Plasminogen activator inhibitor-1 in tumor progression and metastasis

描述(由申请人提供)：纤溶酶原激活物抑制物-1(PAI-1)是内源性丝氨酸蛋白酶抑制物(Serpins)家族的成员，它控制组织和尿激酶型纤溶酶原激活物(分别为tPA和uPA)将纤溶酶原激活为纤溶酶。过去，PAI-1被认为具有抗肿瘤作用。然而，S在20世纪90年代末的多项临床研究发现，uPA和PAI-1含量高的癌症患者的临床预后较差，提示PAI-1可能促进而不是抑制肿瘤的发生。PAI-1在癌症中的矛盾作用的一个解释是它的促血管生成功能。在过去的拨款资助期间，我们发表了两个基本观察结果，进一步阐明了PAI-1的促肿瘤功能。我们首次报道PAI-1部分通过一种新的机制发挥其促血管生成活性，即PAI-1通过抑制纤溶酶对可溶性mFasL促凋亡片段的脱落来保护内皮细胞免受Fas介导的凋亡。其次，我们证明了PAI-1也能保护肿瘤细胞免于凋亡，但其机制仅部分依赖于纤溶酶和Fas。强调肿瘤微环境的作用，我们还证明了间质来源的PAI-1弥补了肿瘤来源的PAI-1的缺失，并且PAI-1有助于肿瘤细胞对巨噬细胞的募集。这一应用的主要假设是，PAI-1通过对内皮细胞(促血管生成活性)、巨噬细胞(促进迁移)和肿瘤细胞(促进存活)的组合作用发挥其促肿瘤活性，这涉及到特定的PAI-1受体相互作用。我们将在几个不同来源和产生不同数量PAI-1的人类癌细胞株中体外测试这一假说，并通过结合遗传方法和药理学方法在体内验证这一假说。在目标1中，我们将研究PAI-1与其细胞表面受体之一低密度脂蛋白受体样蛋白1(LRP1)相互作用的机制，以保护肿瘤细胞免受自发和药物诱导的凋亡。在目标2中，我们将确定PAI-1是否是允许肿瘤促进血管生成、招募巨噬细胞和退出休眠所必需的。在免疫缺陷PAI-1基因缺失的小鼠中，移植了人肿瘤细胞，其中PAI-1表达的抑制由多西环素控制。我们还将研究PAI-1抑制对PAI-1缺失小鼠与转基因NB-Tag小鼠的肿瘤启动的影响，这些转基因小鼠在神经母细胞瘤形成中具有100%的外显率。在目标3中，我们将测试新开发的PAI-1小分子抑制剂在临床前小鼠肿瘤发生和肿瘤启动模型中对PAI-1的药理抑制作用。这些研究不仅将更好地从根本上了解PAI-1促肿瘤活性的机制，而且还将更明确地回答靶向PAI-1作为癌症治疗策略的一部分的潜在治疗价值。

DESCRIPTION (provided by applicant): Plasminogen activator inhibitor-1 (PAI-1) is a member of the family of endogenous serine protease inhibitors (serpins) that controls the activation of plasminogen into plasmin by tissue and urokinase type plasminogen activators (tPA and uPA, respectively). In the past, PAI-1 was considered to be anti-tumorigenic. However, in the late 1990's multiple clinical studies revealed that cancer patients whose tumors have a high content of uPA and PAI-1 have a poor rather than a favorable clinical outcome, suggesting that PAI-1 may promote rather than inhibit tumorigenesis. One explanation for the paradoxical role attributed to PAI-1 in cancer has been its pro-angiogenic function. During the past funding period of the grant, we have published two fundamental observations that further elucidate the pro-tumorigenic function of PAI-1. We first reported that PAI-1 exerts its pro-angiogenic activity in part through a novel mechanism whereby PAI-1 protects endothelial cells from Fas-mediated apoptosis through inhibition of the shedding by plasmin of a soluble mFasL pro-apoptotic fragment. Secondly, we demonstrated that PAI-1 also protects tumor cells from apoptosis, but that the mechanism is only partially dependent on plasmin and Fas. Emphasizing the role of the tumor microenvironment, we also demonstrated that stromal-derived PAI-1 compensates for a lack of tumor-derived PAI-1, and that PAI-1 contributes to the recruitment of macrophages by tumor cells. The overarching hypothesis of this application is that PAI-1 exerts its pro-tumorigenic activity through a combination of effects on endothelial cells (pro-angiogenic activity), macrophages (promotion of migration) and tumor cells (promotion of survival) that involves specific PAI-1-receptor interactions. We will test this hypothesis in vitro in several human cancer cell lines of different origins and producing variable amounts of PAI-1, and in vivo by combining genetic approaches with a pharmacologic approach. In Aim 1, we will investigate the mechanism by which the interaction between PAI-1 and one of its cell surface receptors, the low density lipoprotein receptor-like protein 1 (LRP1) signals and protects tumor cells from spontaneous and drug-induced apoptosis. In Aim 2, we will determine whether PAI-1 is necessary to allow tumors to promote angiogenesis, recruit macrophages and exit dormancy in immunodeficient PAI-1 null mice implanted with human tumor cells in which the suppression of PAI-1 expression is controlled by doxycycline. We will also investigate the effect of PAI-1 suppression on tumor initiation in PAI-1 null mice crossed with transgenic NB-Tag mice that have a 100% penetrance in neuroblastoma tumor formation. In Aim 3, we will test the effect of pharmacological inhibition of PAI-1 by newly developed small molecule inhibitors of PAI-1 in pre-clinical mouse models of tumorigenesis and tumor initiation. These studies will provide not only a better fundamental understanding of the mechanisms responsible for the pro-tumorigenic activity of PAI-1, but also a more definitive answer on the potential therapeutic value of targetin PAI-1 as part of cancer treatment strategies.