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肿瘤，特别是胶质母细胞瘤侵袭行为的酶。在初步研究中，TFPI-2在胶质母细胞瘤和已建立的胶质母细胞瘤细胞系中均未检测到，而胶质母细胞瘤细胞系在体外和体内均具有高度侵袭性。相比之下，在正常人脑中检测到TFPI-2蛋白，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中检测到的程度较低。这些发现表明TFPI-2水平与胶质瘤的进展呈负相关。我们假设TFPI-2表达的调节将改变胶质瘤在体内和体外的侵袭行为。核心假设是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。为了验证这一假设，我们首先检查具体目标1：构建由独立巨细胞病毒（CMV）启动子和多聚腺苷化信号牛生长激素（BGHpA）驱动的人TFPI-2在e1缺失区意义取向上的0.7 kb片段表达载体盒，研究其（Ad-TFPI-2）对胶质瘤细胞中TFPI-2及其他蛋白酶水平的影响及其在体外和体内模型中的侵袭行为，以及对胶质瘤生长的影响。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)与模拟构建体和Ad-CMV构建体比较，确定Ad-TFPI-2构建体对胶质瘤细胞生长、粘附和迁移的影响；1c)在体外模型中研究TFPI-2构建体对人胶质瘤细胞侵袭行为的影响。1d)确定Ad-TFPI-2构建体对裸鼠皮下和脑内注射人胶质瘤细胞系侵袭和生长的抑制作用。1e)评价Ad-TFPI-2构建体与Ad-CMV构建体脑内注射的毒性。特异性目标2：确定在体外和体内内皮细胞和胶质瘤细胞共培养中与TFPI-2产生有关的调节脑血管生成的分子机制。2a)测定TFPI-2、VEGF、TF和其他蛋白酶以及内皮细胞与正/反义TFPi-2稳定克隆或胶质母细胞瘤细胞系和原代胶质母细胞瘤细胞感染Ad-CMV和Ad-TFPI-2意义构建物或存在抑制剂（2b）共培养时形成的毛细血管样结构的长度。确定正和反义稳定转染物或感染Ad-TFPI-2意义构建物或存在特定目的2a中指定的抑制剂对肿瘤的影响血管生成。我们相信，通过确定TFPI-2在胶质母细胞瘤侵袭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.