Sirtuins are nicotinamide adenine dinucleotide oxidized form (NAD 1 )-dependent deacetylases and function in cellular metabolism, stress resistance, and aging. For sirtuin7 (SIRT7), a role in ribosomal gene transcription is proposed, but its function in cancer has been unclear. In this study we show that SIRT7 expression was up-regulated in a large cohort of human hepatocellular carcinoma (HCC) patients. SIRT7 knockdown influenced the cell cycle and caused a significant increase of liver cancer cells to remain in the G 1 /S phase and to suppress growth. This treatment restored p21, induced Beclin-1, and repressed cyclin D1. In addition, sustained suppression of SIRT7 reduced the in vivo tumor growth rate in a mouse xenograft model. To explore mechanisms in SIRT7 regulation, microRNA (miRNA) profiling was carried out. This identified five significantly down-regulated miRNAs in HCC. Bioinformatics analysis of target sites and ectopic expression in HCC cells showed that miR-125a-5p and miR-125b suppressed SIRT7 and cyclin D1 expression and induced p21 WAF1/Cip1 -dependent G 1 cell cycle arrest. Furthermore, treatment of HCC cells with 5-aza-2 0 -deoxycytidine or ectopic expression of wildtype but not mutated p53 restored miR125a-5p and miR-125b expression and inhibited tumor cell growth, suggesting their regulation by promoter methylation and p53 activity. To show the clinical significance of these findings, mutations in the DNA binding domain of p53 and promoter methylation of miR125b were investigated. Four out of nine patients with induced SIRT7 carried mutations in the p53 gene and one patient showed hypermethylation of the miR-125b promoter region. Conclusion: Our findings suggest the oncogenic potential of SIRT7 in hepatocarcinogenesis. A regulatory loop is proposed whereby SIRT7 inhibits transcriptional activation of p21 WAF1/Cip1 by way of repression of miR-125a-5p and miR-125b. This makes SIRT7 a promising target in cancer therapy. (HEPATOLOGY 2013;57:1055-1067 S irtuins, also designated as class III histone deacetylases, are nicotinamide adenine dinucleotide oxidized form (NAD þ )-dependent deacetylases that target histone and nonhistone proteins and are implicated in the control of a wide range of biological processes such as apoptosis, stress responses, DNA repair, cell cycle, metabolism, and senescence. 1 The importance of sirtuins is demonstrated by their role in several major human pathologic conditions, including cancer, diabetes, cardiovascular disease, and neurodegenerative disease. 2 Mammals express seven sirtuins (denoted SIRT1-7) that have considerably different functions and catalytic activities. 3 The most closely related to yeast Sir2 and the best-characterized sirtuin, Abbreviations: 5-aza-dC, 5-aza-2 0 -deoxycytidine; CDKN1A, cyclin dependent kinase 1A;

