Genetic Dissection of Context-Dependent and Opposing Roles of Androgen Receptor

描述（由申请人提供）：雄激素受体（AR）调节男性生理，是前列腺癌个体发生和进展的核心。通过识别遗传风险因素和新的治疗靶点，了解AR对人类健康的重要影响。在正常和病理细胞生长中，AR通过基因靶点指导不同的行为，如增殖或分化，这些靶点随启动子、细胞类型和阶段而变化。AR的这些环境依赖性和相反功能在前列腺癌中很明显，AR最初抑制肿瘤发展，但最终促进肿瘤发生。AR基因的种系和体细胞突变突出了AR的双重性质。我们假设变异AR的差异基因调控机制可能揭示了对抗野生型AR病理行为的更有效方法。为了利用遗传工具，我们通过同源重组将小鼠AR基因转化为人类序列。我们首先测试了与男性表型变异相关的多态性谷氨酰胺(Q)通道的影响。在转基因模型中，人源化AR小鼠Q束长度影响雄激素轴，并影响前列腺癌的发生、进展和治疗反应。这些肿瘤的体细胞AR突变证明了治疗的选择性。此外，这在一组精选的人类患者中得到了证实。选择用于进一步研究的影响n端结构域的突变影响配体依赖性和非依赖性激活。突变的ar使用多种机制最终改变靶基因的选择性以促进疾病。我们将使用多管齐下的遗传和基因组分析来定义上游和下游机制，通过这些机制，Q通道变体子集和突变ar（“变体”）赋予差异基因调控。我们的目标是在新的细胞和小鼠模型中定义变异的AR活性，并通过下一代深度测序识别异常功能的下游效应物和上游激活物。在Aim I中，一小部分变体ar将在正常细胞和癌细胞中稳定表达，以观察它们在体外和异种移植肿瘤中不同的生物学程序。在Aim II中，变异ARs的病理活性将在具有前列腺靶向转基因的小鼠、一个PTEN等位基因缺失易患癌症的小鼠以及具有不同雄激素轴强度的小鼠中进行测试。在Aim III中，由变体ARs指导的基因表达程序将通过高通量转录本测序（RNA-tag-Seq）来确定直接和间接的AR靶标。染色质免疫沉淀和高通量测序（ChIP-Seq）将探测不同启动子识别并突出指导不同生物学结果的共调节因子。除了独特的作用外，AR变体之间的共同差异将突出野生型AR的关键下游靶点和上游调节因子。对这些AR变体的综合遗传和基因组研究将揭示野生型AR功能的各个方面，以便将来我们可以在不同背景下选择性地阻断AR的疾病促进作用，同时保留AR的疾病抑制作用。这些结果对今后的研究具有直接的应用价值和广泛的意义。

DESCRIPTION (provided by applicant): The androgen receptor (AR) regulates male physiology and is central to the ontogeny and progression of prostate cancer. Understanding AR's role strongly impacts human health by identifying genetic risk factors and new therapeutic targets. In normal as well as pathologic cell growth, AR directs divergent actions, such as proliferation or differentiation, through gene targets that vary with promoter, cell type and stage. These context- dependent and opposing functions of AR are evident in prostate cancer where AR initially suppresses tumor development but ultimately promotes oncogenesis. AR's dual nature is highlighted by germ-line and somatic mutations of the AR gene. We hypothesize that mechanisms underlying differential gene regulation by variant ARs may reveal more effective ways to combat pathological actions of the wild type AR. In order to use genetic tools, we converted the mouse AR gene to the human sequence by homologous recombination. We first tested the effect of a polymorphic glutamine (Q) tract associated with male phenotypic variation. Q tract length in humanized AR mice impacts the androgen axis, and affects initiation, progression and therapy response of prostate cancer in a transgenic model. Somatic AR mutations in these tumors evince selection by treatment. Moreover, this was substantiated in a select set of human patients. Mutations chosen for further study affecting the N-terminal domain influence ligand-dependent and -independent activation. The mutant ARs use multiple mechanisms that ultimately alter target gene selectivity to promote disease. We will use a multipronged genetic and genomic analysis to define upstream and downstream mechanisms by which a subset of Q tract variants and mutant ARs ("the variants") confer differential gene regulation. Our aims will define variant AR activity in novel cell and mouse models and identify the downstream effectors and upstream activators of aberrant function by Next Generation deep sequencing. In Aim I, a small set of variant ARs will be stably expressed in normal and cancer cells to view their distinct biological programs in vitro and in xenograft tumors. In Aim II, pathologic activity of variant ARs will be tested in mice with prostate- targeted transgenes, in mice predisposed to cancer by deletion of one PTEN allele, and in mice with varying androgen axis strength. In Aim III, gene expression programs directed by the variant ARs will be defined by high-throughput sequencing of transcripts (RNA-tag-Seq) to identify direct and indirect AR targets. Chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) will probe differential promoter recognition and highlight coregulators directing different biological outcomes. Differences shared amongst AR variants, in addition to unique actions, will highlight critical downstream targets and upstream regulators of wild type AR. Combined genetic and genomic study of these AR variants will reveal aspects of wild type AR function so that in future we may selectively block disease-promoting while retaining disease-inhibiting effects of AR in diverse contexts. These results will have immediate applications and broad significance for future studies. PUBLIC HEALTH RELEVANCE: As demonstrated by recent advances in prostate cancer therapy aimed at the steroid signaling pathway, the androgen receptor remains the high impact target. Our humanized mouse model provides a powerful means to compare the functions of a set of variant androgen receptors, using molecular, genetic and genomic approaches. This will identify common features that drive cell pathways towards proliferation versus differentiation and provide a comprehensive basis for targeting the functions of wild type androgen receptor, rather than the receptor itself, for more effective cancer treatments that are tailored to specific contexts.