Does the Hepatitis B Antigen HBx Promote the Appearance of Liver Cancer Stem Cells?

Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease and hepatocellular carcinoma (HCC). HBV-encoded X antigen, HBx, and pathways implicated in the self-renewal of stem cells contribute to HCC, but it is not clear whether HBx expression promotes stemness. Thus, experiments were designed to test the hypothesis that HBx triggers malignant transformation by promoting properties that are characteristic of cancer stem cells (CSC). To test this hypothesis, HepG2 cells were stably transduced with HBx and then assayed for phenotypic and molecular characteristics of stemness. The relationship between HBx and stemness-associated markers was also evaluated by immunohistochemical staining of liver and tumor tissue sections from HBV-infected patients. The results showed that Oct-4, Nanog, Klf-4, beta-catenin, and epithelial cell adhesion molecule (EpCAM) were activated by HBx in vitro and in vivo. EpCAM was detected in the nuclei of human HCC cells from infected patients. HBx promotes stemness by activating beta-catenin and epigenetic upregulation of miR-181, both of which target EpCAM. HBx expression was also associated with depressed levels of E-cadherin. Moreover, HBx stimulated cell migration, growth in soft agar, and spheroid formation. This work is the first to propose that HBV promotes stemness in the pathogenesis of HCC. HBx-associated upregulated expression of multiple stemness markers supports the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristics of CSCs. Cancer Res; 71(10); 3701-8. (C) 2011 AACR.