Project 2: Mechanisms Driving AR Full Length and Splice Variant Activities and Antagonist Resistance

我们的总体目标是剖析雄激素受体(AR)对转录的激活作用 和AR剪接变异体，以及在前列腺癌(PCa)中驱动这种活动的机制 对包括阿比特龙和阿比特龙在内的第二代AR靶向治疗产生耐药性 苯扎鲁胺。我们最近发现，AR招募蛋白磷酸酶1(pp1α)，它可以 然后去磷酸化CDK9并动员P-TEFb复合体。在目标1中，我们重点关注 与AR配体结合区相互作用的PP1调节蛋白的鉴定 (独立于雄激素)，调节这种相互作用，并确定是否/如何 AR-V7剪接变异体介导PP1TEFb募集和随后的P-α动员。我们的 以往和目前的研究也表明，AR N端的S81磷酸化 结构域(NTD)在推动转录方面起着重要作用，也是积极反馈的中枢 在低雄激素水平或在AR拮抗剂存在时可能放大AR活性的环。 因此，目标2侧重于确定直接或间接的辅助激活物相互作用 受S81磷酸化的调节，S81磷酸化在AR-V7活性中的作用，以及 CDK9抑制剂的治疗潜力。然后，目标3进一步专注于AR剪接变体。而当 AR剪接变体同源二聚体可以驱动转录，来自其他人和我们的证据表明 AR-FL和AR剪接变异体之间的异源二聚体起主要作用。因此，我们将测试 AR-FL/V7异源二聚体是脑内AR活性的主要介体假说 苯扎鲁胺耐药的PCA模型。然后，我们将重点介绍LBD在这些方面的角色 异二聚体，以及它们是否仍可能是治疗的靶点。最后，我们将确定 除了增加表达，似乎还增强了AR-V7的活性 对苯扎鲁胺耐药的PCA细胞。具体的目标是：1)确定分子基础 通过AR全长和剪接变异体招募Pp1α，2)决定AR S81的功能 驱动AR全长和剪接变异体的磷酸化，3)通过 哪一种AR-V7在耐ENZ的模型中驱动AR活动。

Our overall objective has been to dissect transcriptional activation by androgen receptor (AR) and AR splice variants, and mechanisms driving this activity in prostate cancer (PCa) that becomes resistant to second generation AR targeted therapies including abiraterone and enzalutamide. We recently found that AR recruits protein phosphatase 1 (PP1α), which can then dephosphorylate CDK9 and mobilize the P-TEFb complex. In Aim 1 we focus on identification of the PP1 regulatory protein that interacts with the AR ligand binding domain (independently of androgen) that mediates this interaction, and on determining whether/how the AR-V7 splice variant mediates PP1α recruitment and subsequent P-TEFb mobilization. Our previous and current studies also indicate that phosphorylation of S81 in the AR N-terminal domain (NTD) plays a major role in driving transcription, and is a hub for a positive feedback loop that may amplify AR activity at low androgen levels or in the presence of AR antagonists. Therefore, Aim 2 focuses on identification of coactivator interactions that are directly or indirectly regulated by S81 phosphorylation, the role of S81 phosphorylation in AR-V7 activity, and the therapeutic potential of CDK9 inhibitors. Aim 3 then focuses further on AR splice variants. While AR splice variant homodimers can drive transcription, evidence from others and us indicate that heterodimers between AR-FL and AR splice variants play a major role. Therefore, we will test the hypothesis that AR-FL/V7 heterodimers are the major mediators of AR activity in enzalutamide-resistant PCa models. We then will focus on the role of the LBD in these heterodimers, and whether they it may still be a therapeutic target. Finally, we will identify mechanisms in addition to increased expression that appear to be enhancing AR-V7 activity in enzalutamide-resistant PCa cells. The Specific Aims are 1) Determine the molecular basis for PP1α recruitment by AR full length and splice variants, 2) Determine the function of AR S81 phosphorylation in driving AR full length and splice variants, 3) Identify mechanisms through which AR-V7 drives AR activity in ENZ-resistant models.