基于网络筛选到的PARD6A基因团簇在卵巢癌的作用机制研究

Ovarian cancer is the leading cause of death from gynaecological cancers, and studies about targeted therapies used as single agents have so far documented little efficacy in this disease. The significant mortality associated with epithelial ovarian cancer (EOC) suggests a great need to identify unique signaling hubs and their corresponding proteins that may play critical roles in the pathogenesis of this disease and thus, improve our understanding of ovarian cancer and its therapeutic strategies. The proposed studies offer an opportunity to employ a functional approach—the signaling network combined with RNAi synthetic lethality screens to identify novel signaling hubs of interesting tumor core regulators in EOC. We identified Src, ranked No. 2 tumor core regulator in our earlier studies published on Clinical Cancer Research, through data-mining meta-analyses integrating results from our multiple independent siRNA screens. We have used siRNA screening with a targeted library to identify a set of gene candidates that influence the response of EOC cells to the Src-targeting contemporary agent saracatinib, a clinically promising agent for treatment of EOC. Mapping the pattern of hits back to the network revealed not only well-validated cancer-related signaling hubs, such as EGFR, implying we have identified key ovarian cancer signaling hubs and the methodology is highly effective and practical; we also identified a few potential signaling hubs in EOC, such as PARD6A. Analysis of clinical relevance of the PARD6A gene cluster in EOC and the mode of action of the gene cluster will be a priority. Finally, novel therapeutic combinations involving validated hits and saracatinib in orthotopic xenograft models will be tested. We believe this cutting-edge approach-RNAi will help to nominate new targets for drug development, and to generate biomarkers of treatment response, and initiating clinical trials using combined molecular targeted agents. We hope this work will yield a paradigm that can subsequently be applied for multiple therapeutic applications in EOC.

卵巢癌是最致命的一种妇科癌症，靶向药物的单独用药往往收效甚微。本项目拟用功能研究的方法—信号网络结合RNA干扰筛选的方法来鉴定肿瘤核心调控基因在卵巢癌有协同作用的新的信号中心。在我们之前的工作中已经筛选和分析出了Src作为排位第二的肿瘤核心调控基因。以临床上有希望的Src激酶家族拮抗剂saracatinib(SAR)对候选靶向基因筛选和验证后，不仅筛选出了EGFR等众所周知的肿瘤靶点，证明了方法的高度有效和实用性；还筛选出了许多有潜力的卵巢癌致癌信号通路中心。我们选择了极性蛋白基因RAPD6A团簇进行进一步的功能研究，考察它们是否和卵巢癌临床上紧密相关、它们对卵巢癌细胞功能的影响及小鼠原位异种移植模型中与SAR联合用药的效果。相信用这种目前最先进的方法之一—RNA干扰，将会筛选出全新的卵巢癌信号中心，希望用以启动新的靶向药物和生物标记的研发，发展新的联合用药组合，最终改善卵巢癌的治疗。

关于分割缺陷蛋白6同源物α（Partitioning defective 6 homolog alpha，PARD6A）基因的研究，目前的主要聚焦在它作为细胞极化和细胞不对称分裂的一个重要的分子支架蛋白的作用。尽管已有少量文献报道了该基因可能与乳腺癌及肺癌的发生发展有关，并且多篇文献报道，极性蛋白很有可能影响肿瘤的迁移和侵润。但是，这个基因在卵巢癌中还未曾研究过，对这个基因在卵巢癌的作用和机制的研究，有可能定义出全新的卵巢癌靶向基因，用以启动新的靶向药物研究。..本项目首先考察PARD6A基因与卵巢癌的相关性，研究PARD6A基因的蛋白表达量与临床卵巢癌肿瘤相关性；其次，通过特异性沉默或过表达PARD6A基因，检测了该基因对卵巢癌细胞株功能的影响；此外，进一步研究了抑制PARD6A基因在小鼠卵巢癌转移模型中的作用和机制。..研究中发现，经免疫印迹技术检测到，PARD6A基因在浆液性卵巢癌细胞株SKOV3和A2780是异常高表达的；经免疫组织化学技术检测到PARD6A基因的蛋白在浆液性和粘液性卵巢肿瘤临床组织样本中高表达（其中浆液性是卵巢癌中最常见的组织学类型），比在正常卵巢组织中的表达显著增加。经细胞划痕实验、Transwell小室法、吸附实验及免疫荧光技术等检测到，沉默PARD6A基因，抑制了卵巢癌细胞株迁移、侵润能力和减弱吸附能力等；同时，PARD6A过量表达促进了锚定非依赖性生长、细胞的迁移和侵润等。经卵巢癌的小鼠肺转移模型实验，验证出以小发卡RNA（short hairpin RNA，shRNA）沉默PARD6A基因，可以在小鼠体内抑制卵巢癌细胞SKOV3的转移；对于PARD6A对卵巢癌抑制迁移和侵润的机制，本研究检测到PARD6A基因可能影响了Cdc42和RhoA介导的细胞迁移和侵润相关的信号通路。..综上所述，本研究旨在考察PARD6A基因在卵巢癌的作用和机制，希望籍此发现全新的卵巢癌靶向基因，用以改善卵巢癌的治疗和预后，防止肿瘤的恶化和复发。