EGFR受PKGI磷酸化调控的分子机制及其作为卵巢癌诊断标志物的临床研究

Early diagnosis of ovarian cancer is one of major clinical problems, so it is important to find effective biomarkers for early diagnosis of ovarian cancer. Epithelial ovarian cancer (EOC) is the most common type of ovarian cancer. Our previous work found that PKG I inhibited the activation of EGFR and downstream signaling pathways (e.g. MAPK/ERK and PI3K/AKT pathways) in ovarian cancer. The serum EGFR level of ovarian cancer patients is also abnormal. This study was to investigate whether PKG I inhibited EGF-induced phosphorylation of EGFR tyrosine in EOC, then effected the biological functions, such as cell proliferation, migration and invasion. And we will study the molecular mechanism of this inhibitory effect. At the same time, we will study the change of serum EGFR concentration in EOC patients, and explore the possibility of serum EGFR as a new specific biomarker for early diagnosis and prognosis evaluation of EOC. The study is expected to provide effective and specific new biomarkers for early diagnosis, real-time monitoring and prognosis evaluation of EOC. Also, the molecular mechanism of PKG I inhibits EGFR tyrosine phosphorylation and the determination of phosphorylation sites will provide a new therapeutic target for ovarian cancer, which has important scientific significance and potential clinical application value.

卵巢癌早期诊断是临床一大难题，因此寻找有效的早期诊断标志物至关重要。卵巢上皮癌（Epithelial ovarian cancer, EOC）是最常见的卵巢癌类型。申请人前期发现卵巢癌中 PKGI抑制EGFR激活及下游信号转导通路（MAPK/ERK和PI3K/AKT通路），同时卵巢癌患者血清中的EGFR水平中也存在异常。本研究旨在探究EOC中PKGI是否抑制EGF引起的EGFR酪氨酸磷酸化，进而影响细胞增殖、迁移和侵袭等生物学效应，并深入探讨该抑制效应的分子机制；同时研究血清中EGFR在EOC患者血清中浓度改变，探讨这种改变作为EOC早期诊断、预后评估的特异性新型标志物的可能性。本项目将有望为EOC的早期诊断、实时监控和预后评价提供有效而特异的新标志物；同时PKGI抑制EGFR酪氨酸磷酸化分子机制的阐明以及磷酸化位点的确定可为卵巢癌提供新的治疗靶点，具有重要的科学意义和潜在的临床价值。

卵巢癌早期诊断是临床一大难题，因此寻找有效的早期诊断标志物至关重要。卵巢上皮癌 （Epithelial ovarian cancer, EOC）是最常见的卵巢癌类型。本项目只要探讨8-Br-cGMP激活的PKG I对EGF引起的EGFR相关的信号通路卵巢癌的抑制作用，并探讨sEGFR作为卵巢癌的标志物的可能。（1）卵巢癌组织中PKG I的活性低于癌旁组织，8-Br-cGMP激活PKG I之后有效地抑制了卵巢癌细胞A2780和SKOV3的增殖、迁移和侵袭，并且通过EGFR/ERK信号通路。之后我们通过皮下移植瘤和腹腔移植瘤模型证实8-Br-cGMP激活的PKG I抑制卵巢癌的生长和转移。（2）通过免疫共沉淀和激光共聚焦结果显示卵巢癌细胞中PKG I与EGFR相互结合。PKG I作为丝/苏氨酸蛋白激酶在8-Br-cGMP激活之后可以引起EGFR的丝/苏氨酸磷酸化。通过LC-MS结果发现PKGI可以使EGFR的T693位点磷酸化。以上结果表明8-Br-cGMP激活的PKG I通过使EGFR的T693位点磷酸化从而抑制了其酪氨酸的磷酸化和下游通路的激活，从而抑制卵巢癌的增殖和转移。（3）收集30例卵巢癌患者、30例卵巢囊肿以及17例健康女性的血清，通过ELISA检测发现血清中sEGFR的水平在卵巢癌患者中高于卵巢囊肿和健康人。并且通过分析发现虽然单独的sEGFR与CA125和HE4相比其特异性和灵敏度不高。但是通过三者联合可以有效地提高灵敏度和特异度。为卵巢癌诊断提供新的标志物和治疗靶点。本项目按计划顺利执行，发表SCI文章4篇，申请发明专利一项，培养硕士研究生两名。