Mechanisms of Renal Carcinogenesis

描述（由申请人提供）：透明细胞肾癌（RCC）是肾癌的主要形式。转移性肾癌患者的药物治疗通常无效，中位生存期仅为1年。RCC源于von Hippel-Lindau （VHL）基因的突变。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，下调HIF-2 α是阻断肿瘤形成的必要和充分条件。活性氧（ROS）如超氧阴离子和过氧化氢参与介导许多应激和生长反应的信号通路。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依赖性氧化酶亚基Nox4和p22phox增加ROS的产生，在没有VHL的情况下维持HIF-2 α蛋白的表达，在RCC的发病机制中起着关键作用。我们的研究目的是阐明vhl缺乏、NAD(P)H氧化酶的表达和激活增加以及HIF-2 α和靶基因表达增加在体外和体内参与RCC发生和进展的分子机制。AIM1将确定VHL调控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泛素连接酶复合物的一部分，该复合物通过26S蛋白酶体途径对特定蛋白质进行多泛素化，以调节蛋白质降解。这个过程需要将VHL结合到底物上。将确定Nox4或p22phox蛋白是否在蛋白酶体抑制剂存在下稳定，以及Nox4和p22phox是否在体外和体内以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。另外，Nox4可能在vhl缺陷细胞中通过增强mrna的转录而上调。我们将通过Northern Blotting和定量实时PCR来确定Nox4 mRNA水平是否在vhl缺陷细胞中升高。我们还将研究Nox4启动子的活性，以探索HIF-21对Nox4启动子的反馈环作为Nox4表达的潜在机制。本研究的第二个目的是阐明Nox氧化酶成分在vhl缺陷细胞中维持HIF-2 α蛋白表达的机制。我们将明确确定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AIM将在体外和体内研究稳定沉默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性（与VHL-缺乏相关的其他特征）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依赖性氧化酶增加活性氧的产生来阐明vhl缺陷肾癌发生和进展的致病机制，这是维持HIF-21蛋白表达所必需的，HIF-21蛋白是vhl缺陷肿瘤发生进展的必要和充分的关键因素。这个项目可能会导致新的和特殊的治疗方案的发展，以治疗肾细胞癌。

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.