Plasminogen activator inhibitor-1 in tumor progression and metastasis

描述(申请人提供)：血管生成是肿瘤进展的关键。这一过程是由肿瘤细胞产生的血管生成因子启动的。结果，内皮细胞(EC)处于静止状态，令人惊讶的是，它对细胞凋亡变得更加敏感。这就提出了一个根本的问题：EC如何绕过在血管生成转换过程中启动的促凋亡力量？多年来，有报道称，在癌症的晚期，纤溶酶原激活物抑制物-1(PAI-1)的水平升高是矛盾的，PAI-1是临床预后不良的指标。PAI-1在血管生成中可能起刺激而不是抑制作用。在我们的实验室中，我们已经做了两个观察，表明PAI-1可能提供了一条保护EC免受细胞凋亡的关键途径。首先，我们报道了PAI-1在没有细胞凋亡的情况下阻止EC与玻璃体连接蛋白的附着，并刺激其向纤维连接蛋白迁移。第二，我们最近证明，PAI-1通过其细胞外和抗蛋白水解性活性，阻止了纤溶酶对细胞膜相关Fas配体(MFasL)的切割和21.5 kDa的可溶性促凋亡FasL片段的释放，从而诱导EC细胞凋亡。在这些观察的基础上，将在这项研究提案中检验的中心假设是，在刺激的EC中PAI-1表达的增加对于保护EC免受FasL/Fas介导的凋亡至关重要。这一假设将通过三个具体目标进行检验。在目标1中，我们将研究21.5 kDa纤溶酶产生的sFasL诱导细胞凋亡的机制，探讨PAI-1是否也能独立于Fas保护EC的凋亡，并检测缺氧(HIF-11)或血管内皮生长因子(VEGF)上调PAI-1是否对刺激的EC的生存至关重要。在目标2中，我们将在体内测试PAI-1通过阻止sFasL的释放来控制血管生成，以及这一过程如何受到缺氧的影响。我们还将确定骨髓源性细胞和肿瘤细胞产生的PAI-1在血管生成和转移中的作用。在目标3中，我们将使用药理学方法来检测PAI-1抑制对肿瘤生长和血管生成的影响。通过这些研究，我们期望对EC在血管生成刺激过程中逃脱FasL/Fas介导的细胞凋亡的关键机制有一个基本的了解。公共卫生意义：纤溶酶原激活物抑制物-1(PAI-1)是一种控制血栓溶解的蛋白质。这种蛋白一直被发现在癌症患者，特别是乳腺癌患者中升高，是临床结果不佳的指标。这项应用将研究PAI-1如何积极促进肿瘤血管生成，并检查抗体或小分子对PAI-1的抑制是否具有治疗价值。由于PAI-1在癌症以外的其他疾病中起着重要作用，如糖尿病视网膜病变和血栓性疾病，我们的研究将为靶向PAI-1不仅治疗癌症，而且治疗其他几种人类疾病提供有价值的信息。

DESCRIPTION (provided by applicant): Angiogenesis is critical for tumor progression. This process is initiated by angiogenic factors produced by tumor cells. As a result, endothelial cells (EC) leave a state of quiescence and surprisingly become more sensitive to apoptosis. This raises the fundamental question of how can EC circumvent the pro-apoptotic forces that are initiated during the angiogenic switch? For many years it has been reported that the levels of plasminogen activator inhibitor-1 (PAI-1) are paradoxically elevated in more advanced stages of cancer and that PAI-1 is an indicator of poor clinical outcome. That PAI-1 could play a stimulatory rather than an inhibitory role in angiogenesis has also been suggested. In our laboratory we have made two observations indicating that PAI-1 may provide a critical pathway to protect EC from apoptosis. First we reported that PAI-1 prevents EC attachment to vitronectin and stimulates migration toward fibronectin in the absence of apoptosis. Second, we recently demonstrated that by its extracellular and anti-proteolytic activity, PAI-1 prevents the cleavage by plasmin of cell membrane-associated Fas ligand (mFasL) and the release of a 21.5 kDa soluble pro-apoptotic FasL fragment that induces apoptosis in EC. On the basis of these observations, the central hypothesis that will be tested in this research proposal is that an increase in PAI-1 expression in stimulated EC is critical to protect EC from FasL/Fas-mediated apoptosis. This hypothesis will be tested by 3 specific aims. In Aim 1 we will study the mechanism by which the 21.5 kDa plasmin-generated sFasL induces apoptosis, explore whether PAI-1 could also protect EC from apoptosis independently of Fas and test whether the up-regulation of PAI-1 by hypoxia (HIF-11) or VEGF is critical for the survival of stimulated EC. In Aim 2, we will test in vivo that PAI-1 controls angiogenesis by preventing the release of sFasL and how this process is affected by hypoxia. We will also determine the contribution of PAI-1 produced by bone marrow-derived cells and tumors cells to angiogenesis and metastasis. In Aim 3, we will use a pharmacological approach to examine the effect of PAI-1 inhibition on tumor growth and angiogenesis. From these studies we anticipate a fundamental understanding of a critical mechanism used by EC to escape FasL/Fas mediated apoptosis during angiogenic stimulation. PUBLIC HEALTH RELEVANCE: Plasminogen activator inhibitor-1 (PAI-1) is a protein that controls the dissolution of blood clots. This protein has been consistently found to be elevated in patients with cancer and in particular breast cancer, and is an indicator of poor clinical outcome. This application will study how PAI-1 positively contributes to tumor angiogenesis and examine whether inhibition of PAI-1 by antibodies or small molecules can be of therapeutic value. Because PAI-1 plays a contributory role in other conditions than cancer, like diabetic retinopathy and thrombotic diseases, our studies will provide valuable information in regard to targeting PAI-1 in the treatment not only of cancer but of several other human diseases.