

Summary of SIRT1 and Cancer

All the data so far suggest that SIRT1 has both pro and anti cancer roles. We propose that SIRT1 may act as a genome caretaker in normal cells and therefore suppress tumorigenesis. However, upon oncogenic events, tumor cells co-opt SIRT1-regulated cellular pathways to promote unabated proliferation, progression, resisting death signals, and genetic/epigenetic evolution. Such dual roles in tumorigenesis are not unprecedented. For examples, TERT can both hinder and promote tumor formation;¹⁶ TGFβ can be antiproliferative but is redirected towards promoting EMT and high-grade malignancy at the later stages of tumorigenesis;¹²⁷ aberrant DNA hypermethylation by DNMT1 promotes tumorigenesis by silencing tumor suppressor genes but DNMT1 deletion or expression of a hypomorphic allele in mice results in tumorigenesis.^{128, 129} As such, the precise role of SIRT1 in tumorigenesis may be dependent on cellular and molecular contexts.

SIRT2

Biochemical Overview

SIRT2 shares certain biochemical parallels with SIRT1. SIRT2 has thus far been characterized primarily as an NAD⁺ dependent protein deacetylase. Although being mostly cytoplasmic, SIRT2 can translocate to the nucleus where it deacetylates H4K16 during mitosis.^{130, 131} SIRT2 has also been shown to deacetylate H3K56.¹³² Much fewer non-histone substrates have thus far been identified for SIRT2. Alpha-tubulin is the first known SIRT2 substrate,¹³³ but the exact biological role of tubulin acetylation/deacetylation remains elusive. Sharing substrates with SIRT1, SIRT2 also deacetylates FOXO1,^{134, 135} FOXO3,^{115, 136} and p53^137-139 for regulating autophagy, apoptosis and differentiation. Most recently, SIRT2 is shown to regulate programmed necrosis by targeting receptor-interacting proteins.¹⁴⁰ Mitotic regulation is another biological function frequently ascribed to SIRT2 as detailed in a previous review.¹⁴¹ Kim et al. described that SIRT2 regulates mitosis by deacetylating components of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase machinery to enhance its activity, directing degradation of Aurora kinases as cells exit mitosis.¹⁴² In the dietary obesity setting, the intra-adipose tissue hypoxia and activation of HIF-1α results in transcriptional repression of SIRT2, leading to increased acetylation of PGC-1a and affecting β-oxidation and mitochondrial gene expression.¹⁴³

SIRT2 and Cancer

Analogous to the bifurcated results with SIRT1 in cancer, SIRT2 has both roles in tumor suppression and promotion. SIRT2 is reported to be a tumor suppressor in gliomas ^{144, 145} and melanomas¹⁴⁶ with chromosomal loss at the SIRT2 locus and point mutations within the catalytic domain that abrogates its enzymatic activity. SIRT2 expression is reduced in certain cancers,^{142, 145, 147} and gene silencing by histone deacetylation is associated with SIRT2 downregulation in glioma cells.¹⁴⁸ However, forced SIRT2 expression only moderately reduces proliferation of glioma cells.¹⁴⁹ The direct support of SIRT2’s tumor suppressor role is provided by Kim et al. showing that SIRT2 knockout mice develop tumors in various organs due to abnormal chromosomal segregation and aneuploidy caused by increased expression of mitotic regulators including Aurora kinases upon SIRT2 knockout.¹⁴²

On the other hand, it is observed that SIRT2 may promote oncogenic phenotypes. SIRT2 is increased in acute myeloid leukemia (AML) cells compared to normal bone marrow cells, and SIRT2 inhibition causes apoptosis of AML cells in vitro.¹⁵⁰ Hou et al. showed that SIRT2 expression positively correlates with Cortactin in high-grade prostate cancer samples and predicts poor prognosis.¹⁵¹ Knockdown of SIRT2 induces p53 accumulation and promotes apoptosis of cancer cells.^{138, 139} Recently, Liu et al showed that SIRT2 is activated by N-MYC in neuroblastoma cells and by c-MYC in pancreatic cancer cells. Activated SIRT2 stabilizes NMYC and c-MYC protein and promotes cell proliferation through downregulating ubiquitin-protein ligase NEDD4.¹⁵² Liu et al also found that SIRT2 increases Aurora A expression in these cells, in contrast to the finding by Kim et al.¹⁴² In this regard, it is worthy to note that Aurora A has bifurcated roles in cancer with either gene over-expression or knockout promoting tumorigenesis.^{153, 154} More studies are needed to further delineate the precise roles of SIRT2 in cancer.

