Hepatocyte Nox proteins in the pathogenesis of hepatocellular carcinoma

描述(申请人提供)：丙型肝炎病毒是肝细胞癌的主要病原体。丙型肝炎病毒诱发的肝细胞癌通常先于慢性肝炎和肝硬变。研究表明，丙型肝炎病毒具有直接致癌潜力，并增加靠近细胞核的羟基自由基和过氧亚硝酸盐的生成，造成DNA损伤，可能在癌症的发展过程中产生突变。然而，丙型肝炎病毒继发于慢性炎症/肝硬变的氧化应激，以及丙型肝炎病毒如何通过氧化应激诱导肝细胞癌，其来源和机制在很大程度上仍未解决。最近，我们的实验室发现，肝细胞NAD(P)H氧化酶1和4(NOX1和NOX4)是产生感染性丙型肝炎病毒的肝细胞以及感染丙型肝炎病毒的人肝脏中ROS的主要来源。此外，NOX4在这些细胞的细胞核中显著存在，特别是在丙型肝炎病毒存在的情况下，并增加了细胞核中ROS和硝基酪氨酸的生成，这可能增加了活性物质在致癌过程中破坏宿主核DNA的可能性。丙型肝炎病毒通过一种促纤维化的细胞因子--转化生长因子β1使NOX4升高。此外，丙型肝炎病毒感染的特点是慢性、低水平的炎症。Toll样受体4(TLR4)和肿瘤坏死因子α(TNF？)在丙型肝炎患者中升高。TLR4信号转导需要NOX4，而肿瘤坏死因子？可以诱导NOX4。因此，在丙型肝炎病毒感染过程中，肝细胞NOX4可能是炎症、纤维化和癌症之间的纽带。重要的是，调控NOx酶的能力使它们成为潜在的治疗靶点。然而，丙型肝炎病毒持续诱导NOX4的致病后果以及丙型肝炎病毒与炎症的联合致癌效应还没有明确的描述。因此，本研究的目的是确定NOx酶在丙型肝炎病毒和炎症诱导的DNA损伤中的作用，重点是肝细胞NOX4。这项研究将使用具有感染性的丙型肝炎病毒产生细胞培养系统和丙型肝炎病毒动物模型。我们假设，在慢性丙型肝炎感染期间，肝细胞NOX4与炎症驱动的NOX和其他来源的ROS一起增加宿主DNA的突变，以促进肝细胞癌的发展。其具体目的是：1)确定肝细胞NOX1和NOX4在丙型肝炎病毒诱导的宿主DNA氧化修饰/突变中的作用。NOX1和NOX4的亚细胞定位以及NOX4的核周/核定位的分子决定因素将继续被表征，因为它们与活性物种破坏宿主DNA的可能性有关；2)确定NOX4在体内丙型肝炎病毒诱导的自发肝肿瘤中的作用。将现有的丙型肝炎病毒核心转基因小鼠与NOX4基因敲除小鼠杂交，获得丙型肝炎病毒转基因/NOX4基因敲除小鼠，以检测NOX4基因敲除后的丙型肝炎病毒转基因小鼠的DNA损伤和肝脏肿瘤发生率是否降低；3)检测丙型肝炎病毒和炎症对肝细胞NOX酶、DNA损伤和肝癌发生的联合影响。本研究结果将加深我们对肝细胞NOX4以及炎症在癌症中的作用的认识，为治疗的发展提供参考。

DESCRIPTION (provided by applicant): Hepatitis C virus is a major etiologic agent of hepatocellular carcinoma (HCC). HCV-induced HCC is typically preceded by chronic hepatitis and cirrhosis. Studies suggest that HCV has a direct oncogenic potential and increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage that can be mutagenic in the development of cancer. However, the source of reactive oxygen species (ROS) and how HCV induces HCC through oxidative stress, secondary to chronic inflammation/cirrhosis, are still largely unresolved. Recently, our laboratory discovered that hepatocyte NAD(P)H oxidases 1 and 4 (Nox1 and Nox4) are prominent sources of ROS in hepatocytes producing infectious HCV as well as in the HCV-infected human liver. Furthermore, Nox4 was prominent in the nucleus of these cells, particularly in the presence of HCV, and increased the generation of ROS and nitrotyrosine in the nucleus which is likely to increase the probability of reactive species damaging the host nuclear DNA in carcinogenesis. HCV elevated Nox4 through a fibrogenic cytokine, transforming growth factor beta 1. In addition, HCV infection is characterized by chronic, low level of inflammation. Toll-like receptor 4 (TLR4) and tumor necrosis factor alpha (TNF?) are elevated in hepatitis C patients. Nox4 is required for TLR4 signaling, and TNF? can induce Nox4. Thus, hepatocyte Nox4 may serve as a link between inflammation, fibrosis, and cancer during HCV infection. Importantly, the ability to regulate Nox enzymes makes them potential targets for therapy. Nevertheless, the pathogenic consequence of persistent Nox4 induction by HCV and combined oncogenic effects of HCV plus inflammation have not been clearly delineated. Therefore, the goal of this study is to determine the role of Nox enzymes in the HCV- and inflammation-induced DNA damage in hepatocarcinogenesis, focusing on hepatocyte Nox4. The study will employ infectious HCV-producing cell culture systems and an HCV animal model. We hypothesize that hepatocyte Nox4, together with inflammation-driven Noxes and other sources of ROS, increases mutations to the host DNA to facilitate the development of HCC during chronic HCV infection. The specific aims are: 1) to determine the role of hepatocyte Nox1 and Nox4 in the HCV-induced oxidative modification/mutations to the host DNA in hepatocarcinogenesis. Subcellular localization of Nox1 and Nox4 and molecular determinants of perinuclear/nuclear location of Nox4 will continue to be characterized as they relate to the probability of reactive species damaging the host DNA; 2) to determine the role of Nox4 in the spontaneous, HCV-induced hepatic tumors in vivo. HCV transgenic/Nox4 knockout mice will be generated by crossing HCV core transgenic mice with Nox4 knockout mice, which are available, to examine whether the rate of DNA damage and hepatic tumors decreases in the HCV transgenic mice with genetic ablation of nox4 gene; 3) to examine the combined effects of HCV and inflammation on the hepatocyte Nox enzymes, DNA damage, and hepatocarcinogenesis. Results from this study will increase our understanding of hepatocyte Nox4 and the role of inflammation in cancer, for the development of therapy.