Mechanisms of Renal Carcinogenesis

描述（由申请人提供）：肾透明细胞癌（RCC）是肾癌的主要形式。RCC的药物治疗通常无效，转移性疾病患者的中位寿命仅为1年。RCC源自von Hippel-Lindau（VHL）基因突变。在VHL缺陷细胞中，缺氧诱导转录因子HIF-2 α稳定并上调支持肿瘤生长的几个基因。在动物模型中，HIF-2 α的下调对于阻断肿瘤形成是必要的和足够的。活性氧（ROS），如超氧阴离子和过氧化氢参与的信号转导途径介导的许多胁迫和生长反应。NAD（P）H氧化酶是参与肾氧化应激的ROS的主要来源。我们有强有力的证据表明，VHL缺乏通过NAD（P）H依赖性氧化酶亚基Nox 4和p22 phox增加ROS的产生在RCC的发病机制中起着关键作用，Nox 4和p22 phox在缺乏VHL的情况下维持HIF-2 α蛋白表达至关重要。本研究的目的是阐明VHL缺乏、NAD（P）H氧化酶表达和/或活化增加以及HIF-2 α和靶基因表达增加与肾细胞癌体外和体内发生和进展相关的分子机制。AIM 1将决定VHL调节NAD（P）H氧化酶Nox 4及其膜结合伴侣p22 phox的催化单位的机制。VHL是E3泛素连接酶复合物的一部分，其通过26 S蛋白酶体途径聚泛素化特定蛋白以调节蛋白质降解。该过程需要VHL与底物结合。将确定Nox 4或p22 phox蛋白在蛋白酶体抑制剂存在下是否稳定，以及Nox 4和p22 phox是否以VHL依赖性方式在体外和体内后泛素化。将进行体外和体内结合研究以确定VHL是否结合Nox 4和p22 phox。或者，Nox 4可以在VHL缺陷细胞中通过mRNA的转录增强而上调。我们将通过北方印迹和定量真实的时间PCR确定Nox 4 mRNA水平是否在VHL缺陷细胞中增加。还将检查Nox 4启动子活性以探索HIF-21在Nox 4启动子上的推定反馈环作为Nox 4表达的潜在机制。该建议的第二个目的将阐明Nox氧化酶组分在VHL缺陷细胞中维持HIF-2 α蛋白表达的机制。我们将特别确定在VHL缺陷细胞中ROS对HIF-2 α的维持是否需要持续的转录、mRNA稳定或介导翻译的信号转导途径的激活。第三个目的将在体外和体内检查Nox 4和p22 phox或超氧化物抗氧化剂的稳定沉默对HIF-2a转录活性、肿瘤生长和细胞侵袭性（与VHL-缺陷相关的其他特征）的影响。公共卫生相关性：VHL缺乏在肾透明细胞癌（RCC）的发病机制中起着关键作用。该项目的目标是阐明VHL缺陷型肾细胞癌发生和进展的致病机制，通过增加NAD（P）H依赖性氧化酶产生的活性氧簇，这些酶对于维持HIF-21蛋白表达至关重要，而HIF-21蛋白表达是VHL缺陷型肿瘤发生进展所必需且充分的关键因素。该项目可能导致开发新的和特定的治疗方案来治疗RCC。

DESCRIPTION (provided by applicant): Clear-cell renal carcinoma (RCC) is the predominant form of renal carcinoma. Medical treatment of RCC is generally ineffective with a median life span of only 1 year in patients with metastatic disease. RCC is derived from mutations in the von Hippel-Lindau (VHL) gene. In VHL-deficient cells, the hypoxia inducible transcription factor, HIF-2 alpha, is stabilized and up-regulates several genes that support tumor growth. Down-regulation of HIF-2 alpha is necessary and sufficient to block tumor formation in animal models. Reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide are involved in the signaling pathways mediating many stress and growth responses. NAD(P)H oxidase(s) are a major source of ROS implicated in renal oxidative stress. We have strong evidence that VHL-deficiency plays a critical role in the pathogenesis of RCC through increased generation of ROS by the NAD(P)H-dependent oxidase subunits, Nox4 and p22phox, which are critical in maintaining HIF-2 alpha protein expression in the absence of VHL. The goal of our studies is to elucidate the molecular mechanisms linking VHL-deficiency, increased expression and or activation of NAD(P)H oxidases and increased expression of HIF-2 alpha and target genes involved in the development and progression of RCC in vitro and in vivo. AIM1 will determine the mechanisms by which VHL regulates the catalytic unit of the NAD(P)H oxidase Nox4 and its membrane binding partner, p22phox. VHL is part of an E3 ubiquitin ligase complex that poly-ubiquitinates specific proteins for regulated protein degradation through the 26S proteasome pathway. This process requires the binding of VHL to the substrate. It will be determined if Nox4 or p22phox protein are stabilized in the presence of proteasome inhibitors and if Nox4 and p22phox are post translationally ubiquitinated in vitro and in vivo in a VHL-dependent manner. In vitro and in vivo binding studies will be performed to determine if VHL binds Nox4 and p22phox. Alternatively, Nox4 may be up- regulated in VHL-deficient cells through enhanced transcription of the mRNAs. We will determine if Nox4 mRNA levels are increased in VHL-deficient cells by Northern Blotting and quantitative real time PCR. Nox4 promoter activity will also be examined to explore a putative feed back loop of HIF-21 on the Nox4 promoter as a potential mechanism of Nox4 expression. The second AIM of this proposal will elucidate the mechanisms by which the Nox oxidase components maintain HIF-2 alpha protein expression in VHL-deficient cells. We will specifically determine if the maintenance of HIF-2 alpha by ROS in VHL-deficient cells requires on-going transcription, mRNA stabilization or activation of signal transduction pathways that mediate translation. The third AIM will examine the effect of stable silencing of Nox4 and p22phox or superoxide antioxidants on HIF-2a transcriptional activity, tumor growth and cell invasiveness (other characteristics associated with VHL- deficiency) in vitro and in vivo. PUBLIC HEALTH RELEVANCE: VHL-deficiency plays a critical role in the pathogenesis renal clear cell carcinoma (RCC). The goal of this project is to elucidate pathogenic mechanisms of development and progression of VHL-deficient renal cell carcinoma, through increased generation of reactive oxygen species by the NAD(P)H-dependent oxidases which are essential in maintaining HIF-21 protein expression, a critical factor necessary and sufficient for the progression of VHL-deficient tumorigenesis. This project may result in the development of novel and specific therapeutic regiments to treat RCC.