Regulation of Adhesion and Proteolysis in Ovarian Cancer

描述（由申请人提供）：卵巢癌（OvCa）的高死亡率是由于癌细胞在腹腔内广泛扩散，造成了显著的肿瘤负担。转移性OvCa细胞首先遇到的细胞是腹膜和腹腔网膜的间皮细胞。我们使用原代人成纤维细胞、间皮细胞和OvCa细胞建立了腹膜和网膜的器官型3D培养，使我们能够在细胞培养中模拟转移的早期步骤。随着OvCa细胞附着在腹膜上，癌细胞衍生的MMP-2将纤维连接蛋白切割成更小的片段，从而促进？？？？-整合素介导的粘附和转移。c-Met通过其与？？？？的物理关系支持OvCa细胞的转移-整合素，它允许不依赖hgf的c-Met/Src/Fak细胞内信号的激活。促进OvCa传播的其他机制包括？？？？的相互作用-整合素与尿激酶受体（uPAR）在之前的资助期间获得的大多数结果都指向？？？？的突出作用-整合素-纤维连接蛋白在卵巢癌转移中的相互作用。我们的临床研究也证实了这一点，90%的OvCa转移瘤在基质中表达纤维连接蛋白。由于我们的初步数据表明，间皮细胞是纤维连接蛋白的来源，因此这项应用的主要假设是OvCa细胞刺激人间皮细胞分泌纤维连接蛋白，从而增加OvCa的侵袭和转移。在目的1中，我们将阐明间皮细胞与OvCa细胞的相互作用通过分泌纤维连接蛋白促进侵袭和转移的机制。实验将包括研究纤维连接蛋白在体内的作用，使用固定的纤维连接蛋白敲除小鼠，允许在间皮细胞中删除纤维连接蛋白，分析间皮细胞中纤维连接蛋白启动子的调节，

DESCRIPTION (provided by applicant): The high mortality of ovarian cancer (OvCa) is caused by the wide dissemination of cancer cells within the abdominal cavity, which results in significant tumor burden. The first cells which metastatic OvCa cells encounter are the mesothelial cells which line the peritoneum and omentum of the abdominal cavity. We established an organotypic 3D culture of the peritoneum and omentum using primary human fibroblasts, mesothelial, and OvCa cells that allowed us to mimic the early steps of metastasis in a cell culture. Following the attachment of OvCa cells to the peritoneum, cancer cell derived MMP-2 cleaves fibronectin into smaller pieces which promotes ????-integrin mediated adhesion and metastasis. c-Met supports the metastasis of OvCa cells through its physical with ????- integrin, which allows the HGF-independent activation of c-Met/Src/Fak intracellular signaling. Additional mechanisms which promote OvCa dissemination involve the interaction of ????-integrin with the urokinase receptor (uPAR) Most of the results obtained during the previous funding period point towards a prominent role for the ????-integrin - fibronectin interaction in ovarian cancer metastasis. This is also supported by our clinical studies, which established that 90% of all OvCa metastases express fibronectin in the stroma. Because our preliminary data suggest that mesothelial cells are the source of fibronectin, the primary hypothesis underlying this application is that OvCa cells stimulate human mesothelial cells to secrete fibronectin, which increases OvCa invasion and metastasis. In Aim I we will elucidate the mechanisms with which the interaction of mesothelial cells with OvCa cells promotes invasion and metastasis through secretion of fibronectin. Experiments will include studying the role of fibronectin in vivo using a floxed fibronectin knock-out mouse which allows deletion of fibronectin in mesothelial cells, analysis of fibronectin promoter regulation in mesothelial cells, and a siRNA screen to understand how OvCa cells promote fibronectin secretion in the mesothelial cells. In Aim II we will perform a detailed study to identify which fibronectin fragments promote the association between ????-integrin and c-Met. This will be followed by deletion of functional domains in ??-integrin and c-Met to define the binding sites where the two proteins interact and the biological significance of this protein-protein interaction for the intra cellular signaling, invasion, and metastasis of OvCa cells. Based on the findings in Aims I and II, in Aim III, we will combine a c-Met inhibitor with a fibronectin peptide known to block the effect of fibronectin binding integrins and test them in pre-clinical treatment studies using an orthotopi xenograft model (bursal injection of primary cells) as well as in a genetic model of OvCa. Successful inhibition of fibronectin function could result in a novel and clinically relevant approach to the treatment of metastatic ovarian cancer.