Hepatitis B Virus Core Promoter Mutations Contribute to Hepatocarcinogenesis by Deregulating SKP2 and Its Target, p21

Abstract

Clinical studies have associated hepatitis B virus core promoter (CP) mutations with an increased risk of hepatocellular carcinoma. The CP region overlaps with the HBV X (HBx) gene, which has been implicated in hepatocarcinogenesis. The cyclin kinase inhibitor p21WAF1/CIP1 is an important regulator of cell cycle progression and proliferation. We determined whether HBx mutants that result from mutations in the CP deregulate p21 and these processes. We constructed a series of HBx mutants with changes in the CP region that correspond to A1762T/G1764A (TA), T1753A, T1768A, or a combination of these (combo) and expressed them, along with wild-type HBx under control of its endogenous promoter, in primary human hepatocytes (PHHs) and HepG2 cells. We then analyzed the effects of CP mutations on expression and degradation of p21 and the effects on cell cycle progression and proliferation. The combo mutant decreased levels of p21 and increased cyclin E expression in PHHs and HepG2 cells. The combo mutant, but not HBx with single or double CP mutations, accelerated p21 degradation in HepG2 cells. The combo mutant increased expression of S-phase kinase-associated protein 2 (SKP2) in PHHs and Huh7 cells. Silencing of SKP2 abrogated the effects of CP mutations on p21 expression. The kinetics of p21 expression correlated with changes in cell cycle distribution. The combo mutant accelerated cell cycle progression; p21 overexpression restored G1 arrest. HBx mutants with changes that correspond to a combination of CP mutations up-regulate SKP2, which then down-regulates p21 via ubiquitin-mediated proteasomal degradation. CP mutations might increase the risk of hepatocellular carcinoma via this pathway.