Hepatocyte Nox proteins in the pathogenesis of hepatocellular carcinoma

描述（由申请方提供）：丙型肝炎病毒是肝细胞癌（HCC）的主要病原体。HCV诱导的HCC通常在慢性肝炎和肝硬化之前发生。研究表明，HCV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产生，造成DNA损伤，在癌症的发展中可能是诱变性的。然而，活性氧（ROS）的来源以及HCV如何通过氧化应激诱导HCC，继发于慢性炎症/肝硬化，在很大程度上仍然没有解决。最近，我们的实验室发现，肝细胞NAD（P）H氧化酶1和4（Nox 1和Nox 4）是产生感染性HCV的肝细胞以及HCV感染的人肝脏中ROS的主要来源。此外，Nox 4在这些细胞的细胞核中是突出的，特别是在HCV的存在下，并且增加了细胞核中ROS和硝基酪氨酸的产生，这可能增加反应性物质在致癌作用中损害宿主细胞核DNA的概率。HCV通过纤维化细胞因子转化生长因子β 1升高Nox 4。此外，HCV感染的特征在于慢性、低水平的炎症。Toll样受体4（TLR 4）和肿瘤坏死因子α（TNF？）在丙型肝炎患者中升高。Nox 4是TLR 4信号传导所必需的，而TNF？可以诱导Nox 4。因此，肝细胞Nox 4可能是HCV感染过程中炎症、纤维化和癌症之间的联系。重要的是，调节Nox酶的能力使其成为潜在的治疗靶点。然而，HCV持续诱导Nox 4的致病后果和HCV与炎症的联合致癌作用尚未明确描述。因此，本研究的目的是确定Nox酶在HCV和炎症诱导的DNA损伤中的作用，重点是肝细胞Nox 4。该研究将采用感染性HCV产生细胞培养系统和HCV动物模型。我们假设肝细胞Nox 4，与炎症驱动的Noxes和其他ROS来源一起，增加了宿主DNA的突变，以促进慢性HCV感染期间HCC的发展。具体目标是：1）确定肝细胞Nox 1和Nox 4在HCV诱导的宿主DNA氧化修饰/突变中的作用。Nox 1和Nox 4的亚细胞定位以及Nox 4的核周/核定位的分子决定因素将继续被表征，因为它们与反应性物种损伤宿主DNA的概率有关; 2）确定Nox 4在体内自发性HCV诱导的肝肿瘤中的作用。通过将HCV核心转基因小鼠与现有的Nox 4敲除小鼠杂交，产生HCV转基因/Nox 4敲除小鼠，以检查在基因切除nox 4基因的HCV转基因小鼠中DNA损伤和肝肿瘤的发生率是否降低; 3）检查HCV和炎症对肝细胞Nox酶、DNA损伤和肝癌发生的联合作用。本研究的结果将增加我们对肝细胞Nox 4和炎症在癌症中的作用的理解，为治疗的发展。

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.