Hepatitis B virus X protein upregulates HSP90alpha expression via activation of c-Myc in human hepatocarcinoma cell line, HepG2

Abstract

Background: The Hepatitis B Virus X protein (HBx) plays a major role in hepatocellular carcinoma (HCC) development, however, its contribution to tumor invasion and metastasis has not been established so far. Heat shock protein 90 alpha (HSP90alpha) isoform is an ATP-dependent molecular chaperone that maintains the active conformation of client oncoproteins in cancer cells, which is abundantly expressed in HCC, especially in hepatitis B virus (HBV)-related tumors, might be involved in tumor progression.

Methods: The levels of HSP90alpha, extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2) and c-Myc in HBx-transfected HepG2 cells were determined by western blots analysis. The endogenous ERKs activity was demonstrated by ELISA assay. The regulation of c-Myc-mediated HSP90 alpha promoter transactivation by HBx was evaluated through electrophoretic mobility shift analysis (EMSA). The c-Myc-mediated HSP90alpha transcription was analysed by promoter assay. The HBx-expressing cells were transfected with specific small interference RNA (siRNA) against c-Myc. The in vitro invasion potentials of cells were evaluated by Transwell cell invasion assay.

Results: HBx induces HSP90alpha expression at the transcription level. The induction effect of HBx was inhibited after treatment with c-Myc inhibitor, 10058-F4. In addition, the luciferase activity of the HSP90alpha promoter analysis revealed that the HBx is directly involved in the c-Myc-mediated transcriptional activation of HSP90alpha. Furthermore, HBx induces c-Myc expression by activation of Ras/Raf/ERK1/2 cascades, which in turn results in activation of the c-Myc-mediated HSP90alpha promoter and subsequently up-regulation of the HSP90alpha expression. Overexpression of HSP90alpha in HBx-transfected cells enhances tumor cells invasion. siRNA-mediated c-Myc knockdown in HBx-transfected cells significantly suppressed HSP90alpha expression and cells invasion in vitro.

Conclusion: These results demonstrate the ability of HBx to promote tumor cells invasion by a mechanism involving the up-regulation of HSP90alpha and provide new insights into the mechanism of action of HBx and its involvement in tumor metastasis and recurrence of HCC.

Figure 1

Upregulation of Hsp90α expression by HBx. (A) HepG2 cells were transiently transfected with the indicated amount (1 μg, 2 μg, 3 μg) of pcDNA3-X and Western blotting of HSP90α was performed. (B) Total RNA purified from the HBxtransfected HepG2 cells was subjected to RT-PCR for measuring HSP90α transcripts. (C) Increasing amount (1 μg, 2 μg, 3 μg) of pcDNA3-X was cotransfected with 2 μg of HSP90α promoter-luciferase reporter constructs (Hsp90α-Luc1430), and 2 μg of β-galactosidase reporter plasmid into HepG2 cells and luciferase assay was performed. Luciferase activity was normalized with the β-alactosidase activity in cell lysate. Error bars indicate standard deviations (SD) obtained from three different experiments prepared in triplicate.

Figure 2

HBx upregulates Hsp90α expression by activating c-Myc. (A) Lysates from stable cell lines, HepG2-pcDNA3 (lane 1) and HepG2-pcDNA3-X (lanes 2-4) after either mock treatment (lanes 1-3) or treatment with 200 nM U0126 (lane 4) for 4 h were prepared, and the protein levels of c-Myc, ERK1/2 and phosphor-ERK-1/2 were detected by Western blot analysis. GAPDH was included as an internal control. (B) Total RNA purified from HepG2-pcDNA3 (lane 1) and HepG2-pcDNA3-X (lanes 2-4) after either mock treatment (lanes 1-3) or treatment with 5 mM 10058-F4 (lane 4) for 24 h was subjected to RT-PCR to measure the RNA level of c-Myc, HBx and GAPDH. (C) Western blot analysis was performed to measure the levels of c-Myc, HBx and GAPDH in the cells prepared as described above. (D) HepG2-pcDNA3-X cells were transfected with 100 nM of either control siRNA, c-Myc siRNA or in a combination and Western blotting analysis was performed.

Figure 3

Regulation of c-Myc-mediated Hsp90α promoter transactivation by HBx. (A) Schematic illustration of the genomic region encompassing the 5' flanking region of the human the Hsp90α promoter and the reporter constructs used. (B) Cells were cotransfected with or without 1 μg of pcDNA3 or pcDNA3-X plasmid, 2 μg of Hsp90α promoter-luciferase reporter construct, and and 2 μg of β-galactosidase reporter plasmid by the LipofecAMINE method. Cells were cultured in 10% FBS medium for 24 h. Luciferase activity and β-galactosidase activity were assayed by using the luciferase and β-galactosidase enzyme assay system. Luciferase activity was normalized with the β-galactosidase activity in cell lysates. Error bars indicate standard deviations (SD) obtained from three different experiments prepared in triplicate. (C) The nuclear extract isolated from HepG2-pcDNA3 (lane 1-3) and HepG2-pcDNA3-X (lanes 4-6) cells incubated with [γ^-32P]ATP-labeled oligonucleotides corresponding to the wt c-Myc binding site of the Hsp90α promoter. Competition was performed using unlabeled wt oligo or mutant oligo. Each sample was electrophoresed in a 4% nondenaturing polyacrylamide gel in 0.5 × Tris-borate EDTA buffer at 250 V for 20 min. The gel was dried and subjected to autoradiography.

Figure 4

HBx increases cell invasion ability. Matrigel invasion assay of HepG2-pcDNA3 and HepG2-pcDNA3-X cells. Cells (1 × 10⁶) were resuspended in conditioned medium and added to the upper compartments of matrigel invasion chambers supplemented with or without 5 mM 10058-F4 for 24 h. The total number of cells that invaded to the underside of the filters was counted. The values obtained were calculated by averaging the total number of cells from three filters. As a control, a parallel experiment was performed using the device with uncoated membrane. The results represent means of triplicates; * statistically different from control at P < 0.05.