Mechanisms of Renal Carcinogenesis

描述(申请人提供)：肾透明细胞癌(RCC)是肾癌的主要形式。肾癌的内科治疗通常无效，转移性疾病患者的中位生存期仅为1年。肾癌是由von Hippel-Lindau(VHL)基因突变而来。在缺乏VHL的细胞中，缺氧诱导转录因子HIF-2α是稳定的，并上调了几个支持肿瘤生长的基因。在动物模型中，HIF-2α的下调是必要的，也是阻止肿瘤形成的充分条件。超氧阴离子和过氧化氢等活性氧物种参与了介导多种胁迫和生长反应的信号转导通路。NAD(P)H氧化酶(S)是参与肾脏氧化应激的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依赖的氧化酶亚基NOX4和p22Phox增加ROS的产生在RCC的发病中起关键作用，这两个亚单位在没有VHL的情况下维持HIF-2α蛋白的表达是至关重要的。我们的研究目的是阐明VHL缺乏、NAD(P)H氧化酶表达和/或激活以及HIF-2α和靶基因表达增加在体外和体内肾癌发生发展中的分子机制。AIM1将确定VHL调节NAD(P)H氧化酶NOX4及其膜结合伙伴p22Phox的催化单元的机制。VHL是E3泛素连接酶复合体的一部分，它通过26S蛋白酶体途径使特定的蛋白质泛素化，从而调节蛋白质的降解。这一过程需要VHL与底物的结合。将确定在蛋白酶体抑制剂存在的情况下，NOX4或p22Phox蛋白是否稳定，以及NOX4和p22Phox在体外和体内是否以VHL依赖的方式翻译后泛素化。将进行体外和体内结合研究，以确定VHL是否与NOX4和p22Phox结合。或者，在VHL缺陷细胞中，NOX4可能通过增强mRNAs的转录而上调。我们将通过Northern印迹和实时定量聚合酶链式反应来确定在VHL缺陷细胞中NOX4的mRNA水平是否增加。还将检测NOX4启动子的活性，以探索HIF-21在NOX4启动子上可能的反馈环作为NOX4表达的潜在机制。该建议的第二个目的将阐明在VHL缺乏的细胞中，NOx氧化酶组分维持HIF-2α蛋白表达的机制。我们将具体确定在VHL缺陷细胞中，ROS维持HIF-2α是否需要持续的转录、mRNA稳定或激活介导翻译的信号转导通路。第三个目的是在体外和体内检测稳定沉默NOX4和p22Phox或超氧化物抗氧化剂对HIF-2a转录活性、肿瘤生长和细胞侵袭力(与VHL缺乏症相关的其他特征)的影响。公共卫生相关性：VHL缺乏在肾透明细胞癌(RCC)的发病机制中起关键作用。本项目的目的是通过NAD(P)H依赖的氧化酶增加活性氧物种的产生来阐明VHL缺陷性肾癌的发生和发展的致病机制。NAD(P)H依赖的氧化酶是维持HIF-21蛋白表达的关键因素，HIF-21是VHL缺陷性肿瘤发生发展的必要和充分的关键因素。该项目可能导致开发新的、特异的治疗方案来治疗肾癌。

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.