These results indicate that inactivation of ASPP1 and ASPP2 by hypermethylation is a frequent event in the early development of HCC. The ASPP2 gene was found more frequently down-regulated and methylated than the ASPP1 gene in HCC tissues. Moreover, HCCs harboring wildtype p53 more frequently had decreased expression of ASPP2. Knock-down of ASPP2 was more effective in promoting the growth of HCC cells in soft-agar and in nude mice. Thus, ASPP2 might play a more important role in the regulation of tumor development in HCC. ASPP2 was first identified as 53BP2, which contains the C-terminus part of ASPP2.30

The importance of ASPP2 in tumor suppression was recently identified BI 6727 cost in ASPP2-deficient mice.31 ASPP2 heterozygous mice had a 45% tumor incidence over their lifespan, which was three times that in wildtype mice. ASPP2 heterozygous learn more mice also had an increased susceptibility to γ-irradiation-induced tumor development. Besides p53, several proteins have been found to interact with ASPP2, such as Bcl-2, RelA/p65, and hepatitis C virus core protein.32–35 A recent study has found that Drosophila ASPP (dASPP) could interact physically with C-terminal Src kinase (Csk).36 These interactions might contribute to ASPP2-induced cell

survival and proliferation. However, the biological significance of these interactions needs to be explored further. HBx has been found to promote hypermethylation of tumor suppressor genes like find more IGFBP-3 and E-cadherin by activation of DNMTs, and recruitment of DNMTs and methyl-CpG binding proteins to the promoters.21, 23 Recently, HBx was found to have a direct interaction with DNMT3A to regulate gene expression epigenetically.24 It has been found that the methyl-CpG-binding domain (MBD) protein, MBD1, formed a complex with histone H3-K9 methylase SETDB1 and chromatin assembly factor CAF-1 to regulate ASPP2 expression.37 Here we found that ASPP1 and ASPP2 were differentially regulated by HBx. Overexpression

of HBx induced methylation of ASPP2, but not ASPP1. Further analysis revealed that DNMT1 and DNMT3A were recruited to the ASPP2 promoter, but not to the ASPP1 promoter. Thus, the differential regulation of ASPP1 and ASPP2 methylation by HBx might be due to the lacking of DNMTs binding with the ASPP1 promoter. Overexpression of HBx also recruited MeCP2 and MBD1 to the ASPP2 promoter, and released acetylated histone H3 from the ASPP2 promoter. Therefore, HBx might repress ASPP2 expression through regulating the binding of DNMTs and MBD proteins on the ASPP2 promoter. In this study, we demonstrate that methylation-induced ASPP1 and ASPP2 silence play important roles in the development of HCC, which might serve as potent targets for the development of anti-HCC therapy.