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

描述(申请人提供)：肾癌是发病率和死亡率的主要原因。今年，大约54,000名美国人将被诊断为肾癌，大约13,000人将死于肾癌。在大多数肾癌中都发现了von Hippel-Lindau病基因VHL的缺陷，这使得对pVHL蛋白的研究成为了解这种疾病的关键起点。我们已鉴定JADE-1为pVHL相互作用蛋白。JADE-1是具有植物同源结构域(PhD)和害虫蛋白降解序列的JADE蛋白家族的第一个成员。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发挥作用。翡翠一号拥有所有这些活动。此外，JADE-1是由野生型pVHL稳定的，而不是由与肾癌相关的突变形式的pVHL稳定的。因此，pVHL和JADE-1之间的关系与肾癌的风险相关。作为一种受肿瘤抑制因子上调的蛋白质，JADE-1本身就具有肿瘤抑制作用。与这一提议至关重要的是，我们发现Jade-1结合并促进了β-连环素的泛素化和破坏，并抑制了典型的Wnt信号。β-连环蛋白是一种关键的癌蛋白。此外，pVHL通过JADE-1抑制β-连环素和Wnt信号转导。此外，β-连环素活性的抑制剂抑制肾癌细胞的生长。因此，在肾癌中发现了一条新的肿瘤抑制通路，即pVHL/JADE-1/β-连环素轴。目的1：JADE-1在肾癌中的作用。作为这项提案的主要目标，JADE-1在pVHL介导的肾癌和一般情况下在肾癌中的直接作用将被确定。Jade-1基因缺失的小鼠将与获得肾囊肿的条件性Vhlh缺失小鼠杂交，以确定这些途径之间的遗传相互作用。JADE-1小鼠将与患肾囊腺瘤的TSC2杂合子小鼠杂交。Jade-1小鼠将接受化学致癌物治疗。Jade-1在肾癌生长中的作用也将在细胞培养模型中进行检测。为Jade-1介导的肾癌泛素化寻找更多的底物。目的2：β-连环蛋白在肾癌中的作用。将在体外和体内研究中评估β-连环蛋白在肾脏肿瘤发生中的作用。重要的是，Jade-1和β-catenin之间的遗传相互作用将在Jade-1基因敲除小鼠和多发性肠道肿瘤(Min)小鼠身上进行测试，这些小鼠在腺瘤性息肉病结肠(APC)基因中存在缺陷，并增加了β-catenin的活性。还将在体外和裸鼠体内测试β-连环蛋白途径抑制剂对肾癌细胞的影响。目的3：pVHL对JADE-1蛋白稳定性的影响。JADE-1的翻译后修饰负责pVHL介导的稳定将被确定。这种方法将重点放在质谱学上。

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.