Our long-term objective is to elucidate the mechanism of androgen-independent growth of prostate cancer. Ligand-independent nuclear localization of the androgen receptor (AR) is essential in androgen-independent growth. In androgen-sensitive prostate cancer cells, AR is localized to the cytoplasm in the absence of ligand and translocates to the nucleus in the presence of ligand. In contrast, in androgen-refractory prostate cancer cells, AR is localized to the nucleus even in the absence of ligand. These observations led to our hypothesis that the nuclear export signal (NES-AR) is inactive or no longer dominant over the nuclear import signal (NLS), NL1, in the absence of ligand in androgen-refractory prostate cancer cells. Our preliminary studies have identified nuclear export signal (NES-AR) in the ligand-binding domain (LBD). NES-AR is dominant over NL1, located in the DMA-binding domain (DBD) and hinge region, in the absence of ligand. Androgen binding to LBD represses NES-AR. Interestingly, NES-AR-mediated nuclear export is modulated by heat shock protein 90 (HSP90).
Five specific aims are proposed to further characterize NES-AR. 1. Confirm that NES causes nuclear export. GST-GFP-NES-AR fusion protein will be microinjected into the nuclei to test directly that NES-AR is a nuclear export signal instead of a cytoplasmic retention signal. 2. Identify amino acid residues in NES-AR that are necessary for the nuclear export and/or inhibition of the NL1 in the absence of ligand. Deletion and linker-scanning substitution mutagenesis will be employed to define the functionally important amino acid residues in NES. 3. Test the hypothesis that NES-AR is inactive or no longer dominant over NL1 in the absence of ligand in androgen-refractory prostate cancer cells. AR-positive androgen-refractory prostate cancer cells in culture and xenograft tumors will be used as models. 4. Identify and characterize proteins that bind to NES-AR. Yeast 2-hybrid screening of a human prostate cDNA library has identified 4 candidate NES-AR-binding proteins. Gain- and loss-of-function analysis will be carried out to determine their roles in the NES-AR-mediated nuclear export. 5. Characterize the role of HSP90 in NES-AR action. We will test if HSP90 directly interacts with NES-AR and/or influences the nuclear export machinery.
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