Role of TFPI-2 in human gliomas invasiveness

描述（由申请人提供）：我们实验室的长期目标是了解TFPI-2在胶质母细胞瘤侵袭性和恶性行为中的调节作用。TFPI-2抑制六种不同的丝氨酸蛋白酶，包括纤溶酶，一种似乎直接参与原发性肿瘤，特别是胶质母细胞瘤的侵袭行为的酶。在初步研究中，TFPI-2在成胶质细胞瘤和已建立的成胶质细胞瘤细胞系中均检测不到，该细胞系在体外和体内具有高度侵袭性。相反，TFPI-2蛋白在正常人脑中检测到，在较小程度上，在低级别胶质瘤和间变性星形细胞瘤以及来自这些肿瘤的细胞系。这些发现表明TFPI-2水平与胶质瘤的进展之间呈负相关。我们推测TFPI-2表达的调节将改变胶质瘤在体外和体内的侵袭行为。中心假设是TFPI-2是促进胶质瘤侵袭和血管生成的蛋白酶的关键负调节因子。为了验证这个假设，我们首先检查具体目标1：构建了一个含有人TFPI-2的0.7-kb片段的表达载体盒，该片段位于E1缺失区，由一个独立的巨细胞病毒（CMV）启动子和牛生长激素（BGHpA）多聚腺苷酸化信号驱动，并研究了其表达特性。（Ad-TFPI-2）对胶质瘤细胞中TFPI-2、其他蛋白酶水平及其体外和体内模型侵袭行为的影响及其对体内胶质瘤生长的影响。1a）构建并确定Ad-TFPI-2构建体对TFPI-2和蛋白酶水平的影响，1b）确定Ad-TFPI-2构建体与模拟物和Ad-CMV构建体对胶质瘤细胞生长、粘附和迁移的影响，1c）研究TFPI-2构建体对体外模型中人胶质瘤细胞侵袭行为的影响，1d）确定Ad-TFPI-2构建体抑制皮下和脑内注射到裸鼠中的人神经胶质瘤细胞系的侵袭和生长的效果。1 e）用脑内注射给予的Ad-CMV构建体评价Ad-TFPI-2构建体的毒性。具体目标二：确定调节脑血管生成的分子机制，与内皮细胞和胶质瘤细胞体外和体内共培养物中TFPI-2的产生有关。2a）测定TFPI-2、VEGF、TF和其他蛋白酶以及在内皮细胞与有义/反义TFPi-2稳定克隆或胶质母细胞瘤细胞系和用Ad-CMV和Ad-TFPI-2有义构建体感染的原代胶质母细胞瘤细胞共培养期间或在抑制剂存在下形成的毛细血管样结构的长度，以及2b）确定有义和反义稳定转染子或Ad-TFPI-2有义构建体的感染或在特定目标2a中指定的抑制剂存在下对肿瘤血管生成的影响。我们相信，通过确定TFPI-2在胶质母细胞瘤侵袭性中的作用及其在血管生成中的作用，将有可能设计进一步的研究，以评估这种新型蛋白酶抑制剂在脑肿瘤中的治疗作用。

DESCRIPTION (provided by applicant): The long-term objective of our laboratory is to understand the regulatory roles of TFPI-2 in the invasive and malignant behavior of glioblastomas. TFPI-2 inhibits six different serine proteases, including plasmin, an enzyme that appears to be directly involved in the invasive behavior of primary tumors, particularly glioblastomas. In preliminary studies, TFPI-2 was undetectable in both glioblastomas and an established glioblastoma cell line that is highly invasive in vitro and in vivo. In contrast, the TFPI-2 protein was detected in normal human brain and, to a lesser extent, in low-grade gliomas and anaplastic astrocytomas as well as in cell lines derived from these tumors. These findings show an inverse correlation between the TFPI-2 levels and the progression of gliomas. We postulate that modulation of TFPI-2 expression will alter the invasive behavior of gliomas in vitro and in vivo. The central hypothesis is that TFPI-2 is a key negative regulator of proteases that promote glioma invasion and angiogenesis. To test this hypothesis, first we examine Specific Aim 1: Construct an expression vector cassette containing a 0.7-kb fragment of human TFPI-2 in the sense orientation in an E1-deleted region driven by an independent cytomegalovirus (CMV) promoter and the polyadenylation signal bovine growth hormone (BGHpA) and study its (Ad-TFPI-2) effect on the levels of TFPI-2, other proteases in glioma cells and their invasive behavior both in vitro and in vivo models and its effect on glioma growth in vivo. 1a) Construct and determine the effect of the Ad-TFPI-2 construct on the levels of TFPI-2 and proteases, 1b) Determine the effect of the Ad-TFPI-2 construct on glioma cell growth, adhesion and migration with that of mock and Ad-CMV construct, 1c) Investigate the effect of the TFPI-2 construct on the invasive behavior of human glioma cells in vitro models, 1d) Determine the effect of Ad-TFPI-2 construct to inhibit the invasion and growth of human glioma cell lines injected subcutaneously and intracerebrally in nude mice. 1e) Evaluate the toxicity of Ad-TFPI-2 construct with that of Ad-CMV construct given as intracerebral injections. Specific Aim 2: Identify the molecular mechanisms that regulate cerebral angiogenesis in relation to the production of TFPI-2 in co-cultures of endothelial and glioma cells both in vitro and in vivo. 2a) Determine that levels of TFPI-2, VEGF, TF and other proteases and the length of capillary-like structures formed during cocultures of endothelial cells with sense/antisense TFPi-2 stable clones or glioblastoma cell lines and primary glioblastoma cells infected with Ad-CMV and Ad-TFPI-2 sense constructs or in the presence of an inhibitor and 2b) Determine the effect of sense and antisense stable transfectants or infection of Ad-TFPI-2 sense construct or in the presence of an inhibitor specified in specific aim 2a on tumor angiogenesis. We believe that by determining the role of TFPI-2 in the invasiveness of glioblastomas and its role in angiogenesis, it will be possible to design further studies that will evaluate the therapeutic role of this novel protease inhibitor in brain tumors.