HBx mutations promote hepatoma cell migration through the Wnt/β-catenin signaling pathway

Abstract

HBx mutations (T1753V, A1762T, G1764A, and T1768A) are frequently observed in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Aberrant activation of the Wnt/β-catenin signaling pathway is involved in the development of HCC. However, activation of the Wnt/β-catenin signaling pathway by HBx mutants has not been studied in hepatoma cells or HBV-associated HCC samples. In this study, we examined the effects of HBx mutants on the migration and proliferation of HCC cells and evaluated the activation of Wnt/β-catenin signaling in HBx-transfected HCC cells and HBV-related HCC tissues. We found that HBx mutants (T, A, TA, and Combo) promoted the migration and proliferation of hepatoma cells. The HBx Combo mutant potentiated TOP-luc activity and increased nuclear translocation of β-catenin. Moreover, the HBx Combo mutant increased and stabilized β-catenin levels through inactivation of glycogen synthase kinase-3β, resulting in upregulation of downstream target genes such as c-Myc, CTGF, and WISP2. Enhanced activation of Wnt/β-catenin was found in HCC tissues with HBx TA and Combo mutations. Knockdown of β-catenin effectively abrogated cell migration and proliferation stimulated by the HBx TA and Combo mutants. Our results indicate that HBx mutants, especially the Combo mutant, allow constitutive activation of the Wnt signaling pathway and may play a pivotal role in HBV-associated hepatocarcinogenesis.

Keywords: GSK-3β; HBx mutants; Wnt/β-catenin signaling pathway; hepatitis B virus; hepatocellular carcinoma.

Figure 1

HBx mutations (T1753V, A1762T, G1764A, and T1768A) promote migration and proliferation of hepatoma cells. (a) SMMC‐7721 and SK‐Hep1 cells were infected with recombinant adenoviruses Ad‐GFP, Ad‐HBx WT, or Ad‐HBx mutants (T, A, TA, or Combo mutants). At 24 h after infection, cells were subjected to Transwell assay. (b) Quantitative evaluation of cell migration activity is presented as the mean ± SD of five randomly selected microscopic fields from three independent experiments. *P < 0.05 versus GFP control; **P < 0.01 versus WT. (c) Cell growth curves. Cells were treated as described in (a) then plated into a 96‐well plate at 2 × 10³ cells/well. The number of viable cells was counted for 4 days. Data are presented as the mean ± SD. **P < 0.05 versus WT.

Figure 2

HBx mutations (T1753V, A1762T, G1764A, and T1768A) activate the Wnt/β‐catenin pathway in hepatocellular carcinoma cells. (a) HBx mutations increase TOP‐luc luciferase reporter activity. SMMC‐7721 cells were transfected with the Transcription Factor 4/Liver‐Enriched Factor 1 reporter, pTOP‐luc. At 24 h after transfection, cells were infected with recombinant adenoviruses Ad‐GFP (control), Ad‐HBx WT, or Ad‐HBx mutants. At 36 h after infection, cells were collected for luciferase assays. Results are presented as the mean relative luciferase activity relative to the activity of the GFP‐treated control sample ± SD of three independent experiments. **P < 0.01 versus WT; ^## P < 0.01 versus GFP control. (b) β‐Catenin levels in nuclear and cytoplasmic fractions. SMMC‐7721 and normal liver LO2 cells were infected with Ad‐GFP, Ad‐HBx WT, or Ad‐HBx mutants (T, A, TA, or Combo mutants). At 48 h after infection, cytosolic and nuclear proteins were extracted. Subsequently, both the cytosolic and nuclear fractions were immunoblotted with an anti‐β‐catenin antibody. The purity of the cytosolic and nuclear fractions was verified by immunoblot analysis using anti‐β‐tubulin and anti‐lamin B1 antibodies, respectively. All data were acquired from three independent experiments, and representative results are shown. (c) HBx mutants inactivate glycogen synthase kinase‐3β (GSK‐3β) activity in hepatoma and liver cells. At 48 h following infection, whole cell lysates were subjected to immunoblotting with anti‐GSK‐3β and anti‐phospho‐ (p‐)GSK‐3β antibodies. β‐Actin was used as the loading control. Integrated density of GSK‐3β and p‐GSK‐3β was quantitatively analyzed using ImageJ software. **P < 0.01 versus WT; ^## P < 0.01 versus GFP control.

Figure 3

HBx mutations upregulate Wnt/β‐catenin target genes. (a) Semiquantitative RT‐PCR analyses of mRNA expression for selected Wnt target genes. Hepatocellular carcinoma SMMC‐7721 and normal liver LO2 cells were infected with recombinant adenoviruses Ad‐GFP, Ad‐HBx WT, or Ad‐HBx mutants for 36 h. Total RNA was isolated for RT‐PCR analyses using primers specific for WISP2, CCND1,CTGF, and c‐Myc. All samples were normalized to GAPDH. (b) mRNA expression levels of c‐Myc, cyclin D1, CTGF, and WISP2 were determined by quantitative real‐time PCR. Data are shown as means ± SD of three independent experiments. *P < 0.05 versus WT; **P < 0.01 versus WT. (c) HBx mutations enhance the expression of Wnt/β‐catenin downstream genes at the protein level. Cells were infected with Ad‐GFP, Ad‐HBx WT, or Ad‐HBx mutants. Total cell lysates were obtained 48 h after infection. β‐Actin was used as the loading control. Experiments were carried out in triplicate, and representative results are shown.

Figure 4

HBx T1753A/A1762T/G1764A/T1768A (Combo) mutant upregulates β‐catenin in hepatocellular carcinoma (HCC) tissue. (a) Western blot analyses of β‐catenin expression in paired hepatitis B virus‐related HCC samples. Tissue lysates were collected and blotted with a β‐catenin antibody to determine the expression of β‐catenin in HCC tumor (T) and paired adjacent non‐tumorous tissues (N).*P < 0.05 versus WT in tumor tissues; ^# P < 0.05 versus WT in non‐tumorous tissues. (b) Immunohistochemical staining of β‐catenin in tumor (T) and paired adjacent non‐tumorous tissues (N). Tissue samples were incubated with a β‐catenin antibody and then visualized with 3,3′‐diaminobenzidine. β‐Catenin‐positive cells stained brown. Representative images are shown (magnification, ×400).

Figure 5

HBx A1762T/G1764A (TA) and T1753A/A1762T/G1764A/T1768A (Combo) mutant‐stimulated migration and proliferation are partially blocked by Wnt antagonists. (a) Hepatoma cells were infected with an optimal titer of β‐catenin siRNA vector (siBC) and a scrambled shRNA control (siControl). After 24 h, the infected cells were replated and infected with recombinant adenoviruses Ad‐GFP, Ad‐HBx WT, or Ad‐HBx mutants. Transwell and MTS assays were carried out. (b) Quantitative evaluation of cell migration activity. **P < 0.01, siBC+TA mutant versus siControl+TA mutant; ^## P < 0.01, siBC+Combo mutant versus siControl+Combo mutant. (c) Cell growth curves. **P < 0.01, siBC+HBx mutants versus siControl+HBx mutant.