Ubiquitin-binding histone deacetylase 6 (HDAC6) is uniquely endowed with tubulin deacetylase activity and plays an important role in the clearance of misfolded protein by autophagy. In cancer, HDAC6 has become a target for drug development due to its major contribution to oncogenic cell transformation. In the present study we show that HDAC6 expression was down-regulated in a large cohort of human hepatocellular carcinoma (HCC) patients, and that low expression of HDAC6 was significantly associated with poor prognosis of HCC patients in 5-year overall, disease-free, and recurrence-free survival. Notably, we observed that ectopic overexpression of HDAC6 suppressed tumor cell growth and proliferation in various liver cancer cells, and elicited increased LC3B-II conversion and autophagic vacuole formation without causing apoptotic cell death or cell cycle inhibition. In addition, the sustained overexpression of HDAC6 reduced the in vivo tumor growth rate in a mouse xenograft model. It was also found that HDAC6 mediated autophagic cell death by way of Beclin 1 and activation of the LC3-II pathway in liver cancer cells, and that HDAC6 overexpression activated c-Jun NH2-terminal kinase (JNK) and increased the phosphorylation of c-Jun. In contrast, the induction of Beclin 1 expression was blocked by SP600125 (a specific inhibitor of JNK) or by small interfering RNA directed against HDAC6. Conclusion: Our findings suggest that loss of HDAC6 expression in human HCCs and tumor suppression by HDAC6 occur by way of activation of caspase-independent autophagic cell death through the JNK/Beclin 1 pathway in liver cancer and, thus, that a novel tumor suppressor function mechanism involving HDAC6 may be amenable to nonepigenetic regulation. (HEPATOLOGY 2012;56:644-657) H epatocellular carcinoma (HCC) is an aggressive form of cancer, the fifth most common cancer, and the third leading cause of cancer death worldwide. 1 Surgery with curative intent is feasible for only 15% to 25% of patients and most HCC patients die from locally advanced or metastatic disease in a relatively short period of time. 2 Hepatitis B virus, hepatitis C virus, and aflatoxin B1 are well-known major causes of HCC. However, the overall survival of patients with HCC has not improved significantly over the past two decades, and the mechanisms responsible for the development and progression of HCC remain poorly understood. 3 To date, molecular targeted therapy has shown promise for the treatment of advanced Abbreviations: 3-MA, 3-methyladenine; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HAT, histone acetyltransferase; HCC, hepatocellular carcinoma; HDAC, histone deacetylases; JNK, c-Jun NH2-terminal kinase; LC3, microtubule-associated protein 1 light chain 3; mRNA, messenger RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly (ADP-ribose) polymerase; siRNA, small interfering RNA; TEM, transmission electron microscopy; TMA, tissue microarray.From the

Histone deacetylase 2 (HDAC2) is crucial for embryonic development, affects cytokine signaling relevant for immune responses and is often significantly overexpressed in solid tumors; but little is known about its role in human hepatocellular carcinoma (HCC). In this study, we showed that targeted-disruption of HDAC2 resulted in reduction of both tumor cell growth and de novo DNA synthesis in Hep3B cells. We then demonstrated that HDAC2 regulated cell cycle and that disruption of HDAC2 caused G1/S arrest in cell cycle. In G1/S transition, targeted-disruption of HDAC2 selectively induced the expression of p16INK4A and p21WAF1/Cip1, and simultaneously suppressed the expression of cyclin D1, CDK4 and CDK2. Consequently, HDAC2 inhibition led to the down-regulation of E2F/DP1 target genes through a reduction in phosphorylation status of pRb protein. In addition, sustained suppression of HDAC2 attenuated in vitro colony formation and in vivo tumor growth in a mouse xenograft model. Further, we found that HDAC2 suppresses p21WAF1/Cip1 transcriptional activity via Sp1-binding site enriched proximal region of p21WAF1/Cip1 promoter. In conclusion, we suggest that the aberrant regulation of HDAC2 may play a pivotal role in the development of HCC through its regulation of cell cycle components at the transcription level providing HDAC2 as a relevant target in liver cancer therapy.

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Histone deacetylase 2 (HDAC2) is crucial for embryonic development, affects cytokine signaling relevant for immune responses, and is often significantly overexpressed in solid tumors, but little is known of its role in human lung cancer. In this study, we demonstrated the aberrant expression of HDAC2 in lung cancer tissues and investigated oncogenic properties of HDAC2 in human lung cancer cell lines. HDAC2 inactivation resulted in regression of tumor cell growth and activation of cellular apoptosis via p53 and Bax activation and Bcl2 suppression. In cell cycle regulation, HDAC2 inactivation caused induction of p21WAF1/CIP1 expression, and simultaneously suppressed the expressions of cyclin E2, cyclin D1, and CDK2, respectively. Consequently, this led to the hypophosphorylation of pRb protein in G1/S transition and thereby inactivated E2F/DP1 target gene transcriptions of A549 cells. In addition, we demonstrated that HDAC2 directly regulated p21WAF1/CIP1 expression in a p53-independent manner. However, HDAC1 was not related to p21WAF1/CIP1 expression and tumorigenesis of lung cancer. Lastly, we observed that sustained-suppression of HDAC2 in A549 lung cancer cells attenuated in vitro tumorigenic properties and in vivo tumor growth of the mouse xenograft model. Taken together, we suggest that the aberrant regulation of HDAC2 and its epigenetic regulation of gene transcription in apoptosis and cell cycle components play an important role in the development of lung cancer.