Role of TFE3 in Renal Cell Carcinoma

描述（由申请人提供）：本提案的目标是确定由正常和致癌形式的TFE3调节的细胞过程和基因表达模式，TFE3是一种参与乳头状肾细胞癌（PRCC）和TGFb反应性的转录因子。TFE3是MiT转录因子家族的一员，包括在小眼症中发生突变的基因Mitf，它与TGFa、Wnt和c-kit信号通路有关。我们的初步实验表明，TFE3的异位表达，而不是其他MiT蛋白，触发凋亡细胞死亡，暗示TFE3参与细胞周期控制。TFE3基因易位存在于散发性幼年型PRCC的一个亚群中，支持TFE3突变在转化过程中的关键作用。已经确定了四个不同的TFE3易位伙伴，它们产生了将TFE3的c端部分（包含DNA结合、二聚化和转录激活结构域）与其他基因产物连接起来的融合蛋白。这些嵌合蛋白如何促进肾上皮细胞转化过程的分子基础，以及TFE3在肾脏中的正常功能和细胞因子反应性的分子基础尚不清楚。我们在Aim 1中提出鉴定和表征MiT蛋白（包括TFE3）在肾上皮细胞中的活性。为了实现这一目标，我们创造了一种跨显性阴性（TDN）蛋白，可以特异性地阻断所有MiT家族成员的活性。在Aim 2中，我们将监测TDN和TFE3癌蛋白表达对肾上皮细胞细胞因子反应性的影响。作为这一目标的一部分，我们将检验TFE3是c-met信号传导介质的假设；肝细胞生长因子（HGF）受体c-met在遗传性PRCC的一个亚群中发生突变。在Aim 3中，我们将确定TFE3负责细胞凋亡的部分和活性。在Aim 4中，我们将在肾上皮细胞和非肾细胞中表达TDN、癌蛋白和正常TFE3蛋白，进行比较基因表达分析，以鉴定受TFE3蛋白调控的靶基因。包括TAT-TDN的评估，这是一种HIV TAT蛋白与TDN蛋白的融合。这将允许TDN自主渗透细胞膜，使我们能够抑制静止细胞中的TFE3活性。一个重要的结果将是建立TDN阻断TFE3癌蛋白活性的能力。我们的研究将加深我们对TFE3突变如何促进肾细胞转化的理解，并为PRCC的新干预策略提供见解。

DESCRIPTION (provided by applicant): The goal of this proposal is to identify cellular processes and gene expression patterns regulated by normal and oncogenic forms of TFE3, a transcription factor involved in papillary renal cell carcinoma (PRCC) and TGFb responsiveness. TFE3, a member of the MiT transcription factor family that includes Mitf, the gene mutated in microphthalmia, has been implicated in TGFa, Wnt, and c-kit signaling pathways. Our preliminary experiments indicate that ectopic expression of TFE3, but not other MiT proteins, triggers apoptotic cell death, implicating TFE3 in cell cycle control. Translocations of the TFE3 gene are found in a subset of sporadic juvenile forms of PRCC, supporting a key role for TFE3 mutation in transformation. Four distinct TFE3 translocation partners have been identified, creating fusion proteins linking the C-terminal portion of TFE3, which contains the DNA binding, dimerization, and a transcriptional activation domain, with the other gene product. The molecular basis for how these chimeric proteins contribute to the transformation process of renal epithelial cells, and the normal function of TFE3 in the kidney and in cytokine responsiveness, are not known. We propose in Aim 1 to identify and characterize the activity of MiT proteins, including TFE3, in renal epithelial cells. Towards this goal, we have created a transdominant negative (TDN) protein that specifically blocks the activity of all MiT family members. In Aim 2, we will monitor the impact of the TDN and TFE3 oncoprotein expression on cytokine responsiveness of renal epithelial cells. As part of this aim, we will test the hypothesis that TFE3 is a mediator of c-met signaling; c-met, the hepatocyte growth factor (HGF) receptor, is mutated in a subset of hereditary PRCC. In Aim 3, we will identify portions and activities of TFE3 responsible for apoptosis. In Aim 4, we will express the TDN, oncoproteins, and normal TFE3 proteins in renal epithelial and non-kidney cells for comparative gene expression analyses to identify target genes regulated by the TFE3 proteins. Included will be the evaluation of TAT-TDN, a fusion of the HIV TAT protein to the TDN protein. This will allow the TDN to autonomously permeate the cell membrane and enable us to inhibit TFE3 activity in quiescent cells. An important outcome will be to establish the ability of the TDN to block TFE3 oncoprotein activity. Our studies will enhance our understanding of how TFE3 mutation promotes transformation of renal cells, and provide insight into new interventive strategies for PRCC.