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Epigenetic control of metastasis-associated protein 1 gene expression by hepatitis B virus X protein during hepatocarcinogenesis.

ABSTRACT: Expression of metastasis-associated protein 1 (MTA1) gene correlates with the degree of invasion and metastasis in hepatocellular carcinoma (HCC). Expression of MTA1 is induced by hepatitis B virus X protein (HBx); however, little is known about the transcriptional regulation of MTA1 gene expression. Here, we report that the 5'-flanking region of the human MTA1 promoter contains two CpG islands. Transient expression of HBx in Chang liver cells increased the methylation of the CpG island1 from 18 to 49% when measured by bisulfite-modified direct sequencing. Chromatin immunoprecipitation showed that HBx recruited DNA methyltransferase 3a (DNMT3a) and DNMT3b to the CpG island1. In silico analysis of CpG island1 predicted the existence of putative p53-binding sequences. p53 was pulled down by a DNA probe encoding the p53-binding sequences but not by the methylated DNA probe. The mouse MTA1 promoter also contains a CpG island encoding a p53-binding sequence of which p53 binding was decreased in the presence of HBx, and the expression of MTA1 and DNMT3 was increased in the liver of HBx-transgenic mice. Comparison of MTA1 and DNMT3a expression in the human normal liver and HCC specimens produced a significant correlation coefficient >0.5 (r=0.5686, P=0.0001) for DNMT3a, and a marginally significant coefficient (r=0.3162, P=0.0103) for DNMT3b. These data show that HBx induces methylation of CpG island in the MTA1 promoter, which interferes with DNA binding of p53 in the specific DNA region. This result may explain the molecular mechanism responsible for the induction of MTA1 gene expression by HBx.

SUBMITTER: Lee MH

PROVIDER: S-EPMC3503295 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Epigenetic control of metastasis-associated protein 1 gene expression by hepatitis B virus X protein during hepatocarcinogenesis.

Lee M-H MH Na H H Na T-Y TY Shin Y-K YK Seong J-K JK Lee M-O MO

Oncogenesis 20120903

Expression of metastasis-associated protein 1 (MTA1) gene correlates with the degree of invasion and metastasis in hepatocellular carcinoma (HCC). Expression of MTA1 is induced by hepatitis B virus X protein (HBx); however, little is known about the transcriptional regulation of MTA1 gene expression. Here, we report that the 5'-flanking region of the human MTA1 promoter contains two CpG islands. Transient expression of HBx in Chang liver cells increased the methylation of the CpG island1 from 18 ...[more]

PMID: 23552838

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Project description:BackgroundOur previous report suggested that centrosomal P4.1-associated protein (CPAP) is required for Hepatitis B virus (HBV) encoded non-structure protein X (HBx)-mediated nuclear factor kappa light chain enhancer of activated B cells (NF-κB) activation. CPAP is overexpressed in HBV-associated hepatocellular carcinoma (HCC); however, the interaction between CPAP and HBx in HBV-HCC remains unclear.MethodsThe mRNA expression of CPAP and HBx was analyzed by quantitative-PCR (Q-PCR). NF-κB transcriptional activity and CPAP promoter activity were determined using a reporter assay in Huh7 and Hep3B cells. Immunoprecipitation (IP) and in situ proximal ligation assay (PLA) were performed to detect the interaction between CPAP and HBx. Chromatin-IP was used to detect the association of cAMP response element binding protein (CREB) and HBx with the CPAP promoter. Cell proliferation was measured using cell counting kit CCK-8, Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) incorporation, and clonogenic assays. The tumorigenic effects of CPAP were determined using xenograft animal models.ResultsHBx can transcriptionally up-regulate CPAP via interacting with CREB. Overexpressed CPAP directly interacted with HBx to promote HBx-mediated cell proliferation and migration; SUMO modification of CPAP was involved in interacting with HBx. Knocked-down expression of CPAP decreased the HBx-mediated tumorigenic effects, including cytokines secretion. Interestingly, overexpressed CPAP maintained the HBx protein stability in an NF-κB-dependent manner; and the expression levels of CPAP and HBx were positively correlated with the activation status of NF-κB in HCC. Increased expression of CPAP and CREB mRNAs existed in the high-risk group with a lower survival rate in HBV-HCC.ConclusionThe interaction between CPAP and HBx can provide a microenvironment to facilitate HCC development via enhancing NF-κB activation, inflammatory cytokine production, and cancer malignancies. This study not only sheds light on the role of CPAP in HBV-associated HCC, but also provides CPAP as a potential target for blocking the hyper-activated NF-κB in HCC.

Project description:ObjectivesHepatitis B virus X (HBx) is closely associated with HBV-related hepatocarcinogenesis via the inactivation of tumour suppressors. Protein phosphatase 2A (PP2A) regulatory subunit B56 gamma (B56γ), as a tumour suppressor, plays a critical role in regulating cellular phosphorylation signals via dephosphorylation of signalling proteins. However, the underlying mechanism that B56γ involved in regulating HBx-associated hepatocarcinogenesis phenotypes and mediating anti-HBx antibody-mediated tumour suppression remains unknown.Materials and methodsWe used bioinformatics analysis, paired HCC patient specimens, HBx transgenic (HBx-Tg) mice, xenograft nude mice, HBV stable replication in the HepG2.2.15 cells, and anti-HBx antibody intervention to systematically evaluate the biological function of protein kinase B (AKT) dephosphorylation through B56γ in HBx-associated hepatocarcinogenesis.ResultsBioinformatics analysis revealed that AKT, matrix metalloproteinase 2 (MMP2), and MMP9 were markedly upregulated, while cell migration and viral carcinogenesis pathways were activated in HBV-infected liver tissues and HBV-associated HCC tissues. Our results demonstrated that HBx-expression promotes AKT phosphorylation (p-AKTThr308/Ser473 ), mediating the migration and invasion phenotypes in vivo and in vitro. Importantly, in clinical samples, HBx and B56γ were downregulated in HBV-associated HCC tumour tissues compared with peritumor tissues. Moreover, intervention with site-directed mutagenesis (AKTT308A , AKTS473A ) of p-AKTThr308/Ser473 mimics dephosphorylation, genetics-based B56γ overexpression, and intracellular anti-HBx antibody inhibited cell growth, migration, and invasion in HBx-expressing HCC cells.ConclusionsOur results demonstrated that B56γ inhibited HBV/HBx-dependent hepatocarcinogenesis by regulating the dephosphorylation of p-AKTThr308/Ser473 in HCC cells. The intracellular anti-HBx antibody and the activator of B56γ may provide a multipattern chemopreventive strategy against HBV-related HCC.

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Epigenetic control of metastasis-associated protein 1 gene expression by hepatitis B virus X protein during hepatocarcinogenesis.

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Epigenetic control of metastasis-associated protein 1 gene expression by hepatitis B virus X protein during hepatocarcinogenesis.

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