Role of Autotaxin in HCV-associated Hepatocellular Carcinoma

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)感染是发展为肝细胞癌(肝细胞癌)的最重要的危险因素，肝细胞癌是世界范围内主要的肝脏恶性肿瘤和癌症类型。与丙型肝炎病毒相关的肝细胞癌发病机制相关的分子遗传学和信号通路仍然知之甚少。我们的长期目标是阐明慢性丙型肝炎进展为肝细胞癌的分子机制。自体趋化蛋白(ATX)是一种胞外核苷酸焦磷酸/磷酸二酯酶2酶，是一种强大的细胞刺激运动原，在多种恶性肿瘤组织中过表达，并与多种肿瘤细胞的致瘤和转移潜能有关。2007年，两个研究小组首次提出证据表明，慢性丙型肝炎患者血清ATX活性和血浆溶血磷脂酸(LPA)水平升高与肝纤维化相关，肝细胞癌合并丙型肝炎患者ATX及ATX信号通路相关基因表达上调。这是丙型肝炎病毒感染的原因还是后果？ATX的异常调节如何影响丙型肝炎病毒的复制和发病？我们推测“ATX的过度表达和信号转导可能是丙型肝炎病毒复制和致病的重要决定因素”。我们的假设是基于一些新的和重要的线索，将ATX的表达与丙型肝炎病毒感染和发病机制联系起来。在初步实验中，我们观察到在丙型肝炎病毒感染患者的肝组织中ATX mRNA的表达上调，并且在与丙型肝炎病毒相关的肝细胞癌样本中其表达进一步增强，这与2007年的临床研究一致。此外，丙型肝炎病毒复制子细胞与复制子治愈细胞相比，ATX mRNA的表达水平上调，这表明丙型肝炎病毒在诱导ATX表达中发挥了作用。这一观察结果，再加上我们的数据，即丙型肝炎病毒复制子细胞分泌ATX，提示丙型肝炎病毒感染的肝脏可能导致了丙型肝炎病毒感染患者和丙型肝炎病毒相关性肝细胞癌患者ATX的异常表达。我们发现，在丙型肝炎病毒复制子细胞中，ATX启动子被激活和刺激，从而暗示丙型肝炎病毒感染细胞中的丙型肝炎病毒蛋白和宿主因子可能在ATX的异常调节中发挥作用。此外，ATX在携带丙型肝炎病毒RNA的人肝癌细胞中过表达，诱导了RhoA的表达，激活了RhoA，刺激了丙型肝炎病毒RNA的复制。相反，阻断ATX分泌可抑制RhoA诱导和消融丙型肝炎病毒RNA复制。我们认为，在丙型肝炎病毒感染过程中，ATX/LPA的过度表达可能会破坏Rho GTPase信号转导机制，从而参与丙型肝炎病毒的发病。为了检验我们的假设并扩展我们的发现，我们提出了两个独立但互补的具体目标。在目标1中，我们将研究病毒蛋白和宿主因素在丙型肝炎病毒感染和致病过程中ATX表达异常调节中的作用。在目标2中，我们建议解决ATX在丙型肝炎病毒复制和致病机制中的作用。为了研究这些方面，我们建议使用未转化的人肝细胞(HH4细胞)有条件地表达全长丙型肝炎病毒ORF(HH4-丙型肝炎病毒)作为丙型肝炎病毒感染早期阶段的模型，以及在肝癌细胞中复制丙型肝炎病毒RNA的C-5B复制子细胞来解决与丙型肝炎病毒感染的肿瘤微环境中的情景有关的问题。这些目标将通过包括细胞培养、生化和分子遗传学在内的多管齐下的方法来实现。我们预计，我们的发现将为研究ATX在丙型肝炎病毒感染过程中的调控提供重要的见解，并进一步阐明ATX在丙型肝炎病毒复制和发病机制中的作用。这些研究为丙型肝炎的治疗干预提供了合理的靶点。 公共卫生相关性：拟议的研究有可能为自体趋化蛋白(ATX)在丙型肝炎病毒感染和发病机制中的调控和作用提供重要的见解，并可能为丙型肝炎的治疗干预确定新的模式。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) infection is the most significant risk factor for the development of hepatocellular carcinoma (HCC), a major liver malignancy and cancer type worldwide. The molecular genetics and signaling pathways associated with HCV-associated HCC pathogenesis remain poorly understood. Our long-term goal is to elucidate the molecular mechanisms underlying progression of chronic HCV to HCC. Autotaxin (ATX), an ectonucleotide pyrophosphatase/phosphodiesterase 2 enzyme and a potent cell stimulating motogen is over-expressed in various malignant tumor tissues and has been implicated to confer the tumorigenic and metastatic potential of a variety of cancer cells. In 2007, two groups provided the first evidence that the serum ATX activity and plasma lysophosphatidic acid (LPA) levels are increased in chronic hepatitis C in association with liver fibrosis, and that ATX and genes related to ATX signaling pathway were upregulated in HCC patients co-infected with HCV. Is this the cause or consequence of HCV infection? And how does aberrant regulation of ATX impact HCV replication and pathogenesis? We postulate that "ATX over-expression and signaling may be an important determinant in the replication and pathogenesis of HCV". Our hypothesis is based on some novel and vital clues linking ATX expression to HCV infection and pathogenesis. In preliminary experiments we observed that ATX mRNA expression is upregulated in the liver tissues of HCV-infected patients and its expression is further enhanced in HCV- associated HCC samples, consistent with the 2007 clinical study. Further, the expression levels of ATX mRNA were upregulated in HCV replicon cells versus replicon cured cells, suggesting a role for HCV in inducing ATX expression. This observation coupled with our data that HCV replicon cells secrete ATX, suggest that the HCV infected liver may potentially contribute to the aberrant expression of ATX in HCV- infected patients and patients with HCV-associated HCC. We found that the ATX promoter was activated and stimulated in HCV replicon cells, thus implicating HCV proteins and host-factors in HCV-infected cells to potentially play a role in aberrant regulation of ATX. Further, over-expression of ATX in HCV RNA bearing human hepatoma cells induced RhoA expression, activated RhoA and stimulated HCV RNA replication. Conversely, blockage of ATX secretion abrogated RhoA induction and ablated HCV RNA replication. We envision that ATX/LPA over-expression during the course of HCV infection may de-regulate mechanisms involved in Rho GTPase signaling thus contributing towards HCV pathogenesis. To test our hypothesis and to extend our findings two independent but complementary specific aims are proposed. In Aim 1 we will investigate the role of viral proteins and host factors in aberrant regulation of ATX expression during HCV infection and pathogenesis. In Aim 2, we propose to address the role of ATX in HCV replication and pathogenesis mechanisms. To investigate these aspects, we propose to employ nontransformed human hepatocytes (HH4 cells) conditionally expressing the full-length HCV ORF (HH4-HCV) as models for the early stage of HCV infection, and C-5B replicon cells which replicate HCV RNA in hepatoma cells to address questions pertaining to the scenario in the HCV infected tumor microenvironment. These objectives will be achieved via multi-prong approach, including cell culture, biochemical and molecular genetics. We envisage that our findings will provide crucial insights into modulation of ATX during HCV infection and further clarify the role of ATX in HCV replication and pathogenesis. These investigations may provide rational target for therapeutic intervention of HCV. PUBLIC HEALTH RELEVANCE: The proposed studies have the potential to provide crucial insights into regulation and role of autotaxin (ATX), a tumorigenic and metastatic protein, in HCV infection and pathogenesis mechanisms and may likely identify novel modalities for therapeutic intervention of Hepatitis C.