REGULATION OF FGF RECEPTOR SPLICING IN PROSTATE CANCER

描述（申请人提供）：前列腺的正常生长和发育途径受到雄激素的高度调节，而间充质-上皮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的雄激素受体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（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。维持这一通路需要成纤维细胞生长因子受体2 （FGFR2）外显子IIIb和IIIc的互斥剪接，从而在上皮细胞中产生FGFR2-IIIb，在间充质（或基质）细胞中产生FGFR2-IIIc。FGFR2-IIIb（但不是FGFR2-IIIc）对FGF-7和FGF-10有反应，FGF-7和FGF-10由前列腺基质细胞表达，调节上皮细胞的增殖和分化。Dunning模型大鼠前列腺癌的研究表明，FGFR2的选择性剪接途径在雄激素非依赖性癌症的进展中发生了开关。这些研究表明，由于剪接调节异常导致的FGFR2-IIIb缺失可能会切断维持生长控制和分化的基质信号。我们假设细胞类型特异性剪接调节蛋白对于维持上皮细胞中FGFR2- iiib的表达至关重要，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FGFR2前mrna剪接的功能顺式元件，因为人们认为剪接调节所需的RNA顺式元件结合指导剪接装置区分识别剪接位点的蛋白质。2)利用生化方法鉴定与RNA顺式元件结合的调节反式作用蛋白因子。3)利用cDNA文库和FGFR2小基因（可通过荧光标记筛选外显子IIIb包含），利用新的表达克隆策略鉴定功能性剪接调节蛋白。4)通过体内和体外功能剪接试验确定推定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，并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。在前列腺发育和成人前列腺中，FGFR2-IIIb的上皮细胞特异性表达在介导控制上皮细胞增殖和分化的细胞过程中显然是关键的。鉴定调节FGFR2剪接的特定蛋白将有助于我们理解在快速进展的、不依赖雄激素的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.