Regulation of Adhesion and Proteolysis in Ovarian Cancer

卵巢癌的高死亡率是由癌细胞在腹腔内扩散引起的。 腔而肿瘤细胞与腹膜的粘附是通过整合素介导的， 至少部分由蛋白酶尿激酶（uPA）及其受体（u-PAR）的表达介导。我们 初步研究表明β 3-整联蛋白表达抑制uPA和u-PAR转录。这导致 我们的一般假设是粘附受体可以调节uPA/u-PAR蛋白水解系统， 从而调节入侵。 具体目的：我们将研究β 3-整合素表达和抑制对uPA/u-PAR的影响。 调节卵巢癌细胞。腹膜的3D模型将被组装，使用初级人类 腹膜成纤维细胞和间皮细胞，以评估基质细胞对 β 3-整合素介导的侵袭和粘附。我们将使用小鼠模型来评估粘附， 粘附于腹膜的表达β 3整联蛋白的卵巢癌细胞的扩散和生长， 漂浮在腹水中（Aim 1）。我们的假设是，通过了解整合素 调节u-PAR/尿激酶表达，我们将能够提高我们对蛋白酶转录的理解。 由于我们的初步数据显示NF-κ B参与蛋白酶调节，我们将确定（目的2） NF-κ B通路是否在体内调节u-PAR和uPA的粘附、侵袭和表达，以及 还确定β 3整合素如何调节u-PAR和uPA启动子活性。我们的初步研究 涉及与转录因子PEA 3结合的足迹区域（-238/-260），其介导抑制 β 3-整合素对u-PAR的影响揭示PEA 3调控u-PAR的转录机制 我们将（目的3）确定β 3-整联蛋白是否影响PEA 3的合成或涉及β 3-整联蛋白的反式作用活性。 PEA 3中的那些残基，这些残基通过磷酸化而改变，从而影响启动子 活动最后，我们将验证我们已经阐明的卵巢癌细胞系的机制，在体内， 通过测量PEA 3（EMSA，ChIP）与人卵巢癌中uPA/u-PAR启动子的结合， 组织的各个阶段和分化，并与正常卵巢进行比较。 意义：我们研究的长期目标是了解卵巢癌的粘连和侵袭， 为了设计一种合理的方法，使我们能够干扰分子机制， 调节转移。

The high mortality of ovarian cancer is caused by the dissemination of cancer cells within the abdominal cavity. While adhesion of tumor cells to the peritoneum is mediated through integrins, migration and invasion is mediated at least in part by the expression of the protease urokinase (uPA) and its receptor (u-PAR). Our preliminary studies show that beta 3-integrin expression represses uPA and u-PAR transcription. This lead to our general HYPOTHESIS that adhesion receptors can regulate the uPA/u-PAR proteolytic system and thereby modulate invasion. SPECIFIC AIMS: We will study the effect of beta 3-integrin expression and inhibition on uPA/u-PAR regulation in ovarian cancer cells. A 3D model of peritoneum will be assembled, using primary human peritoneal fibroblasts and mesothelial cells, for the purpose of evaluatingthe contributionof stromal cells to beta 3-integrin mediated invasion and adhesion. We will use a mouse model to assess adhesion, dissemination, and growth of beta3 integrin expressing ovarian cancer cells adherent to the peritoneum and floating in ascites (Aim 1). Our hypothesis is that by understanding the signaling events by which integrins regulate u-PAR/urokinase expression we will be able to improve our understanding of protease transcription. Since our preliminary data show that NF-KB is involved in protease regulation, we will determine (Aim 2) whether the NF-KB pathway regulates the adhesion, invasion and expression of u-PAR and uPA in vivo, and also ascertain how beta3 integrin regulates u-PAR and uPA promoter activity. Our preliminary studies implicate a footprinted region (-238/-260), bound with the transcription factor PEA3, that mediates inhibition of u-PAR by the beta 3-integrin. To uncover the transcriptionalmechanism by which PEA3 regulates u-PAR we will (Aim 3) determine if beta3-integrin affects PEA3 synthesis or involves the trans-acting activityof PEA3 and identify those residues in PEA3 which are altered by phosphorylation, thereby affecting promoter activity. Finally, we will validate the mechanism we have elucidated with ovarian cancer cell lines, in vivo, by measuring the binding of PEA3 (EMSA, ChIP) to the uPA/u-PAR promoter in human ovarian cancer tissue of various stages and differentiation, and comparing it to normal ovary. SIGNIFICANCE: The long-term goal of our studies is the understanding of adhesion and invasion in ovarian cancer in order to design a rationale approach enabling us to interfere with molecular mechanisms that regulate metastasis.