HBV Pre-S Mutant-mediated Molecular Events in Precursor Lesions of HBV-related Hepatocellular Carcinoma

• Main Authors: Jui-ChuYang, 楊瑞珠
• Other Authors: Ih-Jen Su
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/93413317329491624180

博士 === 國立成功大學 === 基礎醫學研究所 === 98 === Hepatocellular carcinoma (HCC) is one of the most important causes of cancer death worldwide. Chronic hepatitis B virus (HBV) infection is a major risk factor for HCC, especially with the presence of HBV pre-S mutants. However, the exact oncogenic mechanism for the progression from a benign precursor lesion to HCC remains to be established. In addition to chronic inflammation, fibrosis and immune response cytokines, HBV DNA integration and viral proteins, HBx or HBV pre-S mutants, may play important roles in HBV-related hepatocarcinogenesis. Previously, we found that ground glass hepatocytes (GGHs) contain pre-S delection mutants may induce ER stress and contribute to nodular proliferation and transforming ability, suggesting that GGHs may represent the precursor neoplastic lesions of HBV-associated HCC. This study was aiming at whether pre-S mutants induced cytokine/growth factors and play a role in the progression from GGHs to HCC. In this study, according to our microarray data, we found that pre-S mutants in HuH-7 hepatoma cell line could induce the expression of many growth factors, like TGF-βs and FGFs. Furthermore, cytokine/growth factor array data also showed that the expression of TGF-βs、FGFs and VEGFs were upregulated by pre-S mutants. We examined the secretion of cytokine/growth factor (VEGF-A, IL-6, HGF, EGF and TGF-α) in hepatocytes expressing pre-S mutants by ELISA. The production of VEGF-A was upregulated by pre-S mutants 48 hours post-transfection, but IL-6, HGF, EGF and TGF-α showed no detectable changes by pre-S mutants as compared to control. Thus, VEGF-A was selected for studies in details due to its critical role in angiogenesis at the early stage of tumorigenesis. We confirmed that the expression of VEGF-A was enhanced by pre-S mutants and could be suppressed by ER stress inhibitor vomitoxin. Further, the VEGF-A secreted from pre-S mutant-expressing HuH-7 hepatocytes could not only enhance the proliferation of endothelial cells but also promote the proliferation of HuH-7 cells, which could be inhibited by neutralization of VEGF-A. This indicated that VEGF-A may not only act as paracrine growth factor but also as an autocrine. Furthermore, we observed that pre-S mutants could upregulate the expression of VEGF receptor II and activation of Akt/mTOR signaling, which could be inhibited by neutralization of VEGF-A. Otherwise, we also demonstrated that Ras/Raf-1/ERK signal pathway was independent to the expression of pre-S mutants. By immunohistochemistry, both type I and type II GGHs showed enhanced expression of VEGF-A. Consistent with this notion, the enhanced expression of VEGF-A and the activation of Akt/mTOR signaling were also detected in HBV-related non-tumorous livers as compared to the levels of paired HCC tissues. To further address these issues, we used the transgenic mouse model to study the stepwise progression of ER stress and VEGF-A/Akt/mTOR signals at different time point. By Western blot assay, the data revealed that the expression of GRP-78 and VEGF-A and the activation of Akt were enhanced in 3-month-old transgenic livers harboring pre-S mutants as compared to control livers. Moreover, the activation of Akt was increased at the age of 6 month. Based on these results, the enhanced expression of VEGF-A in GGHs provides potential mechanism to explain the progression from pre-neoplastic GGHs to HCC in chronic HBV infection and may provide novel therapeutical strategies or chemoprevention via this study of the stepwise activation of signaling pathways in the transgenic mice.