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. ? ? ?
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