Does the hepatitis B antigen HBx promote the appearance of liver cancer stem cells?

Abstract

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, β-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 β-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.

Fig. 1

PowerBlot analysis of (A, C) HepG2X and (B, D) HepG2CAT cells. Selected differentially expressed genes are highlighted. Representative western blots with HepG2X and HepG2CAT cells using (E) total (50 μg) or (F) nuclear (100 μg) extracts. β-actin and Lamin A are loading controls.

Fig. 2

Staining for EpCAM in (A) tumor (×400), (B) tumor (×200) and in (C) nontumor (×200). All panels show membranous staining and (C) also shows mixed membranous and nuclear localization. Detection of (D) Oct-4 and (E) Nanog in human HCC tissue sections from HBV carriers (×200).

Fig. 3

Staining for (A) HBx and (B) EpCAM in consecutive HCC sections from a representative patient (×200).

Fig. 4

Expression of miR-181a in tumor (HCC) and non-tumor liver. Negative values mean that the miRNA was expressed more in tumor compared to nontumor from the same patient. Positive values mean the miRNA was expressed more in non-tumor compared to tumor.

Fig. 5

Representative images of spheroids derived from (A) HepG2X cells, (B) HepG2CAT cells, and (C) relative numbers of spheroids for each culture. Data are shown as mean ± SD. D. Soft agar assay. (E) Migration through Matrigel basement membrane. All experiments were performed in triplicate.

Fig. 6

Western blots showing levels of acetyl-H3, E-cadherin, β-catenin and EpCAM after the treatment of HepG2X and HepG2CAT cells with TSA. The blot is representative of three independent experiments.