Regulation of Adhesion and Proteolysis in Ovarian Cancer

卵巢癌的高死亡率是由于癌细胞在腹部扩散所致。 空洞。而肿瘤细胞与腹膜的黏附是通过整合素、迁移和侵袭来调节的 至少部分是由蛋白水解酶尿激酶(UPA)及其受体(u-PAR)的表达所介导的。我们的 初步研究表明，β3-整合素的表达抑制uPA和u-PAR的转录。这导致了 我们的一般假设是，黏附受体可以调节uPA/u-PAR蛋白分解系统，并 从而调节入侵。 具体目的：我们将研究β3-整合素的表达和抑制对uPA/u-PAR的影响 卵巢癌细胞的调控。一个腹膜的3D模型将被组装，使用原始人 腹膜成纤维细胞和间皮细胞，以评估基质细胞对 β3-整合素介导的侵袭和黏附。我们将使用小鼠模型来评估粘附性， 表达β3整合素的卵巢癌细胞在腹膜和腹膜上的扩散和生长 漂浮在腹水中(目标1)。我们的假设是，通过了解整合素 调控u-PAR/尿激酶的表达，我们将能够提高我们对蛋白水解酶转录的理解。 由于我们的初步数据显示，核因子-KB参与了蛋白水解酶的调节，我们将确定(目标2) 核因子-kB通路是否调节体内u-PAR和uPA的黏附、侵袭和表达 还要确定β3整合素如何调节u-PAR和uPA启动子的活性。我们的初步研究 涉及一个足迹区域(-238/-260)，与转录因子PEA3结合，介导抑制 由β-3-整合素产生的u-PAR。揭示PEA3调节u-PAR的转录机制 我们将(目标3)确定β3整合素是否影响PEA3的合成或涉及PEA3的反式作用活性。 并鉴定PEA3中那些被磷酸化改变的残基，从而影响启动子 活动。最后，我们将用卵巢癌细胞株验证我们所阐明的机制，在体内， 通过检测人卵巢癌中PEA3(EMSA、CHIP)与uPA/u-PAR启动子的结合 不同发育阶段和分化程度的卵巢组织，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的黏附和侵袭 为了设计一种基本的方法，使我们能够干扰 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.