Regulation of Adhesion and Proteolysis in Ovarian Cancer

描述（由申请人提供）：卵巢癌（OvCa）的高死亡率是由腹腔内癌细胞的广泛扩散引起的，这导致了显著的肿瘤负荷。转移性OvCa细胞首先遇到的细胞是排列在腹腔腹膜和大网膜上的间皮细胞。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，这使我们能够在细胞培养中模拟转移的早期步骤。在OvCa细胞附着于腹膜后，癌细胞衍生的MMP-2将纤连蛋白切割成更小的碎片，这促进α 5 β 1-整联蛋白介导的粘附和转移。c-Met通过其与α 5 β 1-整联蛋白的物理作用支持OvCa细胞的转移，所述α 5 β 1-整联蛋白允许c-Met/Src/Fak细胞内信号传导的HGF非依赖性活化。促进OvCa播散的其它机制涉及α 5 β 1-整联蛋白与尿激酶受体（uPAR）的相互作用。在先前的资助期间获得的大多数结果指向α 5 β 1-整联蛋白-纤连蛋白相互作用在卵巢癌转移中的突出作用。这也得到了我们的临床研究的支持，该研究证实90%的OvCa转移瘤在基质中表达纤连蛋白。由于我们的初步数据表明间皮细胞是纤连蛋白的来源，因此本申请的主要假设是OvCa细胞刺激人间皮细胞分泌纤连蛋白，从而增加OvCa侵袭和转移。在目的I中，我们将阐明间皮细胞与OvCa细胞的相互作用通过分泌纤连蛋白促进侵袭和转移的机制。实验将包括研究纤连蛋白在体内的作用，使用floxed纤连蛋白敲除小鼠，其允许在间皮瘤细胞中缺失纤连蛋白，分析间皮瘤细胞中纤连蛋白启动子调控，以及siRNA筛选以了解OvCa细胞如何促进间皮瘤细胞中纤连蛋白分泌。在目的II中，我们将进行详细的研究，以确定哪些纤连蛋白片段促进α 5 β 1-整联蛋白和c-Met之间的结合。随后将删除α5-整联蛋白和c-Met中的功能结构域，以确定两种蛋白质相互作用的结合位点以及这种蛋白质-蛋白质相互作用对细胞内的生物学意义。 OvCa细胞的细胞信号传导、侵袭和转移。根据目标一和二的调查结果， 在目的III中，我们将联合收割机与已知阻断纤连蛋白结合整联蛋白的作用的纤连蛋白肽组合，并在使用原位异种移植模型（原代细胞的囊注射）以及OvCa遗传模型的临床前治疗研究中测试它们。成功抑制纤连蛋白功能可能会导致一种新的和临床相关的方法来治疗转移性卵巢癌。

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 α5ß1-integrin mediated adhesion and metastasis. c-Met supports the metastasis of OvCa cells through its physical with α5ß1-integrin, which allows the HGF-independent activation of c-Met/Src/Fak intracellular signaling. Additional mechanisms which promote OvCa dissemination involve the interaction of α5ß1-integrin with the urokinase receptor (uPAR) Most of the results obtained during the previous funding period point towards a prominent role for the α5ß1-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 α5ß1-integrin and c-Met. This will be followed by deletion of functional domains in α5-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.