Hepatitis B virus X protein overcomes stress-induced premature senescence by repressing p16INK4a expression via DNA methylation

Cellular senescence is an important tumor suppression process under diverse oncogenic conditions, entering a state of irreversible growth arrest to prevent damaged cells from undergoing aberrant proliferation. Developing a means of evading senescence thus seems to be a fundamental task that all cancer cells should solve early on. Here, we show that an oncogenic X protein of hepatitis B virus (HBx) overcomes cellular senescence provoked by a universal premature senescence inducer, H2O2, in human hepatoma cells, as demonstrated by impaired induction of senescence-associated biomarkers, including morphological change, G1 arrest, and β-galactosidase activity, in the presence of HBx. HBx induced DNA hypermethylation of p16INK4a promoter and subsequently interfered action of transcription factors like Ets1 and Ets2 activated by H2O2 through the p38MAPK pathway, resulting in inhibition of its transcription. Down-regulation of p16INK4a expression by HBx subsequently led to activation of G1-CDKs, phosphorylation of Rb, activation of E2F1, and finally evasion from G1 arrest induced by H2O2. Levels of another senescence regulator, p21waf1, however, were not affected by HBx under our senescence-inducing conditions. In addition, the potentials of HBx to inactivate Rb and subsequently inhibit cellular senescence almost completely disappeared when levels of p16INK4a were recovered either by exogenous complementation or inhibition of the promoter hypermethylation. To our knowledge, our present study represents the first report that an oncogenic virus evades cellular senescence through epigenetic down-regulation of p16INK4a expression.