REGULATION OF FGF RECEPTOR SPLICING IN PROSTATE CANCER

描述(申请人提供)：正常的生长和发育途径在前列腺中受雄激素高度调节，间充质-上皮的相互作用在这些过程中起着至关重要的作用。前列腺间充质细胞中雄激素受体的激活启动了促进上皮细胞分化的定向旁分泌途径。成纤维细胞生长因子家族的成员被认为是这一定向途径的关键旁分泌调节因子。要维持这一途径，需要相互排斥地剪接成纤维细胞生长因子受体2(FGFR2)外显子IIIb和IIIc，以在上皮细胞中产生FGFR2-IIIb，在间充质(或基质)细胞中产生FGFR2-IIIc。FGFR2-IIIb(而不是FGFR2-IIIc)对前列腺基质细胞表达的、调节上皮细胞增殖和分化的成纤维细胞生长因子-7和成纤维细胞生长因子-10有反应。在Dunning大鼠前列腺癌模型中的研究表明，在进展为雄激素非依赖性癌症的过程中，FGFR2的选择性剪接途径发生了转换。这些研究表明，由于异常剪接调控导致的FGFR2-IIIb的丢失可能会切断维持生长控制和分化的基质信号。我们假设细胞类型的特异剪接调节蛋白对维持上皮细胞中FGFR2-IIIb的表达至关重要，并建议通过以下目的来鉴定和表征这种反式作用蛋白因子：1)表征调控FGFR2前-mRNAs剪接的功能顺式元件，因为人们认为剪接调节所需的RNA顺式元件结合蛋白质，指导剪接装置区分识别剪接部位。2)利用生物化学方法鉴定与RNA顺式元件结合的调节反式作用蛋白因子。3)采用一种新的表达克隆策略，利用可利用荧光标记筛选外显子IIIb的cDNA文库和FGFR2微基因，鉴定功能剪接调节蛋白。4)利用体内和体外功能剪接实验确定可能的蛋白质反式因子的作用。这些假定的蛋白质调节剪接位点选择的能力将在体内进行评估，以及使用一种强大的新的体外剪接试验来概括细胞特异性剪接调节。FGFR2-IIIb的上皮细胞特异性表达在调节前列腺发育和成人前列腺中控制上皮细胞增殖和分化的细胞过程中显然是至关重要的。识别调控FGFR2剪接的特定蛋白质将有助于我们理解在快速进展的雄激素非依赖性癌症的发展过程中可能被破坏的分子途径。

DESCRIPTION (provided by applicant): Normal pathways of growth and development in the prostate are highly regulated by androgens, and mesenchymal-epithelial interactions play a crucial role in these processes. Androgen receptor activation in prostatic mesenchymal cells initiates directional, paracrine pathways that promote epithelial cell differentiation. Members of the fibroblast growth factor (FGF) family are implicated as key paracrine regulators of this directional pathway. Maintenance of this pathway requires mutually exclusive splicing of fibroblast growth factor receptor 2 (FGFR2) exons IIIb and IIIc to yield FGFR2-IIIb in epithelial cells and FGFR2-IIIc in mesenchymal (or stromal) cells. FGFR2-IIIb (but not FGFR2-IIIc) is responsive to FGF-7 and FGF-10, which are expressed by prostatic stromal cells and regulate epithelial cell proliferation and differentiation. Studies in model Dunning rat prostate cancers implicate a switch in the alternative splicing pathway of FGFR2 in progression to androgen independent cancers. These studies suggest that loss of FGFR2-IIIb due to aberrant splicing regulation may sever stromal signals that maintain growth control and differentiation. We hypothesize that cell-type specific splicing regulatory proteins are critical for maintenance of FGFR2-IIIb expression in epithelial cells and propose to identify and characterize such trans-acting protein factors through the following aims: 1) Characterize functional cis-elements that regulate splicing of FGFR2 pre-mRNAs as it is thought that RNA cis-elements required for splicing regulation bind proteins that direct the splicing apparatus to differentially recognize splice sites. 2) Identify regulatory trans-acting protein factors that bind to RNA cis-elements using biochemical approaches. 3) Identify functional splicing regulatory proteins using a novel expression cloning strategy using cDNA libraries and FGFR2 minigenes that can be screened for exon IIIb inclusion using fluorescent markers. 4) Determine the role of putative protein trans-factors using in vivo and in vitro functional splicing assays. The ability of these putative proteins to modulate splice site choice will be assessed in vivo as well as using a powerful new in vitro splicing assay that recapitulates cell-specific splicing regulation. Epithelial cell-specific expression of FGFR2-IIIb is clearly critical in mediating cellular processes that control epithelial cell proliferation and differentiation during prostate development and in the adult prostate. Identification of specific proteins that regulate FGFR2 splicing will contribute to our understanding of molecular pathways that can be disrupted in the development of rapidly progressive, androgen independent cancers.