THERAPEUTIC BLOCKADE OF THE UROKINASE RECEPTOR

点击翻译按钮获取中文摘要

Expression of the plasminogen activator urokinase (u-PA) by human breast cancer is a significant adverse, independent prognostic variable. Several pre-clinical studies suggest that surface localization of u-PA is critical to its putative role in promoting tumor invasiveness. The goal of this proposal is to confirm an SCID mouse model of invasion and metastasis by MCF7 cells expressing recombinant luciferase which we have developed to characterize the role of cell surface-associated u-PA in the growth, neovascularization, and metastasis of breast cancers in vivo. The effects of inhibiting this reaction on the progression of breast cancer will be assessed with the ultimate aim of designing drugs that inhibit the progression of breast cancer. First, the extent to which expression of the urokinase receptor (u-PAR) by human breast cancer cell lines is involved in tumor growth and metastasis in SCID mice following orthotopic implantation will be determined. Four approaches will be used to inhibit the interaction between u-PA and its receptor: 1) expression in tumor cells of mutant human or mouse uPA molecules which are proteolytically-inactive but retain the ability to occupy the u-PAR. 2) expression of soluble forms of the u-PAR in the cancer cells as a competitive inhibitor of the membrane-bound receptor. 3) Infusion of monoclonal anti-human u-PAR antibodies which we have made. 4) Infusion of a recombinant Ecotin molecule which has been modified to inactivate u-PA with high potency and specificity. Metastasis to lymph nodes, liver, lung, and brain will be identified and quantitated by measurement of luciferase activity in organ homogenatres. This provides a highly sensitive and quantitative measurement of micrometastases. The role of the u-PA system in the host angiogenic response will be evaluated by overexpressing an inactive murine u-PA in the tumor cells and determining the effect of inhibiting the uPAR on the developing tumor microvasculature. Similarly, infusing a recombinant mouse fusion protein consisting of the u-PA growth factor domain and Ig Fc will be infused to inhibit mouse u-PAR on vascular endothelium and macrophages. A second aim is to obtain structural information relating to the interaction between u-PA and u-PAR in order to provide a basis for the development of therapeutic agents. Soluble forms of u-PAR protein containing various portion of the ligand binding domain produced in CHO cells as well as Pichia are being produced in large quantities for crystallization and X-ray crystallography and, solution phase NMR. A third aim will be to evaluate the efficacy of u-PA - u-PAR inhibitors developed by collaborators in our animal model of breast cancer. u-PA- based inhibitors of u-PAR developed by our collaborators at CHIRON Inc. will be studied. A fourth aim is to determine the extent to which u-PAR is involved in neovasculaization. To address the question of whether blocking the u-PA - u-PAR interaction inhibits angiogenesis and whether this can be translated into inhibition of growth and metastasis of breast cancer. A fifth aim will be to determine the extent to which cell signaling through the u-PAR occurs in breast cancer cells, and whether interruption of this pathway will inhibit invasion and metastasis. Based on recent evidence of the importance of signaling through u-PAR in cancer cell migration, we believe it is worthwhile to pursue this approach to inhibiting u-PA in cancer cells.