Pin1 interacts with a specific Serine-Proline motif of Hepatitis B Virus X protein to enhance human hepatocarcinogenesis

Abstract

We previously reported that the peptidyl prolyl isomerase (PIN1) is over-expressed in hepatocellular carcinoma (HCC) cases related to hepatitis B virus (HBV), and it plays a functional role in hepatocar-cinogenesis (Pang et al, Oncogene 2004). Hepatitis B virus X protein (HBx), a potent transactivator implicated in HBV-related hepatocar-cinogenesis, has a potential PIN1 binding motif. This study investigated the interaction of PIN1 with HBx and its role in HBV-related hepatocar-cinogenesis. We observed positive correlation of PIN1 and HBx expression (p = 0.024) by immunohistochemistry in HBV-related HCCs. Immunofluorescence microscopy confirmed co-localization of HBx with PIN1 in the nucleus, and co-immuoprecip-itation showed that PIN1 bound to ectopically expressed flag-tagged HBx in Hela cells. GST pull-down assay further validated the binding between the two proteins. Co-transfection of PIN1 and HBx transactivated the NF-AT driven luciferase reporter, with two-fold increase in luciferase activity compared with transfection of HBx alone, or PIN1-HBx mutants with abolition of Ser/Pro residues at the pSer41-Pro motif, which abolished its binding with PIN1. Cell proliferation was markedly increased with stable PIN1-HBx transfectants compared with PIN1-HBx mutant transfectants, stable PIN1 or HBx transfectants alone. Subcutaneous injection of PIN1-HBx transfectants in nude mice resulted in development of larger tumors compared with HBx mutants, PIN1 or HBx transfectants alone. We conclude that PIN1 interacts with a specific Serine-Proline motif of HBx and augments its transactivating activity and oncogenic properties. These findings implicate PIN1 expression as an important step in HBV-related hepatocarcinogenesis and suggest that PIN1 may be a novel molecular target for inhibition of hepatocarcinogenesis.