The role of hepatitis B virus X protein in deregulating chromosome stability in hepatocellular carcinoma

Abstract

Centrosome supernumerary is a hallmark of many types of cancer. Overduplicated centrosomes can lead to chromosome missegregation, and subsequently increasing the chance of chromosome instability and aneuploidy. When HBV genome is integrated into the host during HBV chronic infection, the viral encoded regulatory transactivating protein HBx is reported to induce genomic instability, leading to HCC formation. HBx is a multifunctional transactivator, which is involved in several signaling pathways and interacts with key transcription factors through transiting between cytoplasm and nucleus. Several studies showed that HBx could deregulate the normal functions of regulatory proteins in maintaining centrosome dynamics and spindle formation. The present study aims to explore the mechanism behind the effect of HBx in deregulating chromosome stability in HBV-mediated HCC. First, the mitotic phenotypes of previously established stable HBx overexpressing HepG2 clone was characterized. By immunofluorescence staining, HBx overexpressing cells were observed with several mitotic defect features including lagging chromosomes, chromosome congression defects and multinuclei formation. By flow cytometry and metaphase spread, these HBx overexpressing cells with mitotic errors were found accompanied with increased tetraploidy population with chromosomal gain in DNA content. Further, when HBx overexpressing cells were challenged with cell cycle arrest by spindle depolymerizing poison – nocodazole, the cells lost the mitosis state, and existed mitosis prematurely as indicated by a drop of Cyclin B1 protein expression, therefore speculating that HBx possibly compromised spindle assembly checkpoint. This also results in more clustered micronuclei formation with increase in DNA content.

In second part of the study, the effect of HBx on centrosome integrity and function was explored. By centrosome overduplication assay, HBx was found to induce centrosome overduplication in S-phase arrested cells. The ultra-structural studies by electron microscopy (EM) showed that disrupted centriole structure with disorganized microtubules and more scattered PCM around the centrioles in HBx stable clone. Strong evidence by immunofluorescence and sucrose gradient assays showed that HBx was centrosomal localized. Moreover, the microtubule (MT) nucleation and aster anchoring function of centrosome was also dysregulated in HBx overexpressing cells. By microtubule regrowth assay, increased MT nucleation and larger asters are found in HBx overexpressing cells. Furthermore, more active microtubule dynamics and faster rate of microtubule polymerization were evident by live imaging and tubulin polymerization assay. The possible HBx effect on deregulated centrosome integrity and function in microtubule nucleation and network might affect the mitotic spindle in aligning and moving the chromosomes, thus leading to chromosome missegregation and aneuploidy. To this end, our study showed that HBx could interact with a centrosomal protein TAX1BP2, suggesting that HBx may target TAX1BP2 to promote centrosome amplification.

Taken together, the present study revealed that HBx exploits centrosome integrity and function, causing mitotic errors and chromosome missegregation that lead to CIN. Understanding the role of HBx in centrosome function during mitosis may help to develop target therapies in HCC associated with CIN.