Hepatocyte Nox proteins in the pathogenesis of hepatocellular carcinoma

描述（由申请人提供）：丙型肝炎病毒是肝细胞癌（HCC）的主要病因。hcv诱导的HCC通常以慢性肝炎和肝硬化为先期。研究表明，HCV具有直接的致癌潜力，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，从而在癌症的发展中具有诱变作用。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，在很大程度上仍未得到解决。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HCV的肝细胞以及HCV感染的人肝脏中ROS的重要来源。此外，Nox4在这些细胞的细胞核中非常突出，特别是在HCV存在时，并且增加了细胞核中ROS和硝基酪氨酸的产生，这可能增加了致癌过程中活性物质破坏宿主核DNA的可能性。HCV通过纤维化细胞因子转化生长因子β 1升高Nox4。此外，HCV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。Nox4是TLR4信号转导和TNF？可以诱导Nox4。因此，肝细胞Nox4可能在HCV感染期间充当炎症、纤维化和癌症之间的联系。重要的是，调节氮氧化物酶的能力使它们成为治疗的潜在目标。然而，HCV持续诱导Nox4的致病后果以及HCV加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和炎症诱导的肝癌发生中DNA损伤的作用，重点关注肝细胞Nox4。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Nox4与炎症驱动的nox和其他ROS来源一起增加宿主DNA的突变，从而促进慢性HCV感染期间HCC的发展。具体目的是：1)确定肝细胞Nox1和Nox4在hcv诱导的宿主DNA氧化修饰/突变中在肝癌发生中的作用。Nox1和Nox4的亚细胞定位以及Nox4的核周/核位置的分子决定因素将继续被表征，因为它们与活性物质破坏宿主DNA的可能性有关；2)确定Nox4在体内自发性、hcv诱导的肝脏肿瘤中的作用。将HCV核心转基因小鼠与现有的Nox4基因敲除小鼠杂交，生成HCV转基因/Nox4基因敲除小鼠，检测Nox4基因基因消融后HCV转基因小鼠的DNA损伤率和肝脏肿瘤发生率是否降低；3)探讨丙型肝炎病毒与炎症对肝细胞Nox酶、DNA损伤和肝癌发生的联合影响。这项研究的结果将增加我们对肝细胞Nox4和炎症在癌症中的作用的理解，从而促进治疗的发展。

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. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a growing public health concern and is estimated to have infected nearly four to five million individuals in the U.S. alone. One of the most devastating consequences of HCV infection is hepatocellular carcinoma, which is still on the rise. This study examines newly described hepatocyte NAD(P)H oxidases (Nox proteins) as a persistent and robust, intra/peri-nuclear source of reactive species, which would promote the DNA damage in response to fibrogenic and pro-inflammatory cytokines increased by HCV, thereby linking chronic hepatitis/fibrogenesis to hepatocarcinogenesis during HCV infection. Results from this study will increase our understanding of HCV, Nox proteins, and the relationship between oxidative stress and HCV-induced liver disease for the development of therapy directed at the source of the reactive species.