Interaction of hepatitis B virus X protein with PARP1 results in inhibition of DNA repair in hepatocellular carcinoma.

Hepatitis B virus X protein (HBx) contributes to the development of hepatocellular carcinoma (HCC), probably by regulating activities of many host or viral proteins through protein-protein interactions. In this study, we identified poly(ADP-ribose) polymerase (PARP1), a crucial factor in DNA repair, as an HBx-interacting protein using a proteomics approach. Coimmunoprecipitation and proximity ligation assays confirmed the binding and colocalization of HBx and PARP1 in the nucleus. The carboxyl-terminus of HBx protein bound to the catalytic domain of PARP1, and this binding reduced the enzymatic activity of PARP1 in both in vitro and in vivo assays. HBx interrupted the binding of PARP1 to Sirt6, which catalyzes the mono-ADP-ribosylation required for DNA repair. Consistently, overexpression of HBx inhibited the clearance of γH2AX DNA repair foci generated under oxidative stress in Chang liver cells. Recruitment of the DNA repair complex to the site-specific double-strand breaks was inhibited in the presence of HBx, when measured by laser microirradiation assay and damage-specific chromatin immunoprecipitation assays. Consequently, HBx increased signs of DNA damage such as accumulation of 8-hydroxy-2'-deoxyguanosine and comet formation, which were reversed by overexpression of PARP1 and/or Sirt6. Finally, the interaction between PARP1 and Sirt6 was markedly lower in the livers of HBx-transgenic mice and specimens obtained from HCC patients to compare with the corresponding control. Our data suggest that the physical interaction of HBx and PARP1 accelerates DNA damage by inhibiting recruitment of the DNA repair complex to the damaged DNA sites, which may lead to the onset of hepatocarcinogenesis.

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

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.

Hepatitis B virus X protein induces the expression of MTA1 and HDAC1, which enhances hypoxia signaling in hepatocellular carcinoma cells.

Expression level of metastasis-associated protein 1 (MTA1) is closely related to tumor growth and metastasis in various cancers. Although increased expression level of MTA1 was observed in hepatocellular carcinoma (HCC), role of MTA1 complex containing histone deacetylase (HDAC) in hepatitis B virus (HBV)-associated hepatocarcinogenesis has not been studied. Here, we demonstrated that HBx strongly induced the expression of MTA1 and HDAC1 genes at transcription level. MTA1 and HDAC1/2 physically associated with hypoxia-inducible factor-1 alpha (HIF-1 alpha) in vivo in the presence of HBx, which was abolished by knockdown of MTA1 by short interfering RNA (siRNA). HBx induced deacetylation of the oxygen-dependent degradation domain of HIF-1 alpha, which was accompanied with dissociation of prolyl hydroxylases and von Hippel-Lindau tumor suppressor from HIF-1 alpha. These results indicate that HBx-induced deacetylation is important for proteasomal degradation of HIF-1 alpha. Further, we observed that protein levels of MTA1 and HDAC1 were increased in the liver of HBx-transgenic mice. Also, there was a higher expression of HDAC1 in HCC than in the adjacent non-tumorous cirrhotic nodules in 10 out of 12 human HBV-associated HCC specimens. Together, our data indicate a positive cross talk between HBx and the MTA1/HDAC complex in stabilizing HIF-1 alpha, which may play a critical role in angiogenesis and metastasis of HBV-associated HCC.

6-Mercaptopurine, an activator of Nur77, enhances transcriptional activity of HIF-1alpha resulting in new vessel formation.

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in oxygen homeostasis. Previously, we reported that the orphan nuclear receptor Nur77 functions in stabilizing HIF-1alpha. Here, we demonstrate that 6-mercaptopurine (6-MP), an activator of the NR4A family members, enhances transcriptional activity of HIF-1. 6-MP enhanced the protein-level of HIF-1alpha as well as vascular endothelial growth factor (VEGF) in a dose- and time-dependent manner. The induction of HIF-1alpha was abolished by the transfection of either a dominant-negative Nur77 mutant or si-Nur77, indicating a critical role of Nur77 in the 6-MP action. The HIF-1alpha protein level remained up to 60 min in the presence of 6-MP when de novo protein synthesis was blocked by cycloheximide, suggesting that 6-MP induces stabilization of the HIF-1alpha protein. The fact that 6-MP decreased the association of HIF-1alpha with von Hippel-Lindau protein and the acetylation of HIF-1alpha, may explain how 6-MP induced stability of HIF-1alpha. Further, 6-MP induced the transactivation function of HIF-1alpha by recruiting co-activator cyclic-AMP-response-element-binding protein. Finally, 6-MP enhanced the expression of HIF-1alpha and VEGF, and the formation of capillary tubes in human umbilical vascular endothelial cells. Together, our results provide a new insight for 6-MP action in the stabilization of HIF-1alpha and imply a potential application of 6-MP in hypoxia-associated human vascular diseases.