ZBP-89 Regulates Bak Expression via Epigenetic Mechanism

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, epigenetic therapy may represent an alternative for HCC management. ZBP-89 is known to induce Bak in HCC. However, it is unclear whether epigenetic mechanisms contribute to ZBP-89-mediated Bak.;Histone acetylase 3 (HDAC3) is realized as a chemotherapy target and a biomarker of recurrence in HCC. HDAC3 is frequently overexpressed in HCC and its inhibition enhances the efficacy of anti-HCC chemotherapy. The pilot data have indicated that ZBP-89 reduced HDAC3 in HCC but the mechanism responsible was unknown. The post-translational modification of proteins functions as a key regulatory factor in cellular physiological procedures, such as ubiquitinoylation degradation. As a biomarker of HCC development and recurrence, it is important to understand how ZBP-89 mediates the reduction of HDAC3.;This study focuses on if ZBP-89 regulates Bak expression through epigenetic mechanisms. It is designed to investigate whether DNA methyltransferases (DNMTs), histone acetylases (HDACs) are involved in regulation of ZBP-89-induced Bak expression. The study also elucidates the mechanism how ZBP-89 reduces the level of HDAC3 protein.;Methods and Results: The levels of Bak and ZBP-89 as shown on western blots were reduced but DNMT1 and HDAC3 were increased in HCC cancer tissues compared to the corresponding non-cancer tissues. Co-immunoprecipitation experiments showed that ZBP-89 bound to HDAC3 and DNMT1 but not other epigenetic enzymes, such as HDAC4, DNMT3a and DNMT3b. To clarify if ZBP-89 affects the activities of HDACs and DNMTs, ZBP-89 was overexpressed in HCC cells. Enzyme activities of HDACs and DNMTs were determined using relevant assay kits. Results showed that overexpressed ZBP-89 inhibited the activities of HDACs and DNMTs. Further experiments indicated that ZBP-89-mediated Bak up-regulation might contribute to maintenance of histone H3 and H4 acetylation through inhibition of HDACs activity. In another set of experiments, we also found an increased Bak expression in HCC cells when the cells were treated with HDAC inhibitors (HDACi) VPA and TSA. HDAC3 siRNA also increased Bak expression.;Both knockdown of DNMT1 expression and administration of DNMTs inhibitors (zebularine) induced Bak expression. Chromatin immunoprecipitation (ChIP) showed that ZBP-89 bound to Bak promoter at the region from -3188bp to -3183bp and from -275 to -49. As ZBP-89 inhibits DNMT activity, it is essential to know whether its inhibition affectes DNA CpG methylation status and methyl-CpG binding protein (MeCP) binding. The results showed that ZBP-89 overexpression inhibited MeCP2 binding to genomic DNA. The finding indicated that decreased MeCP2 binding to DNA might be due to decreased methyl-CpG number in Bak promoter, suggesting that ZBP-89 might affect CpG island methylation status. Therefore, the bisulfite modified DNA sequencing method was used to clarify if Bak promoter CpG island methylation status was altered after ZBP-89 overexpression. Results revealed that ZBP-89 overexpression could demethylate the CpG islands in Bak promoter.;ZBP-89 overexpression dose-dependently reduced the expression of HDAC3 at protein level but not at mRNA level. Co-immunoprecipitation and western blot methods were used to analyze Peptidyl-prolyl cis/trans isomerase 1 (Pin1) and HDAC3, phospho-I kappa B (pIkappaB), and the result revealed that HDAC3 could bound with either Pin1 or pIkappaB to promote the reduced expression of HDAC3.;Constructed mU6-siPin1 vector was used to knockdown Pin1 expression in HCC cells. We found that knockdown of Pin1 expression blocked ZBP-89-mediated HDAC3 reduction. Experiments performed in Pin1 allele-knockdown JB6 C141 Pin1 -/- and Pin1+/+ cells showed that the reduction of HDAC3 by ZBP-89 was greater in Pin1+/+ cells than in Pin1 -/- cells, confirming the role of Pin1 in ZBP-89-mediated HDAC3 reduction. Furthermore, the ZBP-89-mediated HDAC3 reduction was suppressed by CAY10576, an IkappaB kinase (IKK) activation inhibitor but not by SN50, a p65/p50 translocation inhibitor, suggesting that HDAC reduction may depend on IkappaB kinase rather than NF-kappaB activity.;HCC xenograft mouse model was used to support the involvement of epigenetic mechanism in ZBP-89-induced Bak expression and its therapeutic effects against HCC. Results showed that ZBP-89 as well as HDAC inhibitor valproic acid (VPA) or/and DNMT inhibitor zebularine stimulated Bak expression and induced apoptosis of tumor cells in an HCC xenograft mouse model, arresting tumor growth. In HCC xenografe model, treatment by injection of Ad-ZBP-89 viral expression vector mediated ZBP-89 expression decreased HDAC3 expression, but not HDAC4.;Conclusions: In conclusion, the study demonstrates a novel mechanism through which ZBP-89 mediates an epigenetic pathway to promote Bak expression, and induce apoptosis in HCC cells. It also reveals the mechanism of HDAC3 reduction by ZBP-89 is dependent on IkappaB, which requires the presence of Pin1. This pathway may help develop future epigenetic therapy against HCC.