SIRT3

Biochemical Overview

SIRT3 possesses NAD⁺ dependent deacetylase as well as ADP ribosyltransferase activity. SIRT3 is the major mitochondrial deacetylase 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 N-terminus results in a smaller protein that localizes to mitochondria.^{157, 158} Within the nucleus, SIRT3 deacetylates H4K16 and H3K9 to repress transcription.¹⁵⁶ In mitochondria, SIRT3 regulates metabolism, energy homeostasis, thermogenesis and mitochondrial biogenesis. For a more thorough review on SIRT3 and metabolic regulation, we refer the readers to other reviews.^{159, 160} SIRT3 increases fatty acid β-oxidation by deacetylating and therefore enhancing the activity of Long Chain Acyl CoA Dehydrogenase.¹⁶¹ The catabolism of fatty acids by β-oxidation yields acetyl CoA, which gets further metabolized by the Krebs cycle. SIRT3 regulates several components of the Krebs cycle and electron transport chain to promote ATP production.^{160, 162} This is punctuated by the phenotype seen in SIRT3 knockout mice that show reduced cellular ATP levels as well as increased reactive oxygen species (ROS).¹⁶² Uncontrolled ROS in the cell results in DNA and protein damage, aging and cancer.¹⁶³

SIRT3 and Cancer

SIRT3 acts like a tumor suppressor as evidenced by increased mammary tumor formation in SIRT3 knockout mice.¹⁶⁴ One major tumor suppression mechanism of SIRT3 is through modulation of ROS. As noted above, SIRT3^−/− cells show increased ROS due to faulty electron transport.^{162, 164, 165} Another means by which SIRT3 regulates ROS is through activation of anti-oxidant enzymes, such as superoxide dismutase that is a major mitochondrial detoxifying enzyme.^{166, 167} Further, Someya et al revealed that SIRT3 lowers ROS by deacetylation and activation of mitochondrial isocitrate dehydrogenase 2, which in turn increases NADPH levels and the abundance of active glutathione, a major cellular anti-oxidant.¹⁶⁸ Deacetylation and activation of FOXO3a by SIRT3 also promotes its activation of anti-oxidant enzymes.¹⁶⁹ Yet another mechanism whereby SIRT3 reduces ROS and therefore tumorigenesis is in its capacity to destabilize HIF-1α.^{165, 170} Because of SIRT3’s effect in regulating oxidative phosphorylation and HIF-1, SIRT3^−/− cells may be further prone to cancer by shifting its metabolism toward aerobic glycolysis, a phenomenon termed the Warburg effect that is thematic of most cancer cells.^{171, 172} The studies with SIRT3 knockout mice lend credence to the notion that SIRT3 acts to protect against tumorigenesis. However, it is unclear why SIRT3 knockout only increases mouse mammary tumors, given that these pathways are not mammary gland specific.

In human cancers, Kim et al. showed that SIRT3 protein and mRNA are reduced in breast cancer.¹⁶⁴ Consistently, Bell et al. showed that knockdown of SIRT3 in human cancer cells increases tumors size and reduces latency when injected into mice.¹⁶⁵ Conversely, forced expression of SIRT3 blocks proliferation and reduces tumor xenografts. Finley et al. found that about 20% of all human cancer samples and 40% of breast and ovarian cancer samples contain deletions of SIRT3.¹⁷⁰

Recently, a novel tumor suppressor function of SIRT3 was uncovered in its ability to deacetylate the proto-oncogene, Skp2.¹⁷³ Skp2 is over-expressed in a wide range of cancers and its expression levels foretell a poor clinical prognosis. Skp2 functions biochemically as an E3 ubiquitin ligase and is responsible for targeting numerous tumor suppressors such as p21 and p27 for proteasome-mediated degradation.¹⁷⁴ Deacetylation of Skp2 by SIRT3 leads to its nuclear import where Skp2 is excluded from targeting E-cadherin. In contrast, acetylated Skp2 gets exported to the cytoplasm where it ubiquitinates E-cadherin for proteasome-mediated degradation.¹⁷³ Reduced E-cadherin is observed in many cancers and is a feature of EMT and cancer metastases.¹⁷⁵ Together, these studies indicate that SIRT3 suppresses tumorigenesis.

