Plasminogen activator inhibitor-1 in tumor progression and metastasis

描述（由申请人提供）：血管生成是肿瘤进展的关键。这个过程是由肿瘤细胞产生的血管生成因子启动的。因此，内皮细胞（EC）离开了静止状态，并出人意料地对凋亡变得更加敏感。这就提出了一个基本问题，EC是如何规避在血管生成转换过程中启动的促凋亡力的？多年来，据报道，纤溶酶原激活物抑制剂-1 （PAI-1）的水平在癌症晚期矛盾地升高，PAI-1是不良临床结果的一个指标。PAI-1可能在血管生成中起刺激作用而不是抑制作用。在我们的实验室中，我们进行了两项观察，表明PAI-1可能提供了保护EC免于凋亡的关键途径。首先，我们报道PAI-1阻止EC附着在玻璃体连接蛋白上，并在没有细胞凋亡的情况下刺激向纤维连接蛋白的迁移。其次，我们最近证明，通过其胞外和抗蛋白水解活性，PAI-1可以阻止纤溶蛋白对细胞膜相关Fas配体（mFasL）的切割，并释放21.5 kDa的可溶性促凋亡FasL片段，从而诱导EC细胞凋亡。基于这些观察结果，本研究计划将验证的中心假设是，受刺激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3个具体目标进行检验。在Aim 1中，我们将研究21.5 kDa纤凝蛋白生成的sFasL诱导凋亡的机制，探索PAI-1是否也能独立于Fas保护EC免于凋亡，并测试缺氧（HIF-11）或VEGF上调PAI-1是否对受刺激EC的存活至关重要。在Aim 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，以及缺氧如何影响这一过程。我们还将确定骨髓源性细胞和肿瘤细胞产生的PAI-1对血管生成和转移的贡献。在Aim 3中，我们将使用药理学方法来检查PAI-1抑制对肿瘤生长和血管生成的影响。从这些研究中，我们期望对EC在血管生成刺激过程中逃避FasL/Fas介导的细胞凋亡的关键机制有一个基本的了解。公共卫生相关性：纤溶酶原激活物抑制剂-1 （PAI-1）是一种控制血凝块溶解的蛋白。这种蛋白质一直被发现在癌症患者，特别是乳腺癌患者中升高，是临床结果不佳的一个指标。本应用将研究PAI-1如何积极促进肿瘤血管生成，并检验抗体或小分子抑制PAI-1是否具有治疗价值。由于PAI-1在癌症以外的其他疾病中也发挥着重要作用，如糖尿病视网膜病变和血栓形成性疾病，我们的研究将为针对PAI-1治疗癌症以及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.