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.
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