THERAPEUTIC BLOCKADE OF THE UROKINASE RECEPTOR

纤溶酶原激活剂尿激酶在人乳腺癌细胞中的表达 癌症是一个重要的不利的、独立的预后变量。几 临床前研究表明，u-PA的表面定位至关重要， 其在促进肿瘤侵袭中的假定作用。这个目标 建议通过以下方法确认侵袭和转移的SCID小鼠模型： 我们已经开发了表达重组荧光素酶的MCF 7细胞， 表征细胞表面相关的u-PA在生长中的作用， 新血管形成和体内乳腺癌转移。的影响 在乳腺癌的发展过程中抑制这种反应， 评估的最终目的是设计药物， 乳腺癌的进展。 首先，尿激酶受体（u-PAR）表达的程度， 人乳腺癌细胞系参与肿瘤生长和转移 将确定原位植入后SCID小鼠中的细胞凋亡。四 方法将被用来抑制u-PA和其 受体：1）突变型人或小鼠uPA在肿瘤细胞中的表达 无蛋白水解活性但保留了 占领U-PAR。2)表达可溶性形式的u-PAR在 作为膜结合受体的竞争性抑制剂。第三章 输注我们制备的单克隆抗人u-PAR抗体。四、 输注已被修饰为 具有高效力和特异性的抗纤溶酶原激活剂。淋巴转移 将通过以下方法识别和定量淋巴结、肝脏、肺和大脑 器官匀浆中荧光素酶活性的测量。这提供了 高灵敏度和定量测量微转移。的作用 将通过以下方法评价u-PA系统在宿主血管生成反应中的作用 在肿瘤细胞中过表达无活性的鼠u-PA， 抑制uPAR对肿瘤发展的影响 微脉管系统类似地，将重组小鼠融合蛋白 将输注由u-PA生长因子结构域和IG Fc组成的 抑制血管内皮细胞和巨噬细胞u-PAR。 第二个目的是获得与这些结构相关的结构信息。 u-PA和u-PAR之间的相互作用，以提供基础， 治疗剂的开发。可溶性u-PAR蛋白 含有CHO中产生的配体结合结构域的不同部分 细胞以及毕赤酵母正在大量生产， 结晶和X射线晶体学以及溶液相NMR。 第三个目标是评估u-PA - u-PAR抑制剂的疗效 由合作者在我们的乳腺癌动物模型中开发。u-PA 基于u-PAR的抑制剂由我们的合作者在CHIRON Inc. 将被研究。 第四个目的是确定u-PAR参与的程度， 新生血管形成为了解决是否阻止u-PA的问题， u-PAR相互作用抑制血管生成，以及这种作用是否可以被翻译成 用于抑制乳腺癌的生长和转移。 第五个目标是确定细胞信号传导在多大程度上 通过u-PAR发生在乳腺癌细胞中， 这条通路的作用将抑制侵袭和转移。根据最近 在癌细胞中通过u-PAR信号传导的重要性的证据 移民，我们认为值得采取这种做法， 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.