Downregulation of SHIP2 by Hepatitis B Virus X Promotes the Metastasis and Chemoresistance of Hepatocellular Carcinoma through SKP2

Abstract

Hepatitis B virus (HBV)-encoded X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). The protein SH2 domain containing inositol 5-phosphatase 2 (SHIP2) belongs to the family of enzymes that dephosphorylate the 5 position of PI(3,4,5)P3 to produce PI(3,4)P2. Expression of SHIP2 has been associated with several cancers including HCC. However, its role in the development of HBV-related HCC remains elusive. In this study, we performed tissue microarray analysis using 49 cases of HCC to explore SHIP2 expression changes and found that SHIP2 was downregulated in HBV-positive HCC. In addition, S-phase kinase-associated protein 2 (SKP2), a component of the E3 ubiquitin-ligase complex, was increased in HCC cell lines that overexpressed HBx, which also showed a notable accumulation of polyubiquitinated SHIP2. Moreover, HCC cells with silenced SHIP2 had increased expression of mesenchymal markers, which promotes cell migration, enhances glucose uptake, and leads to resistance to the chemotherapy drug (5-Fluorouracil, 5-FU). Taken together, our results demonstrate that HBx downregulates SHIP2 through SKP2 and suggest a potential role for SHIP2 in HBx-mediated HCC migration.

Keywords: HBx; SHIP2; SKP2; hepatocellular carcinoma (HCC); migration.

Figure 1

SH2 domain containing inositol 5-phosphatase 2 (SHIP2) is downregulated in human hepatocellular carcinoma (HCC) tissue with hepatitis B virus (HBV) infection. (A) Representative hematoxylin and eosin (H&E)-stained tissue microarray (TMA) slide, containing 98 duplicate core tissue samples from 49 individuals with HCC. Scale bar: 50 μm. (B) Expression of SHIP2 in human HCC tissue without or with HBV infection was examined by immunohistochemistry (IHC) analysis of the TMA, which was stained with human SHIP2 antibody. Representative cores from the human HCC TMA are shown, and the areas within the black squares are enlarged and presented in the lower panels. (C) Quantification of IHC intensity for SHIP2 in cores grouped according to HBV infection status. Data represent the mean ± standard deviation (s.d.), *** p < 0.001.

Figure 2

Hepatitis B virus (HBV)–encoded X protein (HBx) reduces SHIP2 protein expression in HCC cells. (A) SHIP2 was reduced in HBx-expressing Hep3Bx and HepG2x HCC cell lines, as compared with parental lines. Quantification of relative SHIP2 expression is shown (n = 3). Data represent the mean ± s.d., *** p < 0.001. (B) HBx expression vector was transiently transfected into Hep3B and HepG2 cell lines for 48 h. SHIP2 and HBx expression was analyzed at the protein level by western blotting and at the mRNA level by RT-PCR. (C) Transient transfection of HBx and control siRNA was performed in Hep3Bx and HepG2x cells for three days. SHIP2 expression and gene silencing of HBx protein expression were examined by western blotting. In all the above cases, α-tubulin, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), and β-actin are used as the internal control. The whole blot has been provided as Supplemental Materials (Figure S4).

Figure 3

SHIP2 is polyubiquitinated by the E3 ubiquitin ligase SKP2 in HCC cells. (A) Time-dependent study of the effect of bortezomib (10 nM) treatment on SHIP2 expression in HBx-transfected HCC cell lines. SHIP2, HBx, and S-phase kinase-associated protein 2 (SKP2) were analyzed by western blotting. (B) Hep3Bx and HepG2x cells were infected with lentivirus for delivery of short-hairpin RNA (shRNA) against luciferase (shLuc) or SKP2 (shSKP2) for three days, and then whole-protein lysates were analyzed by western blotting. (C) Hep3B and HepG2 cells were transiently transfected with SKP2 or control expression vector for 48 h and then were treated in the absence or presence of bortezomib (50 nM) for 16 h prior to cell harvest. Whole-protein lysates were prepared followed by immunoprecipitation (IP) with anti-SHIP2. The precipitate was then electrophoresed on a polyacrylamide gel followed by immunoblotting (IB) using anti-ubiquitin. The whole blot has been provided as Supplemental Materials (Figure S5).

Figure 4

Inhibition of SHIP2 enhances cell migration and invasion in HCC cell lines. (A) The proliferation ability of Hep3B and HepG2 cells grown in 96-well plates was assessed using a WST-1 assay for a period of 24 h after SHIP2 shRNA knockdown. (B) Representative images of cell morphology in parental, shLuc, and shSHIP2 cells. Magnification, 100×. (C) Cell migration and invasion were measured using Transwell assays for shLuc, and shSHIP2 cells. Representative photographs from at least three different experiments are shown. Magnification, 100× (D) The relative migration and invasion rates of cells as shown in (C) were determined by crystal violet staining and quantification. Statistical analysis was performed with a Student’s t-test. ** p < 0.01; *** p < 0.001 as compared with the shLuc group (n = 3).

Figure 5

SHIP2 regulates cell motility through the epithelial–mesenchymal transition (EMT) in HCC cell lines. (A) An epithelial marker (E-cadherin), mesenchymal markers (N-cadherin and vimentin), and SHIP2 were detected by western blotting in control and SHIP2-knockdown HCC cell lines. (B) The mRNA expression of EMT markers was examined by real-time PCR in control and SHIP2-knockdown HCC cell lines. mRNA expression was normalized to actin expression. (C) Hep3Bx and HepG2x cells were transiently transfected with SHIP2 plasmid for 48 h. The SHIP2 and EMT markers were examined by western blotting. (D) The HBx-expressing HCC cell lines were transfected with SHIP2 plasmid for 48 h. The migration rate was then examined by the Transwell migration assay after a 24 h incubation. Magnification, 100×. Representative images of migrated cells are shown. Statistical analysis was performed by Student’s t-test. ** p < 0.01; *** p < 0.001 as compared with the control group (n = 3). The whole blot has been provided as Supplemental Materials (Figure S6).

Figure 6

Inhibition of SHIP2 activates glucose uptake and 5-FU resistance in HCC cells. (A,B) Time-dependent glucose uptake in HCC cells following SHIP2 knockdown. Glucose uptake was analyzed with the fluorescent deoxyglucose analog 2-NBDG by fluorescence microscopy and flow cytometry. Tumor cells were incubated with 50 nM 2-NBDG for 0.5, 1, and 2 h. The lentivirus containing shRNA against luciferase (shLuc) was used as the control. Representative data from three independent experiments are shown. (C) GLUT1 mRNA expression in SHIP2-knockdown HCC cell lines was examined by real-time PCR. GLUT1 mRNA expression was normalized to actin expression. ** p < 0.01 (D,E) The cytotoxic effect of 5-FU in SHIP2-knockdown HCC cells was analyzed. Hep3B and HepG2 cells were plated in 96-well plates after shLuc or SHIP2 knockdown for 72 h. The parental lines were also plated as additional controls. The cells were treated with the indicated doses (12.5–200 μM) of 5-FU for 48 h or were treated with a time course of 25 μM 5-FU for 24, 48, and 72 h. After 5-FU treatment, cell survival was monitored using the WST-1 assay. Statistical analysis was carried out with Student’s t-test. *** p < 0.001 as compared with each control group (n = 3). (F) Schematic diagram depicting how HBx mediates SHIP2 in HCC cells.