Ecdysoneless, A Novel Regulator of Androgen Receptor

项目摘要/摘要 前列腺癌(PCA)是美国男性最常见的非皮肤癌， 是导致男性癌症死亡的第二大常见原因。雄激素受体(AR)是众所周知的前列腺癌的驱动因素， 而雄激素剥夺疗法是最初的治疗方法，但基本上所有患者最终都会 耐阉割。这类患者使用下一代AR合成抑制剂(如 阿比特龙)或AR拮抗剂(如苯扎鲁胺)，但最终出现对这些药物的耐药性 限制了治疗选择。AR基因剪接缺陷是其耐药机制之一。 两个剪接变异体AR-V1和AR-V7的表达在激素抵抗的PCa中高出20倍。建议数 机制包括AR基因的基因组重排或特定剪接因子的上调。二 剪接因子、前信使核糖核酸加工因子8(PRPF8)和RNA解旋酶DDX39B及其类似物DDX39A 似乎与AR基因的输出和剪接有关。综合起来，大量证据表明 AR处理的作用可能有助于激素抵抗的更具侵袭性的PCa。因此，识别 调节AR处理的新机制是识别潜在未来的一个重要研究领域 前列腺癌患者的治疗靶点。 这份R21提案的重点是一种蛋白质ECD，它是苍蝇无蜕皮素基因的保守同源物 我们发现该产物在大多数前列腺癌细胞系和前列腺上皮内瘤变(PIN)中过表达。 和前列腺癌组织，其过度表达预测患者的生存期较短。AR+PCA中的ECD击倒 细胞AR mRNA和蛋白水平降低，细胞增殖、侵袭、迁移和锚定不受影响 成长。从机制上讲，ECD与扮演关键角色的R2TP辅助伴侣复合体的组件相互作用 在组装大的蛋白质或蛋白质-RNA复合体中的作用。重要的是，R2TP客户端复合体是 参与了RNA的加工。我们发现ECD与PRPF8以及DDX39A和DDX39B结合，这是已知的 规范信使核糖核酸的输出和剪接。基于这些新发现，我们假设一个三元复合体 ECD、R2TP和mRNA处理蛋白(PRPF8和DDX39)促进AR输出和PCa中的剪接 促进细胞增殖、细胞存活和肿瘤形成。靶向这条通路可以提供潜在的PCA 治疗策略。在这里，我们将首先定义含有ECD的mRNA处理机制的组成 在前列腺癌细胞中使用生化和共定位方法的复合体。然后我们将评估 ECD在招募mRNA加工机制来调节AR mRNA在PCa细胞中的输出和剪接。 最后，我们将评估通过ECD基因敲除或使用CK2靶向AR mRNA处理的可能性 使用或不使用苯扎鲁胺的抑制剂。因此，我们的研究将建立一个新的机制范式如何PCA 超增殖、细胞存活和激素抵抗是由一种新的机制驱动的，并以此为靶点 通路与抗雄激素受体药物协同抗肿瘤活性。

PROJECT SUMMARY/ABSTRACT Prostate Cancer (PCa) is the most frequently diagnosed non-cutaneous cancer in men in the US and the second most common cause of cancer deaths in men. Androgen receptor (AR) is well known driver of PCa, and androgen deprivation therapy is the initial management, but essentially all patients eventually become castration-resistant. Such patients are treated with next generation AR synthesis inhibitors (such as abiraterone) or AR antagonists (such as enzalutamide) but emergence of resistance to these agents eventually limits therapeutic choices. One of the mechanisms for such resistance is defect in AR mRNA splicing. Expression of two splice variants AR-V1, and AR-V7 is 20-fold higher in hormone resistant PCa. The proposed mechanisms include genomic rearrangement of the AR gene or upregulation of specific splicing factors. Two splicing factors, pre-mRNA processing factor 8 (PRPF8) and RNA helicase DDX39B and its paralog DDX39A seem to be involved in export and splicing of AR mRNA. Taken together a large body of evidence suggests a role of AR processing may contribute to more aggressive PCa that is hormone resistant. Therefore, identifying novel mechanisms those regulate AR processing is an important area of research to identify potential future therapy targets in PCa patients. This R21 proposal focuses on a protein ECD, a conserved homolog of the fly Ecdysoneless gene product, which we show is overexpressed in most PCa cell lines, and in Prostatic intraepithelial neoplasia (PIN) and PCa tissues, and its overexpression predicts shorter survival in patients. ECD knockdown in AR+ PCa cells reduced AR mRNA and protein levels, cell proliferation, invasion, migration and anchorage independent growth. Mechanistically, ECD interacts with components of the R2TP co-chaperone complex that plays a key role in the assembly of large protein or protein–RNA complexes. Importantly, the R2TP client complexes are involved in RNA processing. We show ECD binds to PRPF8, and DDX39A and DDX39B, proteins known to regulate mRNA export and splicing. Based on these novel findings, we hypothesize that a ternary complex of ECD, R2TP and mRNA processing proteins (PRPF8 and DDX39) promotes AR export and splicing in PCa to increase cell proliferation, cell survival and oncogenesis. Targeting this pathway can provide a potential PCa therapy strategy. Here, we will first define the composition of the ECD-containing mRNA processing machinery complexes in PCa cells using biochemical and co-localization approaches. Then we will assess the role of ECD in recruiting the mRNA processing machinery to regulate AR mRNA export and splicing in PCa cells. Finally, we will assess the potential of targeting AR mRNA processing by ECD knockdown or by use of CK2 inhibitor with or without enzalutamide. Thus, our studies will establish a new mechanistic paradigm of how PCa hyper-proliferation, cell survival and hormone resistance is driven by a novel mechanism and targeting this pathway synergizes with anti-androgen receptor agents for anti-tumor activity.