Androgens are important regulators of cell growth and prostate gland development. Androgens exert their biological effects on target tissues through an intracellular receptor protein named androgen receptor (AR). Recent evidence suggests that transcriptional regulation by AR require a diverse group of proteins termed coactivators. Coactivators portray a growing class of proteins that interact with receptors in a hormone dependent manner and are required for maximal gene activation by the receptors. It is the general belief that coactivators enhance receptor function by acting as a bridge between the DNA-bound receptor and basal transcription factors of the preinitiation complex and also by providing histone acetyl transferase activity, HAT, which disrupts the local repressive chromatin structure and contributes to increase transcriptional activity. Recently, our laboratory has cloned, E6-associated protein (E6-AP), as a coactivator of steroid receptors including that of AR. E6-AP possesses ubiquitin-protein ligase activity, instead of HAT activity. Our preliminary observation suggests that E6-AP act as a coactivator of AR and is overexpressed both in mouse and human prostate tumors. The initial data from E6-AP knockout mice also suggest that E6-AP play a major role in the normal development of prostate gland. The prostate glands of the E6-AP knockout mice are smaller and weigh less compare to that of wild-type normal littermates. Furthermore, p53 levels are elevated in E6-AP knockout prostate glands. Additionally, the AR expression is down in E6-AP knockout prostate. Based on these initial findings, we hypothesize that E6-AP, an important modulator of prostate gland, androgen receptor-mediated signal transduction pathway and cell cycle control, is functionally significant in the development of normal prostate gland and prostate cancer. In order to understand the exact role of E6-AP in the development of normal prostate gland and in the development of prostate tumors, we propose the following three specific aims: A) Development and analysis of animal models for the overexpression of wild type E6-AP, C833S mutant E6-AP (an E3 ubiquitin-protein ligase defective mutant) and loss of E6-AP (E6-AP knockout) in the prostate gland, B), Design and development of stable in vitro models for the overexpression of wild-type and C833S mutant E6-AP proteins in the prostate cancer cell lines, and C) Expression analyses of endogenous E6-AP, p53 and AR in prostate tumor biopsy samples.
