Regulation of Adhesion and Proteolysis in Ovarian Cancer

描述(申请人提供)：卵巢癌(OvCa)的高死亡率是由于癌细胞在腹腔内的广泛扩散造成的，这导致了巨大的肿瘤负担。转移的OvCa细胞首先遇到的细胞是位于腹膜和腹膜的间皮细胞。我们使用原代人成纤维细胞、间皮细胞和卵母细胞建立了腹膜和大网膜的器官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，癌细胞来源的基质金属蛋白酶-2将纤维连接蛋白裂解成更小的片段，从而促进整合素介导的黏附和转移。C-Met通过其与？整合素的物理结合支持OvCa细胞的转移，使c-Met/Src/Fak细胞内信号的激活不依赖于HGF。促进OvCa扩散的其他机制包括整合素同尿激酶型受体(UPAR)的相互作用。大多数研究结果表明，整合素-纤维连接蛋白相互作用在卵巢癌转移中起着重要作用。这也得到了我们的临床研究的支持，我们的临床研究证实，90%的OvCa转移瘤间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，因此支持这一应用的主要假设是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Aim I中，我们将阐明间皮细胞与OvCa细胞之间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：使用FLOXED FN基因敲除小鼠研究FN在体内的作用，允许删除间皮细胞中的FN，分析间皮细胞中FN启动子的调控， 以及siRNA筛选，以了解OvCa细胞如何促进间皮细胞中纤维连接蛋白的分泌。在AIM II中，我们将进行一项详细的研究，以确定哪些纤维连接蛋白片段促进整合素和c-Met之间的联系。随后将删除？整合素和c-Met中的功能结构域，以确定这两种蛋白质相互作用的结合部位以及这种蛋白质-蛋白质相互作用对Intra的生物学意义。 卵巢癌细胞的细胞信号、侵袭和转移。根据AIMS I和II中的研究结果， 在AIM III中，我们将c-Met抑制剂与纤维连接蛋白多肽结合起来，以阻断纤维连接蛋白结合整合素的作用，并在使用原位异种移植模型(法氏囊注射原代细胞)以及卵巢癌遗传模型的临床前治疗研究中对它们进行测试。成功地抑制纤维连接蛋白的功能可能导致一种新的和临床相关的方法来治疗转移性卵巢癌。 与公共卫生的相关性：卵巢癌转移的生物学仍然知之甚少，因此，在其治疗方面取得的进展很小。我们的发现是，腹膜间皮细胞通过分泌纤维连接蛋白促进卵巢癌的转移，这一发现使我们研究了纤维连接蛋白及其受体β？整合素之间的相互作用如何促进肿瘤的生长和转移。研究将包括对抑制这种相互作用的化合物进行临床前测试，目的是将获得的知识转化为治疗这种毁灭性疾病的新方法。

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. PUBLIC HEALTH RELEVANCE: The biology of ovarian cancer metastasis is still poorly understood and, therefore, minimal progress has been made in its treatment. Our findings, that the mesothelial cells lining the abdominal cavity promote ovarian cancer metastasis through the secretion of fibronectin, have led us to examine how the interaction between fibronectin and its receptor, ????-integrin, promotes tumor growth and metastasis. Studies will include pre-clinical testing of compounds that inhibit this interaction, with the goal of translating the knowledge gained into new treatments for this devastating disease.