Androgen Receptor Coregulators in Prostate Cancer

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DESCRIPTION (Provided by the applicant) Regulation of gene expression by the androgen receptor (AR) involves the association and action of transcriptional coregulatory proteins. Although a plethora of AR-interacting proteins have been identified, the physiological and pathological roles fulfilled by these factors in AR-mediated signaling pathways remain poorly understood. The goal of this application is to investigate and characterize the involvement of specific AR-coregulatory factors during normal prostate growth and the progression of prostate tumorigenesis. Given that prostate cancer cells are initially androgen-dependent and eventually progress into androgen-independent cells, we hypothesize that neoplastic changes in AR signaling pathways and/or the AR protein itself can abnormally affect the specific types of coregulatory protein complexes that bind to the receptor. To address these issues, we will generate stable FLAG epitope-tagged AR (f:AR) expressing cell lines from immortalized primary prostate cells (normal and malignant) and from metastatic prostate tumor cells. The lines will serve as biological tools with which we will immunoaffinity purify f:AR from hormone treated (and untreated) cells and subsequently examine and characterize the AR-associated proteins using a number of biochemical techniques. Our specific goals are to: (1) Determine whether distinct types of transcriptional coregulatory proteins are differentially associated with f:AR in normal versus malignant prostate cells. Stable f:AR-expressing prostate lines will be cultured in the presence (or absence) of distinct androgens and anti-androgens; f:AR-cofactor complexes will be purified and characterized by silver stain, Western blotting and mass spectrometry. (2) Determine whether androgen-independent signaling pathways induce f:AR-cofactor complex assembly in normal and malignant prostate cells. These studies will examine whether activation of specific receptor tyrosine kinases (previously implicated in prostate cancer and androgen-independent growth) can trigger specific f: AR-cofactor complex formation in the absence of AR ligands. (3) Determine whether pathologically associated mutations/polymorphisms in the AR gene affect f:AR-cofactor assembly. The f:AR cDNA will be subjected to site-directed mutagenesis and subsequently stably introduced into prostate cells. The mutated f:AR will be purified from ligand-treated cells and the associated cofactors identified and characterized. In summary, the studies outlined here should increase our fundamental understanding of the role of coregulatory factors in AR-mediated signaling pathways and potentially identify and define new targets for therapeutic agents in the treatment of prostate cancer.