Yet, as with SIRT1 and SIRT2, there are opposing studies that propose a tumor promoting role for SIRT3. Alhazzazi et al showed that SIRT3 levels are elevated in oral squamous carcinoma cell lines and patient samples, and that knockdown of SIRT3 in OSCC lines reduces cell viability and proliferation as well as tumors in xenografts.¹⁷⁶ Ectopic SIRT3 expression reverses p53-induced cell cycle arrest and senescence, which would provide a mechanism for a tumor-promoting role of SIRT3.¹⁷⁷ Like SIRT1, SIRT3 can deacetylate Ku70 and thereby facilitate its interaction with Bax to mitigate Bax-mediated apoptosis.¹⁷⁸ These studies, albeit few and contradictory, provide some evidence that SIRT3 may have a cell protective role under stress and may give cancer cells a growth advantage.

SIRT4

SIRT4 is a mitochondrial ADP-ribosyltransferase without recognized deacetylase activity. Its primary function thus far uncovered is in regulation of metabolic function. In vitro, SIRT4 can ADP-ribosylate histones.¹⁷⁹ SIRT4 ADP-ribosylates glutamate dehydrogenase (GDH) and inhibits its catalytic activity. GDH catalyzes the conversion of glutamate to α-ketoglutarate (α-KG).^{179, 180} Yang et al. reported that SIRT3 and SIRT4 are required for cell viability in response to genotoxic stress along with NAMPT to maintain NAD⁺ levels within mitochondria.¹⁸¹

To date, there are no systematic studies evaluating the role of SIRT4 in cancer. Though, Bradbury et al. revealed that SIRT4 levels are consistently down in AML samples.⁴¹ Speculatively, since SIRT4 regulates α-KG production from glutamate, we wonder what role SIRT4 may play on α-KG-dependent enzymes that have been shown to be involved in tumorigenesis, such as the TET enzymes and jmjC histone demethylases.^{182, 183} Metabolic dysfunction leading to abnormal epigenetic regulation and ultimately tumorigenesis has a precedent as is seen with isocitrate dehydrogenase mutations in several types of cancers.^{184, 185}

SIRT5

Mitochondrial SIRT5 is perhaps the most unusual of sirtuins as it possess NAD⁺ dependent deacetylase as well as deacylase (demalonylase and desuccinylase) activities.⁴ The best studied substrate of SIRT5 is carbamoyl phosphate synthetase 1 (CPS1) for the rate-limiting step in urea synthesis. Several groups have demonstrated that SIRT5 can deacetylate, demalonylate and desuccinylate CPS1.^{4, 186, 187} Deacetylation of CPS1 activates its enzymatic activity that promotes the production of urea and clearance of excessive ammonia from the cell. The biological significance of demolonylation and desuccinylation of CPS1 by SIRT5 remains unknown.¹⁸⁸ To date, there are no studies evaluating the role of SIRT5 in tumorigenesis.

SIRT6

Biochemical Overview

SIRT6 is a chromatin bound NAD⁺ dependent deacetylase and ADP-ribosyltransferase.¹⁸⁹ Its major histone substrates are H3K9 and H3K56.^{190, 191} Michisita et al. showed that SIRT6 associates with telomeres and deacetylates H3K9 to maintain proper telomeric chromatin,¹⁹⁰ and disruption of telomere functions likely accounts for the premature aging phenotype observed in SIRT6^−/− mice.¹⁹² SIRT6 plays an integral role in DNA repair pathways including BER,¹⁹² HR,^{193, 194} and NHEJ.^{195, 196} SIRT6 can ADP-ribosylate PARP1 leading to its activation and promotion of double strand break repair.¹⁹⁶ Intriguingly, over-expression of SIRT6, but not SIRT1, increases NHEJ and HR repair efficiency in non-malignant cells.¹⁹⁶ Kawahara et al. demonstrated that SIRT6 is recruited with NF-κB to deacetylate H3K9 and silence its target genes.¹⁹⁷ SIRT6 is also a co-repressor of HIF1 and represses expression of the genes involved in energy metabolism by deacetylating H3K9.¹⁹⁸

