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


CONCLUSIONS
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.
Li
Weihua
W
Department of Infectious disease, the First affiliated Hospital of Guangzhou Medical College, Guangzhou 510102, Guangdong province, PR China. liweihuascu@163.com
Miao
Xiaohui
X
Qi
Zhongtian
Z
Zeng
Wenting
W
Liang
Jianxin
J
Liang
Zengwei
Z
D015153:Blotting, Western; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D018841:HSP90 Heat-Shock Proteins / Q000096:biosynthesis*; D006515:Hepatitis B virus / Q000502:physiology*; D022781:Hepatocytes / Q000737:chemistry / Q000821:virology*; D054884:Host-Pathogen Interactions; D006801:Humans; D048370:MAP Kinase Kinase 2 / Q000032:analysis; D048052:Mitogen-Activated Protein Kinase 3 / Q000032:analysis; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis*; D015534:Trans-Activators / Q000502:physiology*; D014158:Transcription, Genetic; D054334:Viral Regulatory and Accessory Proteins
D018841:HSP90 Heat-Shock Proteins; C533585:HSP90AA2P protein, human; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D048052:Mitogen-Activated Protein Kinase 3; D048370:MAP Kinase Kinase 2