CDK1调节AR-v7诱导前列腺癌去势抵抗的机制研究

It is well accepted that the castration resistance of prostate cancer is mainly due to the reactivation of androgen receptor (AR), but in recent years, researchers found that AR splice variant 7 (AR-v7) was able to induce castration resistance as well. It was also reported that the overexpression of AR-v7 in castration resistant prostate cancer was correlated to the application of the new generation AR antagonists, because setting AR as the only target will increase AR-v7 expression. Thus, it is necessary to target AR and AR-v7 simultaneously. Our previous studies found out that CDK1 could increase AR protein expression and also increase AR phosphorylation, and AR-v7 protein is a part of the AR protein, so AR-v7 is probably regulated by CDK1. Meanwhile, our pre-experiments have shown that CDK1 inhibitor could inhibit the prostate cancer cell line that overexpressed AR-v7. Thus, we get a hypothesis that CDK1 is able to regulate the protein expression, phosphorylation and dimerization of AR-v7 in order to induce the castration resistance of prostate cancer. In this project, we plan to study the mechanisms in CDK1 regulating AR-v7 protein expression, and to figure out whether CDK1 can phosphorylate AR-v7 or not, and also to develop the specific phosphorylation sites, and we are going to check out whether CDK1 could regulate AR-v7 dimerization or not, and to address the mechanism, beside, we will try to evaluate the feasibility of treating the castration resistant prostate cancer with CDK1 inhibitor in the animal model. This project aims to elucidate the mechanism of the regulation on AR-v7 by CDK1, in order to provide theoretical supports for the application of CDK1 inhibitor to treat castration resistant prostate cancer.

长期以来，前列腺癌的去势抵抗主要归因于雄激素受体（AR）的再激活，而近年来学者们又发现AR剪切变异体7（AR-v7，v7）也可诱导去势抵抗。目前新一代的去势药物已广泛应用于临床，它们虽能更有效地拮抗AR，但也会引起v7表达上调而导致治疗失败。因此，探索能同时抑制AR和v7的新药物靶点至关重要。本课题组前期发现CDK1可增加AR蛋白表达与磷酸化，因v7与AR有相似的蛋白结构，故v7可能亦受CDK1的调节；预实验还发现，CDK1抑制剂可抑制高表达v7的前列腺癌细胞系的增殖，故推测：CDK1可能参与上调v7的蛋白表达、磷酸化及二聚化从而诱导去势抵抗。本项目旨在研究并阐明CDK1对前列腺癌细胞系中v7的蛋白表达、磷酸化和二聚化等因素的影响、调节作用及其机制，探讨CDK1抑制剂治疗小鼠体内去势抵抗性前列腺癌异种移植物的可行性，为CDK1抑制剂应用于去势抵抗性前列腺癌的治疗提供重要理论基础和实验依据。

背景：长期以来，前列腺癌的去势抵抗主要归因于雄激素受体（AR）的再激活，新一代的去势药物虽然已广泛应用于临床，但在拮抗AR的同时也会引起AR-v7表达上调而导致治疗失败。因此，探索能同时抑制AR和AR-v7的新药物靶点至关重要。前期发现CDK1可增加AR蛋白表达与磷酸化，因AR-v7与AR有相似的蛋白结构，故AR-v7可能亦受CDK1的调节。本研究将探讨CDK1对前列腺癌细胞系22RV1中AR-v7蛋白表达、磷酸化的影响、调节作用及其机制，以及为CDK1抑制剂的临床应用提供实验依据。.研究内容：在体外实验中，用不同浓度的CDK1抑制剂处理高表达AR-v7的RV1细胞系，检测细胞增殖能力及迁移侵袭能力，并通过Western blot检测AR-v7、AR-v7磷酸化、PSA水平以及AR-v7在核内外表达分布的变化。并且利用ChIP-qPCR技术分别检测AR-v7与下游靶基因增强子序列的结合。为进一步验证上述假设，分别敲减和过表达CDK1，并再次检测上述指标变化。在裸鼠成瘤实验中，用CDK1抑制剂处理裸鼠，监测肿瘤大小，绘制肿瘤生长曲线。给予CDK1抑制剂处理10日后摘取肿瘤负荷组织，利用免疫组化检测AR-v7及磷酸化状态、PSA水平、AR-v7在细胞核内外的分布情况。.重要结果：随着CDK1抑制剂浓度的增加，细胞增殖能力、迁移侵袭能力逐渐下降，且AR-v7及其磷酸化水平、下游PSA表达水平呈阶梯式下调。与对照组相比，AR-v7在核内表达有明显下降趋势同时核外的AR-v7亦有降低。Co-IP及ChIP-qPCR实验表明处理组中AR-v7与P300、增强子序列的结合减少。在分别敲减和过表达CDK1后，实验结果与上述趋势一致。通过动物实验发现，与对照组相比，给药组裸鼠肿瘤生长速度较慢，终止时肿瘤体积较小，AR-v7相关指标呈下调趋势，核内AR-v7分布明显减少。.科学意义：CDK1抑制剂可通过下调AR-v7的表达，抑制AR-v7的转录调控，从而降低了前列腺癌细胞系的增殖和转移能力。由此证实CDK1抑制剂治疗高表达AR-v7去势抵抗性前列腺癌具有可行性，为其成为治疗去势抵抗性前列腺癌的新药提供实验依据和理论基础。