Hepatitis B Virus X Protein Confers Resistance of Hepatoma Cells to Anoikis by Up-regulating and Activating p21-Activated Kinase 1

BACKGROUND & AIMS: Patients with chronic hepatitis B virus (HBV) infection are at risk for metastatic hepatocellular carcinoma (HCC). Metastatic cancer cells develop resistance to anoikis. The serine/threonine p21-activated kinase (PAK) 1 regulates cytoskeletal dynamics and protects cells from anoikis; it also promotes virus replication. We investigated the effects of PAK1 on anoikis resistance in human hepatoma cells and in mice. METHODS: We transfected human hepatoma cells with pHBV1.3 (to mimic HBV replication) or plasmids encoding different HBV proteins; we performed colony formation and anoikis assays. We knocked down levels of PAK1 and Bcl2, or inhibited their activity, in hepatoma cells and quantified anoikis and growth of tumor xenografts in nude mice; we also measured anoikis of tumor cells isolated from ascites of the mice. We performed immunohistochemical analysis of PAK1 levels in HCC samples from patients. RESULTS: Human hepatoma cells transfected with pHBV1.3 expressing hepatitis B virus X protein (HBx) underwent anchorage-independent proliferation, were resistant to anoikis, and had higher levels of Bcl2 than nontransfected cells. Expression of HBx increased mitochondrial levels of Bcl2 and PAK1, which interacted physically. Anoikis resistance of Huh7 and SK-Hep1 cells required PAK1 activity and Bcl2. Expression of HBx promoted growth of Huh7 xenograft tumors in mice; PAK1 knockdown reduced growth of these tumors in mice and anoikis of cells isolated from these tumors. In human HCC samples, increased levels of PAK1 correlated with poor prognosis, HBV infection, and portal vein tumor thrombosis. CONCLUSIONS: The HBV protein HBx up-regulates PAK1, allows hepatoma cells to become resistant to anoikis, and promotes growth of aggressive xenograft tumors in mice. HBx induction of PAK1 might promote progression of HCC in patients with chronic HBV infection.