Regulation of Adhesion and Proteolysis in Ovarian Cancer

描述（由申请人提供）：卵巢癌的高死亡率是由癌细胞在腹腔内扩散引起的。虽然肿瘤细胞与腹膜的粘附是通过整联蛋白介导的，但迁移和侵袭至少部分是通过蛋白酶尿激酶（uPA）及其受体（u-PAR）的表达介导的。我们的初步研究表明β 3-整合素表达抑制uPA和u-PAR的转录。这导致我们的一般假设，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卵巢癌细胞中uPA/u-PAR调节的影响。将使用原代人腹膜成纤维细胞和间皮细胞组装腹膜的3D模型，以评价基质细胞对β 3-整联蛋白介导的侵袭和粘附的贡献。我们将使用小鼠模型来评估粘附于腹膜并漂浮在腹水中的表达β 3整合素的卵巢癌细胞的粘附、播散和生长（目的1）。我们的假设是，通过了解整合素调节u-PAR/尿激酶表达的信号事件，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表明，NF-κ B参与蛋白酶的调节，我们将确定（目的2）是否NF-κ B途径调节粘附，侵入和表达的u-PAR和uPA在体内，并确定β 3整合素如何调节u-PAR和uPA启动子活性。我们的初步研究暗示了一个足迹区域（-238/-260），与转录因子PEA 3结合，介导β 3-整联蛋白对u-PAR的抑制。为了揭示PEA 3调节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合成或涉及PEA 3的反式作用活性，并鉴定PEA 3中被磷酸化改变的那些残基，从而影响启动子活性。最后，我们将通过测量PEA 3（EMSA，ChIP）与不同阶段和分化的人卵巢癌组织中uPA/u-PAR启动子的结合，并将其与正常卵巢进行比较，在体内验证我们用卵巢癌细胞系阐明的机制。重要性：我们研究的长期目标是了解卵巢癌的粘附和侵袭，以便设计一种合理的方法，使我们能够干扰调节转移的分子机制。

DESCRIPTION (provided by applicant): 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 evaluating the contribution of 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 transcriptional mechanism by which PEA3 regulates u-PAR we will (Aim 3) determine if beta3-integrin affects PEA3 synthesis or involves the trans-acting activity of 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.