双重mTORC1/2抑制剂在PTEN缺失介导的去势抵抗性前列腺癌多西他赛获得性耐药中的作用及机制

Metastatic castration resistant prostate cancer (mCRPC) remains the major cause of mortality. Though palliative docetaxel is the standard of care in men with mCRPC, most patients who initially respond to docetaxel inevitably develop resistance to the drug. Treatment options in the post-docetaxel setting are limited. Hyperactivation of the mTOR oncoprotein, which can form two biologically distinct protein complexes, is strongly indicated in prostate cancer and associated with castration-resistance. However its role in docetaxel-mediated acquired resistance remains unclear. Our previous studies have suggested that novel dual mTOR inhibitors may be more effectively than the traditional rapamycin in blocking prostate cancer type characterization. Utilizing a variety of biological detection approaches, the project will be investigated in the following respects: To investigate the impact of mTORCs on the acquired docetaxel resistance of CRPC cells; To examine the potential effects and mechanisms of agents targeting both mTORCs in reversing docetaxel resistance in vivo and in vitro; To explore the possible relationship between the mTOR-associated biomarkers and clinicopathological parameters in prostate cancer patients. The successful completion of the proposed studies will provide strong evidence in support of modifying the current mTOR-based intervention protocols by utilizing the novel dual mTORC1/2 inhibitors instead of rapalogs in the post-docetaxel setting of mCRPC.

转移性去势抵抗性前列腺癌（mCRPC）是临床上导致患者死亡的主要原因。多西他赛为mCRPC一线标准化疗药，但难以避出现耐药，且耐药之后可供选择的治疗方案有限。哺乳动物雷帕霉素靶蛋白(mTOR)的异常激活与前列腺癌的进展和去势抵抗的发生紧密相关，然而该蛋白在多西他赛介导的获得性耐药中的作用及机制尚不清楚。我们以往的研究结果表明新型双重mTOR抑制剂在阻断前列腺癌细胞的癌型表征方面比传统的雷帕霉素更有效。本课题拟采用多种生物学检测手段，围绕以下几方面展开研究：探索mTOR复合物在多西他赛获得性耐药的CRPC细胞中的作用；体内外模型检测不同类型mTOR抑制剂对逆转多西他赛获得性耐药的CRPC的作用及机制；在前列腺癌病人中检测mTOR相关标记物与临床病理指标相关性。该项目的成功完成将为改善现有mTOR为基础的干预策略，采用新型的双重mTORC1/2抑制剂用于多西他赛耐药的mCRPC提供有力证据。

本课题的主要研究目标是针对多西他赛在治疗CRPC 中出现的耐药问题尝试新的靶向治疗手段。本研究从mTOR 信号通路出发，围绕耐药机制，探讨mTORC2 信号途径的激活是否为多西他赛耐药的重要原因。首先在人前列腺癌细胞PC-3细胞系的基础上构建了多西他赛耐药（PC-3/DTX）细胞株。PC-3/DTX细胞蛋白水平结果表明， mTORC2的关键蛋白Rictor和下游p AKT(S473)在多西他赛耐药的前列腺癌细胞中表达异常增高。在病人的前列腺癌组织免疫组化结果中同样观察到了在后续发展为CRPC的病人前列腺癌组织样本中Rictor的表达相对于早期前列腺癌样本呈现异常高表达。通过构建Rictor敲除的慢病毒转染PC-3/DTX 细胞后，发现Rictor表达抑制后，细胞对多西他赛的敏感性增高，且随着Rictor表达的降低，细胞对多西他赛的敏感性逐渐升高，而细胞周期延长，细胞阻滞在G0/G1期，细胞增殖能力减弱。将mTORC1抑制剂Rapamycin和mTORC1/2抑制剂AZD8055加入PC-3/DTX细胞中后，在IC50浓度下检测抑制剂处理后的PC-3/DTX细胞对多西他赛的耐药程度变化，结果显示，AZD8055相对于Rapamycin对PC-3/DTX的多西他赛耐药性呈现更好的逆转效果。将CRPC 亲代细胞接种裸鼠建立多西他赛耐药动物模型，并行mTOR 抑制剂缓解多西他赛介导耐药的疗效评价，AZD8055在一定程度上具有多西他赛耐药体内逆转作用，但与雷帕霉素没有差异。取多西他赛耐药的前列腺癌患者的肿瘤组织样本及血液对照样本进行全外显子组测序，检出10个前列腺癌基因和4个多西他赛药物作用相关的可靠突变。以上研究结果将为今后多西他赛治疗失败的mCRPC 患者开创新的临床干预措施提供有价值的数据。