THERAPEUTIC BLOCKADE OF THE UROKINASE RECEPTOR

纤溶酶原激活物尿激活剂(u-PA)在人乳腺中的表达 癌症是一个重要的不利的独立预后变量。几个 临床前研究表明，u-PA的表面定位是关键 其在促进肿瘤侵袭性方面的假定作用。这样做的目的是 建议通过以下方法确认SCID小鼠的侵袭和转移模型 表达重组荧光素酶的MCF7细胞 表征细胞表面相关的u-PA在生长中的作用， 乳腺癌体内新生血管和转移的研究。其影响 抑制这种反应对乳腺癌进展的作用将是 评估的最终目标是设计抑制 乳腺癌的进展。 首先，尿激酶受体(u-PAR)的表达程度 人乳腺癌细胞系参与肿瘤生长和转移 在SCID小鼠中，将确定原位植入后的情况。四 将使用方法来抑制u-PA与其之间的相互作用 受体：1)突变的人或小鼠uPA在肿瘤细胞中的表达 蛋白水解性不活跃但仍有能力 占领u-par。2)可溶性u-PAR在水稻中的表达 癌细胞作为膜结合受体的竞争性抑制物。3) 我们制作的抗人u-PAR单抗的输注。4) 输注重组Ecotin分子，该分子已被修改为 灭活u-PA具有较高的效价和特异性。淋巴转移 节点、肝、肺和脑将通过以下方式进行识别和量化 器官匀浆中荧光素酶活性的测定。这提供了一种 对微转移的高灵敏度和定量测量。角色 将通过以下方法评估u-PA系统在宿主血管生成反应中的作用 灭活小鼠u-PA在肿瘤细胞中的过表达及鉴定 抑制uPAR对肿瘤发生发展的影响 微血管系统。同样，注入重组小鼠融合蛋白 由u-PA生长因子结构域和Ig Fc组成的Fc将注入 抑制小鼠血管内皮细胞和巨噬细胞的u-PAR。 第二个目标是获取与 U-PA和u-PAR之间的相互作用，以便为 治疗剂的发展。U-PAR蛋白的可溶性形式 含有在CHO中产生的配体结合域的各个部分 细胞和毕赤酵母正在大量生产，用于 结晶学、X射线结晶学和溶液相核磁共振。 第三个目标是评估u-PA-u-PAR抑制剂的疗效 由我们的乳腺癌动物模型的合作者开发的。U-PA- 由我们在CHIRON Inc.的合作者开发的基于u-PAR的抑制剂。 将会被研究。 第四个目标是确定u-par参与的程度。 新生血管形成。为了解决阻止u-PA- U-PAR相互作用抑制血管生成及其是否可以翻译 抑制乳腺癌的生长和转移。 第五个目标是确定细胞信号在多大程度上 通过u-PAR发生在乳腺癌细胞中，并且是否中断 该通路的表达将抑制肿瘤的侵袭和转移。基于最近的 通过u-PAR在癌细胞中传递信号重要性的证据 对于移民，我们认为值得采取这种方法来 抑制癌细胞中的u-PA。

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.