TRPC3膜定位及其下游信号通路影响卵巢癌顺铂耐药的机制

Chemotherapy with platiniferous fomula represented by cisplatin, is essential to treat epithelial ovarian cancer. The acquired chemoresistance brings 80% patients into tumor relapse, which leads to cancer related death. The p53 activated pathway of cisplatin (CDDP) resistance accounts for the major topic of chemoresistant research. Considered the mutant p53 in the high grade serous adenocarcinoma, the largest portion of ovarian cancer, it must be great beneficial to perform research on the mechanism of the formation of chemoresistance through p53 independent pathway and the critical targets for regain the chemosensitivity of cancer. Our previous observation showed that the expression of TRPC3, a membranic ion channel, corrlated to early relapse in ovarian cancer. The preliminary studies revealed that TRPC3 expression was elevated in recurrent tumors compared to that before chemotherapy. TRPC3 expression also increased in CDDP-resistant cell lines. Downregulation of TRPC3 could partially recovered the sensitivity to CDDP. Together with previous reports on the fact of caveolin (Cav)-1 colocalization with TRPC3 on cell membrane, we hypothesized that the regulation on the localization of TRPC3 by Cav-1 or other uperstreaming molecules plays an important roll on the formation of the resistance to cisplatin, and further, the targeted therapy might overcome this resistance. We design the experiments to investigate the upstreaming regulator of the TRPC3 locolization and downstreaming Calcium-related signals in relation to chemoresistance, and to discovery potential molecular target to revitalize the CDDP sensitivity. This research is the first time to explore the mechanisms of TRPC3 in the relation of chemoresistance in ovarian cancer, and will evaluate the effect of TRPC3-targeted cancer therapy.

铂类药物为主的化疗是卵巢癌治疗的重要手段，肿瘤耐药导致约80%的患者因复发而死亡。目前铂类耐药研究主要围绕p53依赖的DNA修复途径，而发病率最多的高级别浆液性癌是p53突变型。因此，在卵巢癌中开拓非p53依赖的耐药机制研究，具有重要的科研及临床意义。申请人以往观察到高表达瞬时受体电位通道蛋白(TRPC)3患者倾向早期复发；且化疗后复发肿瘤TRPC3较化疗前有所增加；耐药细胞株的TRPC3亦有增加；体外抑制卵巢癌细胞TRPC3可部分恢复对顺铂的敏感性。结合文献报道窖蛋白(Cav)-1参与TRPC3细胞膜上定位的调控，假设Cav-1对TRPC3定位的调控参与顺铂耐药。本课题将就调控TRPC3膜定位的相关蛋白及其下游信号分子与顺铂耐药进行深入探讨，寻找有效逆转顺铂耐药的靶分子。这是首次在卵巢癌开展TRPC3相关的耐药机制的研究，将为以TRPC3为靶点的卵巢癌治疗提供理论依据。

铂类药物为主的化疗是上皮性卵巢癌治疗的重要手段，肿瘤耐药性导致约80%左右的患者因复发而死亡。本研究基于以往工作基础，探讨TRPC3与顺铂化疗耐药的关系。本研究体外诱导卵巢癌细胞株HEY，制备耐药细胞株HEYcp，其5 μg/ml和20 μg/ml的顺铂的抑制率降低54.3%和47.4%。并与文献中明确的顺铂敏感株A2780和耐药株A2780cp（5 μg/ml和20 μg/ml抑制率降低29.0%和24.2%）进行体外实验，以证实TRPC3通路与顺铂耐药之间的关系。体外实验观察到，耐药细胞株HEYcp与A2780cp的TRPC3的蛋白表达为敏感株HEY和A2780的1.80和1.74倍；Cav-1和表达为2.66和3.26倍。50例卵巢高级别浆液性癌的术后化疗后复发的标本中可见到，TRPC3与Cav-1的表达与首次手术切除肿瘤标本相比均有增加，且具有统计学差异。采用细胞核、细胞质和细胞膜分离技术处理培养的细胞株，提取蛋白免疫印迹检测，观察到TRPC3与Cav-1蛋白在耐药株与敏感细胞株相比，细胞核和细胞质中的含量均降低，而细胞膜上表达增高。免疫共沉淀观察到TRPC3与Cav-1可相互结合，提示两者相互的调控作用。用Cav-1结合域表达质粒转染HEYcp细胞株，共聚焦显微镜观察，可见TRPC3在细胞膜上的定位减少。在TRPC3稳转株中，抑制TRPC3下游分子Calpain、Akt和Bcl-2可部分削弱TRPC3的诱导耐药作用，说明TRPC3的耐药机制与下游分子相关。使用TRPC3的抑制剂抑制其功能或采用shRNA稳定敲减其表达水平，均能有效恢复细胞对顺铂的敏感性，表现在凋亡水平增加、细胞侵袭和细胞集落形成能力降低。提示以TRPC3-Cav-1-Calpain为靶点的卵巢癌治疗可能逆转肿瘤对顺铂药物的敏感性。