新候选癌基因PPP2R1A在上皮性卵巢癌患者中的突变及差异磷酸化蛋白质组学研究

Epithelial ovarian cancer (EOC) is a group of serious disorders affected by both genetic and environmental factors, which greatly affected mental health in China. Recent studies have found that EOC patients harbored recurrent PPP2R1A mutations, which is a structural subunit of protein phosphatase 2A (PP2A); and these results were also confirmed by our recent work. Regarding the relationship between PPP2R1A mutations and the carcinogenesis of EOC, three key questions remain to be answered, that is, whether PPP2R1A mutations could promote the pathogenesis of EOC? Whether these mutations would contribute to the development of EOC via affecting the catalytic activity of PP2A? Which signaling pathways play synergetic roles with PPP2R1A mutations in the development of EOC? Based on our previously acquired results, we plan to perform the following experiments to answer these questions: we will continue to perform the mutational screen of PPP2R1A gene in Chinese EOC patients; detect the potential carcinogenicity of PPP2R1A mutations and corresponding PP2A activity by functional assays, then we will search for PPP2R1A mutation-associated differentially phosphorylated proteins and corresponding signaling pathway networks using 2D-DIGE and bioinformatic methods; finally we will select a representative signaling pathway and detect its potential role in EOC carcinogenesis with protein antagonist and/or agonist. These results will help us to better understand the role of PPP2R1A gene mutations in the pathogenesis of EOC, and provide valuable guidance and references for the patients with PPP2R1A mutations.

上皮性卵巢癌是一类影响我国女性生命健康的重大疾病，遗传和环境因素都能影响其发病。近期研究发现，蛋白磷酸酶PP2A结构亚基PPP2R1A在上皮性卵巢癌中存在高频突变，我们近期的研究也证实了该结果。目前对于PPP2R1A突变与上皮性卵巢癌发生的关系，有3个主要问题尚未得到解决：PPP2R1A突变能否促进上皮性卵巢癌的发生？该突变是否将影响PP2A磷酸酶活性而促进肿瘤发生？与该突变协同作用致癌的信号通路有哪些？我们将在前期工作基础上，对我国上皮性卵巢癌患者开展PPP2R1A突变检测；功能实验研究该基因突变的潜在致癌性和对PP2A磷酸酶活性的影响；利用2D-DIGE和生物信息学方法，找出突变相关的差异磷酸化蛋白并构建信号通路网络，最后利用蛋白抑制剂和/或激动剂验证代表性信号通路的作用。研究结果将有助于我们更好理解PPP2R1A突变在上皮性卵巢癌发生中的作用，同时对含有该突变病人的临床诊治提供指导。

PPP2R1A是PP2A磷酸酶的重要构成亚基，其突变或表达异常在多种人类疾病中扮演重要作用。本项目首先通过PCR及DNA直接测序的方法，发现上皮性卵巢癌中存在PPP2R1A基因突变。分别构建PPP2R1A基因野生型（PPP2R1A-WT）和突变型（PPP2R1A-MT）慢病毒载体，感染ES-2细胞后检测细胞的增殖、迁移、侵袭、凋亡及平板成瘤能力，发现PPP2R1A突变能够增加细胞迁移、侵袭及成瘤能力并抑制细胞凋亡，同时也将减弱细胞的增殖能力；且PPP2R1A突变将减弱PP2A全酶活性；进一步整体动物实验证实PPP2R1A突变能够促进卵巢癌转移，证实PPP2R1A突变能够促进卵巢癌发病。iTRAQ蛋白质组学分析上述感染不同慢病毒ES-2细胞（eGFP、WT、MT-1和MT-2）的差异磷酸化图谱鉴定出其中存在1360个差异磷酸化蛋白，从中选择与肿瘤转移潜在相关的STMN1蛋白进行证实，发现PPP2R1A突变能够促进STMN1总蛋白及磷酸化蛋白水平，进一步利用shRNA干扰载体敲低SMTN1蛋白表达后发现细胞的迁移和侵袭能力减弱，证实STMN1蛋白参与PPP2R1A基因突变的促卵巢癌发病过程。综上所述，本项目的研究结果表明PPP2R1A突变可通过调控STMN1蛋白的表达而促进卵巢癌的发生，这些结果有望为相关患者的分子靶向药物研究设计提供新思路。