AR intracellular trafficking in prostate cancer

前列腺癌中的 AR 细胞内转运

• 批准号: 6916128
• 负责人: Zhou Wang
• 金额: $ 26.12万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6916128
• 关键词: SDS polyacrylamide gel electrophoresis; androgen receptor; binding proteins; biological signal transduction; cell growth regulation; chimeric proteins; green fluorescent proteins; heat shock proteins; immunocytochemistry; immunoprecipitation; intracellular transport; laboratory mouse; microinjections; molecular cloning; neoplastic growth; nuclear transport; prostate neoplasms; protein protein interaction; tissue /cell culture; transfection /expression vector; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：我们的长期目标是阐明前列腺癌雄激素非依赖性生长的机制。雄激素受体（AR）的配体非依赖性核定位在雄激素非依赖性生长中是必不可少的。在雄激素敏感的前列腺癌细胞中，AR在不存在配体的情况下定位于细胞质，并且在存在配体的情况下易位至细胞核。相比之下，在雄激素难治性前列腺癌细胞中，即使在没有配体的情况下，AR也定位于细胞核。这些观察结果导致我们的假设，核输出信号（NES-AR）是无活性的或不再占主导地位的核输入信号（NLS），NL 1，在雄激素难治性前列腺癌细胞的配体的情况下。我们的初步研究已经确定了核输出信号（NES-AR）的配体结合域（LBD）。在不存在配体的情况下，NES-AR相对于位于DMA结合结构域（DBD）和铰链区的NL 1是显性的。雄激素与LBD的结合抑制NES-AR。有趣的是，NES-AR介导的核输出受到热休克蛋白90（HSP 90）的调节。提出了五个具体目标，以进一步表征NES-AR。1.确认内斯导致核出口。将GST-GFP-NES-AR融合蛋白显微注射到细胞核中，以直接测试NES-AR是细胞核输出信号而不是细胞质滞留信号。 2.鉴定NES-AR中对于核输出和/或在不存在配体的情况下抑制NL 1所必需的氨基酸残基。将采用缺失和接头扫描取代诱变来确定内斯中功能重要的氨基酸残基。 3.在雄激素难治性前列腺癌细胞中，在不存在配体的情况下，检验NES-AR相对于NL 1无活性或不再占优势的假设。培养物中的AR阳性雄激素难治性前列腺癌细胞和异种移植肿瘤将用作模型。4.鉴定和表征与NES-AR结合的蛋白质。酵母双杂交筛选人前列腺cDNA文库，鉴定出4个候选NES-AR结合蛋白。将进行功能获得和丧失分析，以确定它们在NES-AR介导的核输出中的作用。5.描述HSP 90在NES-AR作用中的作用。我们将测试HSP 90是否直接与NES-AR相互作用和/或影响核输出机制。