The pVHL/Jade-1/beta-catenin axis in renal cancer

描述（由申请人提供）：肾癌是发病率和死亡率的主要原因。今年，大约有5.4万美国人将被诊断出肾癌，大约1.3万人将死于肾癌。在大多数肾癌中发现了von Hippel-Lindau病基因VHL的缺陷，这使得pVHL蛋白的研究成为了解该疾病的关键起点。我们已经确定Jade-1是pVHL相互作用蛋白。Jade-1是Jade蛋白家族中第一个具有植物同源结构域（phd）和PEST蛋白降解序列的成员。PHD基序可能在蛋白质相互作用、转录激活、组蛋白乙酰化和泛素连接酶活性中起作用。Jade-1有所有这些活动。此外，Jade-1被野生型pVHL稳定，而不被与肾癌相关的突变型pVHL稳定。因此，pVHL与Jade-1的关系与肾癌风险相关。Jade-1作为一种被肿瘤抑制因子上调的蛋白，本身具有肿瘤抑制特性。与此建议关键相关的是，我们发现Jade-1结合并促进β -连环蛋白的泛素化和破坏，并抑制典型Wnt信号传导。-连环蛋白是一种关键的癌蛋白。此外，pVHL通过Jade-1抑制β -连环蛋白和Wnt信号。此外，β -连环蛋白活性抑制剂可抑制肾癌细胞的生长。因此，在肾癌中发现了一个新的肿瘤抑制通路，pVHL/Jade-1/ β -catenin轴。以下目标将被探讨：目标1:Jade-1在肾癌中的作用。作为本提案的主要目的，将确定Jade-1在pvhl介导的肾癌和一般肾癌中的直接作用。Jade-1缺失小鼠将与获得肾囊肿的条件Vhlh缺失小鼠杂交，以确定这些途径之间的遗传相互作用。将Jade-1小鼠与Tsc2杂合小鼠杂交，得到肾囊腺瘤。Jade-1小鼠将接受化学致癌物的治疗。Jade-1在肾癌生长中的作用也将在细胞培养模型中进行研究。将寻找jade -1介导的肾癌泛素化的其他底物。目的2:β -连环蛋白在肾癌中的作用。β -连环蛋白对肾脏肿瘤发生的作用将在体外和体内研究中进行评估。重要的是，Jade-1和β -连环蛋白之间的基因相互作用将通过Jade-1敲除小鼠和多发性肠瘤（Min）小鼠进行测试，这些小鼠在腺瘤性大肠息肉病（Apc）基因中存在缺陷，并且β -连环蛋白活性增加。β -连环蛋白途径抑制剂的作用也将在体外和裸鼠中对肾癌细胞进行测试。目的3:pVHL对Jade-1蛋白稳定性的影响。Jade-1翻译后的修饰负责pvhl介导的稳定将被确定。这种方法将侧重于质谱分析。

DESCRIPTION (provided by applicant): Renal cancer is a major cause of morbidity and mortality. Roughly 54,000 Americans will be diagnosed and about 13,000 will die from renal cancer this year. Defects in the von Hippel-Lindau disease gene VHL are found in the majority of renal cancers, making study of the pVHL protein a key starting point to understand the disease. We have identified Jade-1 as a pVHL interacting protein. Jade-1 is the first member of a Jade protein family that has plant homeodomains (PHDs) and PEST protein degradation sequences. PHD motifs may function in protein interactions, transcriptional activation, histone acetylation and ubiquitin ligase activity. Jade-1 has all these activities. Moreover, Jade-1 is stabilized by wild-type pVHL, but not by mutated forms of pVHL associated with renal cancer. Thus, the relationship between pVHL and Jade-1 correlates with renal cancer risk. As a protein upregulated by a tumor suppressor, Jade-1 has tumor suppressor properties itself. Of critical relevance to this proposal, we have found that Jade-1 binds and promotes the ubiquitination and destruction of beta-catenin and inhibits canonical Wnt signaling. Beta-catenin is a key oncoprotein. Moreover, pVHL inhibits beta-catenin and Wnt signaling through Jade-1. Furthermore, inhibitors of beta-catenin activity inhibit renal cancer cell growth. Thus, a new tumor suppressor pathway in renal cancer has been identified, the pVHL/Jade-1/beta-catenin axis. The following aims will be explored: Aim 1: The role of Jade-1 in renal cancer. As the major objective of this proposal, the direct role of Jade-1 in pVHL-mediated renal cancer and in renal cancer generally will be determined. Jade-1 null mice will be crossed with conditional Vhlh null mice, which get renal cysts, to determine the genetic interaction between these pathways. Jade-1 mice will be crossed with Tsc2 heterozygous mice, which get renal cystadenomas. The Jade-1 mice will be treated with chemical carcinogens. The role of Jade-1 in renal cancer growth will be examined in cell culture models as well. Additional substrates for Jade-1-mediated ubiquitination in renal cancer will be sought. Aim 2: The role of beta-catenin in renal cancer. The contribution of beta-catenin to renal tumorigenesis will be assessed in in vitro and in vivo studies. Importantly, a genetic interaction between Jade-1 and beta-catenin will tested using Jade-1 knockout mice and multiple intestinal neoplasia (Min) mice, which have a defect in the adenomatous polyposis coli (Apc) gene and have increased beta-catenin activity. The effect of inhibitors of the beta-catenin pathway will also be tested on renal cancer cells in vitro and in nude mice. Aim 3: The effect of pVHL on Jade-1 protein stability. The post-translational modifications of Jade-1 that are responsible for pVHL-mediated stabilization will be identified. This approach will focus on mass spectrometry.