Relationship between Hepatitis B Virus X Protein and Hypoxia-inducible Factors and the Therapeutic Targets of Sorafenib

Chronic HBV infection is the leading cause of hepatocellular carcinoma (HCC) and HBx plays a crucial role in the molecular pathogenesis of HBV-related HCC. Previous investigations have indicated that some variations of HBV or mutations of HBx are associated with higher risk of HCC development, whereas the mutations profiles may be disparate in different regions. In the present studies, we thus aim to screen and identify the HBx mutation hotspots in 120 HCC tissues from Hong Kong, a region with HBV hyper-endemic. HBV DNAs were successfully isolated and amplified in 84.16% (101/120) HCC specimens via nest-PCR, and then subjected to gene sequencing. Three types of HBx mutations, including point mutations, distal carboxyl-terminal truncations and deletion mutations, were discovered. Among the point mutations, 39 mutation hotspots were indentified, with two double mutations (A1630G/G1721A and A1762T/G1764A) occurring in approximate 50% of 101 HCC cases. Distal C-terminal truncated mutations were discovered in 31.68% (32/101) of HCC cases, whereas deletion mutations were detected in 2.97% (3/101) of them. Overall, majority of identified mutations were located at the transactivation domain of HBx, suggesting the crucial roles of these mutations in HCC development.;Hypoxia-inducible factor-1a (HIF-1alpha) also closely involves in the development and progression of HCC. Wild-type HBx has been shown to activate HIF-1alpha. But the relationship between HBx mutants and activation of HIF-1alpha has not been fully elucidated. We here revealed that the transactivaiton domain of HBx was necessary and sufficient to activate HIF-1alpha. Double mutations K130M/V131Z in this domain enhanced the functionality of HBx in upregulating the expression and the activation of HIF-1alpha, whereas C-terminal truncations and deletion mutations weakened this prosperity of HBx. We further uncovered that the C-terminus, especially the region of amino acids 119-140, was essential for the stability and transactivation of HBx. The positive association between the HBx mutants and HIF-1alpha was found in the HCC tissue samples. Therefore, although mutations exerted different effects on the functionality of HBx, the overall activity of HBx mutants was suggested to upregulate HIF-1alpha, whose level is related to poor prognosis of HCC patients.;The therapy targeting a critical molecule in the development of HCC such as HIF-1alpha may be a potential and effective treatment regimen for HCC patients. Sorafenib, a multikinase inhibitor, has demonstrated promising results for the treatment of advanced HCC in clinical trials, but the mechanism that accounts for the anti-angiogenic efficiency of this agent has not been fully elucidated. We here revealed that sorafenib remarkably and dose-dependently decreased the expression and the transcriptional activity of HIF-1alpha, and its target gene, vascular endothelial grow factor (VEGF). Further analysis revealed that this reduction of HIF-1alpha by sorafenib was caused by the inhibition of HIF-1alpha protein synthesis rather than by the promotion of HIF-1alpha protein degradation. Moreover, the phosphorylated levels of mTOR, ERK, p70S6K, RP-S6, eIF4E and 4E-BP1 were significantly suppressed by sorafenib. In vivo studies further confirmed the inhibitory effect of sorafenib on the expression of HIF-1alpha and VEGF proteins, leading to a decrease of tumor vascularisation and growth. Collectively, our data suggest that sorafenib may exhibit anti-angiogenic activity by inhibiting HIF-1alpha synthesis, which is likely to be achieved through suppressing the phosphorylation of mTOR/p70S6K/4E-BP1 and ERK.