Deacetylation of Ku70 by SIRT6 attenuates Bax-mediated apoptosis in hepatocellular carcinoma.

SIRT6 is a class III histone deacetylase that has been implicated in HCC development. We previously reported that SIRT6 potentiated apoptosis evasion in hepatocellular carcinoma by inhibiting both Bax expression and mitochondrial translocalization. However, the mechanism underlying SIRT6-mediated inhibition of Bax mitochondrial localization remains elusive. In this study, we found that although SIRT6 had no effect on the expression level of Ku70, SIRT6 could interact with Ku70 and deacetylate it. The increased acetylation of Ku70 in SIRT6-depleted cells disrupt its interaction with Bax, which finally resulted in Bax mitochondrial translocalization. Furthermore, lysine K542 on Ku70 was the target for deacetylation by SIRT6. Ku70K542Q mutation abolished suppression of association between Ku70 and Bax and caused redistribution of Bax to the cytosol in SIRT6-depleted cells. Finally, Ku70K542Q mutation could reversed the inhibition of growth and apoptosis promotion mediated by SIRT6 silencing. Together, our findings revealed SIRT6 could block the mitochondrial translocation of Bax and decrease the apoptotic ratio of HCC cells by deacetylation of Ku70. SIRT6 may serve as a promising target for developing targeted therapies for HCC in the future.

The SIRT1 inhibitor, nicotinamide, inhibits hepatitis B virus replication in vitro and in vivo.

We previously reported that SIRT1, an NAD(+)-dependent deacetylase belonging to the class III histone deacetylases, enhances hepatitis virus B (HBV) replication by targeting the transcription factor AP-1. However, the potential antiviral effects of nicotinamide, a SIRT1 inhibitor, have not yet been explored. In this study, we show that nicotinamide exhibits potent anti-HBV activity with little cytotoxicity. Nicotinamide suppressed both HBV DNA replicative intermediates and 3.5-kb mRNA expression. Moreover, nicotinamide treatment also suppressed core protein expression and the secretion of the hepatitis B surface antigen (HBsAg) and the hepatitis B e antigen (HBeAg) in HBV-expressing cell models. Importantly, nicotinamide treatment suppressed serum HBV DNA, HBsAg and HBeAg levels and liver HBV DNA in HBV-transgenic mice. Furthermore, using a dual-luciferase reporter assay, it was found that nicotinamide exhibited a marked inhibitory effect on the HBV core, SpI, SpII and X promoters, accompanied by decreased expression of the transcription factors AP-1, C/EBPα and PPARα. Therefore, nicotinamide suppresses HBV replication in vitro and in vivo by diminishing HBV promoter activity. This study highlights the potential application of nicotinamide in HBV therapy.

Sirtuin 1 regulates hepatitis B virus transcription and replication by targeting transcription factor AP-1.

Chronic hepatitis B virus (HBV) infection is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Nevertheless, the molecular mechanism of HBV replication remains elusive. SIRT1 is a class III histone deacetylase that is a structure component of the HBV cccDNA minichromosome. In this study, we found by using microarray-based gene expression profiling analysis that SIRT1 was upregulated in HBV-expressing cells. Gene silencing of SIRT1 significantly inhibited HBV DNA replicative intermediates, 3.5-kb mRNA, and core protein levels. In contrast, the overexpression of SIRT1 augmented HBV replication. Furthermore, SIRT1 enhanced the activity of HBV core promoter by targeting transcription factor AP-1. The c-Jun subunit of AP-1 was bound to the HBV core promoter region, as demonstrated by using a chromatin immunoprecipitation assay. Mutation of AP-1 binding site or knockdown of AP-1 abolished the effect of SIRT1 on HBV replication. Finally, SIRT1 inhibitor sirtinol also suppressed the HBV DNA replicative intermediate, as well as 3.5-kb mRNA. Our study identified a novel host factor, SIRT1, which may facilitate HBV replication in hepatocytes. These data suggest a rationale for the use of SIRT1 inhibitor in the treatment of HBV infection.

SIRT6 Overexpression Potentiates Apoptosis Evasion in Hepatocellular Carcinoma via BCL2-Associated X Protein-Dependent Apoptotic Pathway.

PURPOSE: To characterize the functional role of SIRT6 in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: The expression of SIRT6 in 60 paired paraffin-embedded HCC tissues and adjacent nontumoral liver tissues was examined by immunohistochemistry. The expression of SIRT6 in 101 paired frozen HCC tissues and adjacent nontumoral liver tissues was analyzed by Western blotting analysis and qPCR. The biologic consequences of overexpression and knockdown of SIRT6 in HCC cell lines were studied in vitro and in vivo RESULTS: SIRT6 expression was frequently upregulated in clinical HCC samples, and its expression was highly associated with tumor grade (P = 0.02), tumor size (P = 0.02), vascular invasion (P = 0.004), and shorter survival (P = 0.024). Depletion of SIRT6 from multiple liver cancer cell lines inhibited their growth and induced apoptosis in vitro At the molecular level, we observed that the activation of the BCL2-associated X protein (Bax) signaling pathway, a major pathway that determines cancer cell apoptosis, is regulated by SIRT6 via its deacetylase activity. SIRT6 was recruited to the promoter of Bax, where it deacetylated histone 3 lysine 9 and suppressed its promoter activity. Binding of transcription factors (p53 and E2F-1) to Bax promoter was also generally increased in SIRT6-depleted cells. In mouse xenografts, SIRT6 suppression inhibited tumor growth and induced apoptosis. Finally, there is a negative correlation between SIRT6 and Bax mRNA expressions in human HCC samples. CONCLUSIONS: SIRT6 is an important protumorigenic factor in liver carcinogenesis. Thus, the therapeutic targeting of SIRT6 may offer options for HCC treatment. Clin Cancer Res; 22(13); 3372-82. ©2016 AACR.

Cyclin D2 plays a regulatory role in HBV replication.

Hepatitis B virus (HBV) infection is the leading cause of liver diseases. However, the molecular mechanisms of HBV infection and carcinogenesis have not been fully elucidated. In this study, we found that cyclin D2 was upregualted in HBV-expressing cells and liver tissues of HBV-transgenic mice. Gene silencing of cyclin D2 inhibited HBV DNA replicative intermediates, 3.5 kb mRNA, core protein level, as well as the secretions of HBsAg and HBeAg. On the contrary, overexpression of cyclin D2 promoted HBV replication. Furthermore, cyclin D2 regulated HBV replication by enhancing the activity of HBV core and Sp1 promoters by targeting transcription factor CREB2. Silencing of CREB2 abolished enhancement of HBV replication induced by cyclin D2. Together, our study has uncovered a positive role of cyclin D2 in HBV replication. It is conceivable that therapeutic application of cyclin D2 inhibitor in HBV infection therapy.