HBXIP, Cellular Target of Hepatitis B Virus Oncoprotein, Is a Regulator of Centrosome Dynamics and Cytokinesis

Abstract

Hepatitis B virus accounts for more than 1 million cancer deaths annually, but the mechanism by which this virus promotes hepatocellular carcinoma remains unclear. The hepatitis B virus genome encodes an oncoprotein, HBx, which binds various cellular proteins including HBXIP. We show here that HBXIP is a regulator of centrosome duplication, required for bipolar spindle formation in HeLa human carcinoma cells and primary mouse embryonic fibroblast cells. We found that most cells deficient in HBXIP arrest in prometaphase with monopolar spindles whereas HBXIP overexpression causes tripolar or multipolar spindles due to excessive centrosome replication. Additionally, a defect in cytokinesis was seen in HBXIP-deficient HeLa cells, with most cells failing to complete division and succumbing eventually to apoptosis. Expression of viral HBx in HeLa cells mimicked the effects of HBXIP overexpression, causing excessive centrosome replication, resulting in tripolar and multipolar spindles and defective cytokinesis. Immunolocalization and fluorescent protein tagging experiments showed that HBXIP associates with microtubules of dividing cells and colocalizes with HBx on centrosomes. Thus, viral HBx and its cellular target HBXIP regulate centrosome dynamics and cytokinesis affecting genetic stability. In vivo experiments using antisense oligonucleotides targeting HBXIP in a mouse model of liver regeneration showed a requirement for HBXIP for growth and survival of replicating hepatocytes. Thus, HBXIP is a critical regulator of hepatocyte cell growth in vivo, making it a strong candidate for explaining the tumorigenic actions of viral HBx. (Cancer Res 2006; 66(18): 9099-107)

Global proteome analysis of hepatitis B virus expressing human hepatoblastoma cell line HepG2

“…These results were consistent with previous findings that the HBV X protein is associated with cell cycle checkpoint controls [Fujii et al, 2006b]. Based on proteomic findings, several key steps in the cell cycles are likely to be affected by HBV replication and protein expression.…”

HBV X protein targets hBubR1, which induces dysregulation of the mitotic checkpoint

“…The hBubR1 siRNA duplex significantly reduced BubR1 expression in ChangX cells (Figure 2d) and we found that the kinetochore localization of HBx was diminished in cells lacking kinetochore hBubR1 (Figure 2b, lower panel). In contrast, centrosomal localization of HBx (Fujii et al, 2006) appeared not to be affected by BubR1 siRNA (Figure 2b, arrows). Localization of CDC20 to kinetochores appeared to be stable in cells depleted of BubR1 (data not shown).…”

“…This is likely associated, at least in part, with the observation that integration of the HBV genome causes rearrangement of cellular DNA (Henderson et al, 1988). In addition, our group and others have observed that the HBV X protein (HBx) viral oncoprotein is directly involved in hyperamplification of centrosomes, which leads to chromosome aberrations (Forgues et al, 2003;Yun et al, 2004;Fujii et al, 2006).…”

The Nuclear Import of Oncoprotein Hepatitis B X-interacting Protein Depends on Interacting with c-Fos and Phosphorylation of Both Proteins in Breast Cancer Cells

“…Interestingly, growing evidence shows that HBXIP has broad roles in cancers. HBXIP can suppress apoptosis through survivin/caspase 9 in hepatoma cells (2) and regulates centrosome duplication, causing excessive centrosome production and multipolar mitotic spindles in HeLa cells (3,4). It has been reported that HBXIP is a component of Ragulator, which is a guanine nucleotide exchange factor (GEF) for the Rag GTPases that signal amino acid levels to mTORC1 (5).…”