Modeling androgen receptor in prostate cells

描述(申请人提供)：雄激素受体是一个关键分子，参与正常的前列腺组织发育和前列腺癌的生长和发展。雄激素受体突变与雄激素不敏感、睾丸女性化综合征以及前列腺癌有关。这些突变的特征有助于我们了解雄激素受体的结构和功能关系，以及它们在前列腺细胞发育、分化和生长中的作用。雄激素受体是一种配体诱导的转录因子，在不同的细胞环境中与共调节因子共同诱导分化和生长基因。雄激素受体的活性不仅受其天然配体雄激素的调节，还受生长因子和细胞因子如IL-6、EGF和胃泌素释放肽的调节。这些非类固醇配体激活酪氨酸激酶(如EGF-R和Src)和丝氨酸激酶(如Mark和酪蛋白激酶)，导致雄激素受体的翻译后修饰，包括磷酸化、苏莫化、乙酰化和被蛋白酶切割。前列腺癌最令人不安的方面是它转化为雄激素非依赖性，这是任何有效的治疗方法，包括雄激素消融术。越来越多的证据表明，非类固醇激素对雄激素受体的不适当激活和雄激素受体的基因突变可能是前列腺癌雄激素非依赖性进化的两个主要互补机制。我们的实验室一直在研究这两种机制，并在CWR22异种移植瘤向雄激素非依赖性进化过程中发现了一种新的雄激素受体突变。CWR22异种移植已经被世界各地的许多实验室使用，并成为研究雄激素非依赖性转化的首选模型之一。通过一种新的等位基因置换方法，我们希望证明复制雄激素受体基因外显子3的突变是该异种移植细胞系激素非依赖性转化的遗传基础，并揭示雄激素非依赖性的新机制。该突变使雄激素受体敏感，使其易于被细胞内的蛋白水解酶切割，并产生一个仅携带N-末端结构域的结构性活性受体。前列腺癌组织中常发现钙蛋白酶及其N末端裂解产物的过度表达。调节钙蛋白酶活性的信号，如Src酪氨酸激酶，可能以表观遗传的方式促进这种切割。我们的建议将有助于1)详细了解雄激素受体突变是CWR22.2雄激素非依赖性的基础)导致雄激素受体截短的分子途径，以及3)开发有效的等位基因替换策略来研究与肿瘤相关的雄激素受体突变。这一结果将为雄激素非依赖性的遗传和表观遗传机制提供深入的见解，这超出了CWR22的研究范围。

DESCRIPTION (provided by applicant): Androgen receptor is a critical molecule involved in the normal development of prostate tissues and in the growth and progression of prostate cancer. Mutations of androgen receptor are associated with androgen insensitivity and testicular feminization syndrome as well as prostate cancer. Characterization of these mutations have contributed greatly to our knowledge about the structure-function relationship of androgen receptor, as well as their roles of in development, differentiation and growth of prostate cells. Androgen receptor is a ligand induced transcriptional factor which in combination with co-regulators induce differentiation and growth genes in different cellular context. Androgen receptor activity is modulated not only by its natural ligand, androgen, but also by growth factors and cytokines such as IL-6, EGF and gastrin-releasing peptide. These non-steroid ligands activate tyrosine kinases (e.g., EGF-R and Src) and serine kinases (e.g., MARK and casein kinase), resulting in the post translational modification of androgen receptor including phosphorylation, sumoylation, acetylation and cleavage by proteases. The most troubling aspect of prostate cancer is its conversion to androgen independence, which defies any effective treatment including androgen-ablation. Accumulating evidence suggests that inappropriate activation of androgen receptor by non-steroids and genetic mutation of androgen receptor may represent two major complementary mechanisms responsible for the evolution of androgen independence of prostate cancers. Our lab has been studying both mechanisms and discovered a novel mutation of androgen receptor in CWR22 xenograft, during its evolution to androgen independence. CWR22 xenograft has been used by many laboratories world-wide and become one of the favorite models to study androgen independence conversion. Using a novel allele-replacement approach, we wish to demonstrate that the mutation which duplicates exon 3 of androgen receptor locus is the genetic basis of hormone refractory transformation of this xenograft cell line and uncover a novel new mechanism of androgen independence. This mutation sensitizes androgen receptor toward cleavage by cellular protease calpain and generates a constitutively active receptor carrying only the N-terminal domain. Overexpression of calpain and the cleaved N-terminal domain product are often found in prostate cancer tissues. Signals that modulate calpain activity such as Src tyrosine kinase may contribute to such cleavage in an epigenetic manner. Our proposal will contribute to 1) a detailed understanding of an androgen receptor mutation which underlies androgen independence in CWR22.2) the molecular pathway which contributes to the generation of truncated androgen receptor and 3) the development of an effective allele replacement strategy to study the tumor-associated androgen receptor mutation. The result will provide insights into the genetic and epigenetic mechanisms of androgen independence, which goes beyond the study of CWR22.