SIRT6 and Cancer

Forced expression of SIRT6 induces apoptosis of cancer cells but not in non-transformed cells.¹⁹⁹ SIRT6-mediated apoptosis of cancer cells requires its ADP-ribosyltransferase, but not deacetylase, activity, as well as the intact ATM and p53 pathways.¹⁹⁹ Mice with constitutive SIRT6 knockout display genetic instability and die of a premature aging syndrome approximately 25 days post-natally.¹⁹² Using a conditional gene knockout strategy, Sebastian et al. very recently demonstrated a role of SIRT6 in tumor suppression.²⁰⁰ These authors revealed that loss of SIRT6 is sufficient to transform immortalized mouse embryonic fibroblasts. Conditional knockout of SIRT6 in the intestine of APC^Min/+ mice results in increased tumor incidence, in part by de-repression of Myc activity and activation of aerobic glycolysis. They further showed that SIRT6 is frequently deleted and its expression is significantly reduced in human cancer samples. Together with SIRT6’s roles in genome maintenance, the data so far suggest that SIRT6 may act as a tumor suppressor.

SIRT7

Biochemical Overview

SIRT7 is primarily a deacetylase and is localized to the nucleolus as well as the nucleus.³ Within nucleoli, SIRT7 regulates ribosomal DNA gene expression in part by activating RNA polymerase I.²⁰¹ SIRT7 deacetylates H3K18, specifically, to repress transcription.²⁰² SIRT7 is also shown to deacetylate p53 in vitro and in vivo, and SIRT7 ablation increases p53-mediated apoptosis.²⁰³

SIRT7 and Cancer

Several groups showed that SIRT7 is elevated in human cancers of breast,²⁰⁴ thyroid²⁰⁵ and liver²⁰⁶ origin. Ford et al. demonstrated that knockdown of SIRT7 inhibits cell growth and induces apoptosis.²⁰¹ Barber et al. showed that knockdown of SIRT7 in cancer cell lines reduces cell growth both in vitro and in vivo, and demonstrated a novel oncogenic mechanism of SIRT7 by deacetylating H3K18.²⁰² Interestingly, many different cancers display global hypoacetylation of H3K18, which is associated with a poor prognosis in prostate cancers.^{207, 208} Viral oncogenes can stimulate hypoacetylation of H3K18.^{209, 210} Consistently, Barber et al. showed that knockdown of SIRT7 abolishes adenoviral E1A-mediated proliferation and transformation of fibroblasts.²⁰² These studies point to a growth advantage that SIRT7 renders cancer cells. Further in vivo studies should be undertaken to evaluate roles of SIRT7 in cancer in the future.

Concluding remarks

Studies of sirtuins are rapidly growing in the field of cancer and other diseases. Sirtuins are generally involved in stress response, DNA damage repair and metabolism. SIRT1, SIRT2 and SIRT3 appear to have both roles in tumor inhibition and promotion. Despite the controversy, it appears that SIRT1 has a consistent role in mediating cancer cell survival, in particular for drug resistance, and that SIRT6 has more pronounced roles in tumor suppression. Currently, both sirtuin activators and inhibitors have been developed or under development, and they may be suited for distinct therapeutic purposes.²¹¹ For cancer treatment, inhibitors like tenovin-6²¹² and cambinol¹⁰⁴ that targets both SIRT1 and SIRT2 have shown anti-tumor effects in vivo. However, sirtuin modulators are in general not specific and potent enough. Given the complex roles of each sirtuin, future effort is needed to develop more selective sirtuin modulators for human use to treat cancer or other diseases. We expect to see continued rapid growth in sirtuin research in coming years that will help us better understand functions of this family of proteins for the good of human health.