信号通路中心OCT1在卵巢癌中的作用机制及相关靶向药物研究

Ovarian cancer is one of the most common gynecologic cancers and highly heterogenous. Patients often relapse and develop resistance to anti-cancer therapy. Single-target drugs have so far documented little efficacy in ovarian cancer treatment. Inhibiting a signaling hub, by blocking multiple pathways essential for cancer cell survival, inducing cancer cell death, and preventing occurrence of relapse and resistance, is becoming a more important strategy in ovarian cancer therapy. Though inhibiting heat shock protein 90 (HSP90) as a signaling hub can effectively cause ovarian cancer cell death, HSP90 inhibitors often display insufficient specificity, unacceptable toxicity, and limited clinical efficacy. We have recently built a signaling network highly associated with ovarian cancer pathology and performed different experiments. We found a HSP90 downstream signaling sub-hub, octamer-binding transcription factor 1 (OCT1), which is directly relevant to occurrence and development of ovarian cancer, might be an ideal target for ovarian cancer treatment. Analysis of clinical relevance of OCT1 in ovarian cancer and the mode of action of the gene will be a priority. Then novel and important pathways regulated by OCT1 in ovarian cancer will be discovered by chromatin immunoprecipitation sequencing (ChIP-SEQ) and explored in detail. Finally, OCT1 inhibitors will be identified through small molecule microarray (SMM). We hope this work will nominate novel targets for drug development, and provide theoretical basis and experimental evidences for developing safe and effective targeted drugs and circumventing drug resistance.

卵巢癌是妇科常见恶性肿瘤之一，异质性高，易耐药和复发，单一靶点的靶向药物往往缺乏疗效。以直接抑制信号通路中心为手段，实现同时抑制多条信号通路，杀死肿瘤细胞，防止复发和耐药，正成为靶向药物开发新策略。尽管抑制信号通路中心热休克蛋白90（HSP90）对卵巢癌有极强的杀伤作用，但选择性较差，毒副作用较广泛，临床应用受限。本课题组构建了卵巢癌病理相关的信号网络，经多种实验发现，HSP90下游的、与癌症发生发展直接相关的次级信号通路中心--八聚体转录因子1（OCT1）有潜力成为卵巢癌治疗的理想靶点。本项目拟进行OCT1的临床相关性和生物学验证，以染色质免疫共沉淀测序（ChIP-SEQ）技术发现OCT1调控的、重要的卵巢癌相关信号通路，并利用小分子微阵列（SMM）技术筛选OCT1抑制剂。完成本项目将有助于发现卵巢癌治疗新靶点，为发展安全、高效、抗耐药的卵巢癌靶向药物提供理论指导和实验基础。

卵巢癌异质性高、易耐药和复发、不同分型的卵巢癌治疗方案较单一、死亡率高，靶向药物的单独用药往往收效甚微。同时使用多种药物抑制多个靶点或信号通路或用药物直接抑制信号通路中心正逐渐成为卵巢癌治疗的重要策略，这样有利于同时阻断癌细胞赖以生存的多条信号通路，诱导肿瘤细胞死亡，并尽可能避免耐药发生。我们之前的工作中筛选出了信号通路中心OCT1（八聚体结合转录因子1），OCT1基因作为一个转录因子，参与调控多种生理病理过程。本项目旨在进行这个新靶点的临床相关性和生物学实验确证，探索和发现OCT1调控的卵巢癌信号转导通路及作用机制。首先本项目收集了不同组织学类型的人卵巢癌临床肿瘤样本及正常卵巢上皮组织样本，发现在透明细胞癌和浆液性卵巢肿瘤临床组织样本中的表达量显著高于人正常临床卵巢组织样本。此外，检测到OCT1在浆液性卵巢癌细胞株SKOV3和HO8910中的表达量显著高于人卵巢上皮细胞株HOSEpiC。经Transwell小室法、实时荧光定量PCR技术等检测到，以小干扰RNA（siRNA）敲减OCT1，有效抑制了卵巢癌细胞株迁移和侵润能力；同时，过表达OCT1能够促进癌细胞迁移侵润。本研究还设计了多个短发夹RNA（shRNA）来沉默OCT1，并通过体外细胞实验筛选出能够显著抑制癌细胞迁移侵润的shRNA用于卵巢癌肺转移动物模型，并筛选出在小鼠体内抑制卵巢癌细胞转移的shRNA。为了深入研究OCT1基因在卵巢癌中的作用机制，通过Western blot等技术对迁移、侵润和EMT的标志性蛋白进行筛选和检测，发现敲减或过表达OCT1可以引起紧密连接蛋白ZO-1、人纤维连接蛋白Fibronectin及ERK1/2等蛋白表达的变化。这些结果显示，OCT1可能通过调节ZO-1和Fibronectin、ERK等蛋白相关信号通路调控卵巢癌细胞的迁移和侵润。该项目的实施为卵巢癌的诊疗提供了新的潜在靶点，并将可能用于启动新的靶向药物和生物标记的研发，最终改善卵巢癌的治疗。