HBx T1753A/A1762T/G1764A/T1768A Mutations Promote Hepatoma Cell Tumorigenicity Through The Wnt/β-Catenin Signaling Pathway

Objective: Hepatocellular carcinoma(HCC) is the fifth most common cancer and ranks as the third leading cause of cancer-related death around the world. Chronic infection with hepatitis B virus(HBV) is a major risk factor for cirrhosis and HCC in developing countries. Many viral factors including high virus load, HBV genotype C, and core promoter mutations are associated with a high risk of HCC.HBV is a partially double stranded circular 3.2 kb DNA that belongs to the orthohepadnavirus of the hepadnaviridae family. The HBx protein, encoded by the HBV X gene, is a 154-amino acid polypeptide with a molecular weight of 17 k Da. HBx is a promiscuous transactivator that activate a series of signaling pathways including NFAT, CREB/ATF, Wnt/β-catenin, and NF-κB signaling pathways, which might plays a crucial role in hepatocarcinogenesis. A1762T/T1764A(TA) mutations in the core promoter(BCP), that overlap with the HBx gene, are found commonly in HCC and are independent risk factors in the progression of HCC during chronic HBV infection. In vivo studies suggest that the TA mutant increases the replication capacity of HBV and suppresses HBe Ag serum levels. TA mutations are also predictors of postoperative survival in HBV-related HCC patients. T1753A/A1762T/G1764A/T1768A(Combo) mutations in the basal core promoter of HBV or combinations of multiple point mutations(G1613A, C1653 T, T1753 V, A1762 T, and G1764A) were closely related to HCC among patients infected with HBV. Previous studies have demonstrated that HBx has inhibiting effect in growth of hepatoma cells. Combo mutations result in amino acid substitution(I127N/K130M/V131I/F132Y) that may lead to aberrant biological activity of HBx. However, the potential role of HBx Combo mutations in hepatocarcinogenesis is largely unknown.Aberrant Wnt/β-catenin signaling has been implicated in tumorigenesis of multiple cancers such as colon, breast, and ovarian. Canonical Wnt/β-catenin pathway consists of several parts including Wnt secreted glycoproteins, Frizzled family of transmembrane receptor Dishevelled(Dsh), APC, Axin, GSK-3β, β-catenin and TCF/LEF transcription factor. Mutations in the CTNNB1, AXIN2, and APC genes contribute to aberrant activation of Wnt/β-catenin signaling and HCC development. HBx stabilizes cytoplasmic β-catenin via interaction with Wnt1 or activation of Erk. In the current study, we explored whether HBx mutations play roles in activating the Wnt/β-catenin pathway and are involved in hepatoma cell proliferation and motility. We also investigated β-catenin activation in HBV-related HCC samples within HBx mutants.Methods: HBx mutants including A1762T(T), G1764A(A), A1762T/G1764A(TA), and T1753A/A1762T/G1764A/T1768A(Combo) were constructed and transfected into hepatoma cells. The migration and proliferation capacities of HCC cells were observed by transwell and MTS assays. Luciferase assay and immunofluorescence staining were applied to observe the activation of Wnt/β-catenin by HBx mutants. q RT-PCR and Western Blot were employed to detect the expression levels of Wnt/β-catenin downstream target genes. Furthermore, the expression and location of β-catenin in HBV-related HCC tissues were measured by immunohistochemical staining.Results: Compared with HBx WT, HBx mutants, expecially TA and Combo mutant could significantly strengthen migration and proliferation of hepatoma cells(p<0.01). Knockdown of β-catenin effectively abrogated cell migration and proliferation stimulated by the HBx TA and Combo mutants(p<0.01). Compared with GFP control, the TOP-luc activity of HBx WT was increased by 3.50 fold. The HBx TA and Combo mutants increased luciferase activity by caused 1.97 and 2.25 fold, respectively, compared with HBx WT(p<0.01). The HBx TA and Combo mutant increased expression and nuclear translocation of β-catenin. Moreover, the HBx Combo mutant increased and stabilized β-catenin levels through inactivation of GSK-3β. The increased β-catenin resulted in upregulation of downstream target genes such as c-Myc, CTGF, and WISP2. Next we measured the genetic variations in the core promoter region, the most frequently occurring mutation in this region was A1762T/G1764A(38-40%). The Combo mutations were rarely found in these samples(2%-4%). But we found no significant differences between tumor and non-tumor tissues(p>0.05). Enhanced activation of Wnt/β-catenin was found in the HCC tissues with HBx TA and Combo mutations.Conclusion: HBx TA and Combo mutants can largely enhance the migration and proliferation of hepatoma cells. Wnt antagonists effectively abrogated cell migration and proliferation stimulated by the HBx TA and Combo mutants. Combo mutations increase and stabilize β-catenin levels by inactivating GSK-3β. HBx mutants, especially the Combo mutant, allow constitutive activation of the Wnt signaling pathway and may play a pivotal role in HBV-associated hepatocarcinogenesis. These findings provide detailed information about the roles of HBx TA and Combo mutants in the progression of HCC and suggest a novel therapeutic strategy to prevent HBV-related HCC.