REGULATION OF FGF RECEPTOR SPLICING IN PROSTATE CANCER

描述（由申请人提供）：前列腺生长和发育的正常途径受雄激素高度调节，间充质-上皮相互作用在这些过程中起关键作用。前列腺间充质细胞中雄激素受体激活启动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（FGF）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。该途径的维持需要成纤维细胞生长因子受体2（FGFR 2）外显子IIIb和IIIc的互斥剪接，以在上皮细胞中产生FGFR 2-IIIb，在间充质（或基质）细胞中产生FGFR 2-IIIc。FGFR 2-IIIb（而非FGFR 2-IIIc）对FGF-7和FGF-10有应答，FGF-7和FGF-10由前列腺基质细胞表达并调节上皮细胞增殖和分化。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中，FGFR 2的选择性剪接途径发生了转变。这些研究表明，由于异常剪接调节导致的FGFR 2-IIIb的缺失可能切断维持生长控制和分化的基质信号。我们假设细胞类型特异性剪接调节蛋白对于维持上皮细胞中FGFR 2-IIIb表达至关重要，并提出通过以下目的鉴定和表征此类反式作用蛋白因子：1）表征调节FGFR 2前mRNA剪接的功能性顺式元件，因为认为RNA顺式-剪接调节所需的元件结合指导剪接装置差异识别剪接位点的蛋白质。2)利用生物化学方法鉴定与RNA顺式元件结合的调节性反式作用蛋白因子。3)使用cDNA文库和FGFR 2小基因的新型表达克隆策略鉴定功能性剪接调控蛋白，可使用荧光标记筛选外显子IIIb。4)使用体内和体外功能性剪接分析确定推定的蛋白质反式因子的作用。这些推定的蛋白质的能力，以调节剪接位点的选择将在体内进行评估，以及使用一个强大的新的体外剪接测定法，概括细胞特异性剪接调节。FGFR 2-IIIb的上皮细胞特异性表达在介导前列腺发育期间和成年前列腺中控制上皮细胞增殖和分化的细胞过程中显然是关键的。鉴定调控FGFR 2剪接的特异性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.