Glypican-1 in gliomagenesis

描述(由申请人提供)： 中枢神经系统胶质瘤通过不断生长和局部侵袭导致发病率和死亡率。高级别胶质瘤通常在确诊后几个月内死亡。尽管在治疗方面取得了进展，但这种令人沮丧的预后并没有显著改变。胶质瘤细胞的特征之一是它们的遗传不稳定，这导致了进展和治疗抵抗。发展更有效的胶质瘤治疗策略的一个主要障碍是相对缺乏对胶质瘤发生、发展和遗传不稳定的机制的了解。我们的一项发现可能会为胶质瘤的治疗提供一种新的方法。我们的实验室发现GPC1是一种细胞表面蛋白多糖，在绝大多数胶质瘤中都有过表达。GPC1在体外过表达可激活c-Myc并触发Skp2自身诱导环，其特征是协同诱导E2F、Skp2、细胞周期蛋白E和细胞周期蛋白依赖性蛋白依赖性蛋白2(CDK2)，抑制细胞周期蛋白依赖性蛋白激酶抑制物(CKI)p21和p27。这些细胞周期调节因子的协调变化导致了G1-S-转换和dna复制。在胶质瘤细胞和星形胶质细胞中，GPC1的过表达也会诱导DNA的重新复制，从而导致DNA损伤。初步数据表明，癌基因c-Myc是这些变化的主要上游调节因子。我们假设GPC1是细胞周期的一个强有力的调节器，当在神经胶质细胞中过度表达时，它作为癌基因发挥作用。我们建议通过破译GPC1在胶质瘤细胞和星形胶质细胞中的信号通路，以及通过确定GPC1缺失和过表达在体内胶质瘤发展中的作用来验证这一假说。目的1：分析GPC1在细胞周期调控中的作用机制：分析不同浓度的GPC1对星形胶质细胞和不同胶质瘤细胞DNA复制和细胞周期调节的影响。将特别关注c-Myc激活过程中GPC1的活性。此外，我们将采用发现策略来确定GPC1交互合作伙伴。目的2：分析GPC1转化人脑星形胶质细胞的能力：永生化和原代人脑星形胶质细胞将被转导过表达GPC1。这些细胞的恶性潜能将在体外转化实验和体内肿瘤形成实验中进行评估。目的3：确定GPC1在体内胶质瘤发生发展中的作用：基于RCAS TV-a病毒受体系统的最先进的体内模型将评估GPC1在早期胶质瘤形成中的作用。使用功能增益方法，GPC1将在神经前体细胞和星形胶质细胞中过表达，以确定GPC1是否足以诱导胶质瘤。通过功能丧失的方法，GPC1基因缺陷的小鼠将与转基因小鼠杂交，转基因小鼠以星形胶质细胞特有的方式表达激活的ras癌基因，并发展出高外显率和短潜伏期的高级别胶质瘤。这些实验将揭示GPC1缺陷的动物是否受到保护，免受胶质瘤的发生。此外，我们将询问现有的组织微阵列，以确定GPC1过度表达是否是人脑胶质瘤的独立预后标志。意义：拟议的研究旨在为胶质瘤的发展和进展的生物学以及蛋白多糖GPC1在这些过程中的具体作用提供新的见解。GPC1信号通路可能通过靶向GPC1本身或下游介质为治疗干预提供机会。

DESCRIPTION (provided by applicant): Central nervous system gliomas cause morbidity and mortality by relentless growth and local invasion. High-grade gliomas typically kill patients within months after diagnosis. Despite advances in therapy, this dismal prognosis has not changed significantly. One of the hallmarks of glioma cells is their genetic instability, which contributes to progression and therapy resistance. A major barrier to the development of more effective glioma treatment strategies is the relative lack of understanding of the mechanisms underlying glioma development, progression and genetic instability. We have made a discovery that may offer a novel approach to the treatment of gliomas. Our laboratory discovered that glypican-1 (GPC1), a cell surface proteoglycan, is overexpressed in the vast majority of gliomas. Overexpression of GPC1 in vitro activates c-Myc and triggers the Skp2 autoinduction loop, which is characterized by the coordinated induction of E2F, Skp2, cyclin E and cyclin-dependent kinase 2 (CDK2) and the suppression of the cyclin-dependent kinase inhibitors (CKIs) p21 and p27. These coordinated changes in cell cycle regulators lead to G1-S-transition and DNA replication. In glioma cells and astrocytes, GPC1 overexpression also induces DNA re-replication resulting in DNA damage. Preliminary data indicate that the oncogene c-Myc plays is a major upstream mediator of these changes. We hypothesize that GPC1 is a potent regulator of the cell cycle and acts as an oncogene when overexpressed in glial cells. We propose to test this hypothesis by deciphering the signaling pathway of GPC1 in glioma cells and astrocytes and by determining the effect of GPC1 loss and overexpression on glioma development in vivo. Aim 1: Analyze the mechanism of Gpc1 in cell cycle regulation: We will analyze the effect of different GPC1 concentrations on DNA replication and cell cycle regulators in astrocytes and different glioma cells. A particular focus will be placed on GPC1 activity in the activation of c-Myc. In addition, we will employ a discovery strategy to identify GPC1 interaction partners. Aim 2: Analyze the ability of GPC1 to transform human astrocytes: Immortal and primary human astrocytes will be transduced to overexpress GPC1. The malignant potential of the cells will be assessed in in vitro transformation assay and with in vivo tumorigenesis experiments. Aim 3: Determine the role of GPC1 in glioma development and progression in vivo: The role of GPC1 in early gliomagenesis will be assessed using state-of-the-art in vivo models based on the RCAS tv-a viral receptor system. Using a gain-of-function approach, GPC1 will be overexpressed in neural progenitors and astrocytes to determine whether GPC1 is sufficient for glioma induction. Using a loss-of-function approach, genetically GPC1-deficient mice will be crossed with transgenic mice, which express activated ras oncogene in a astrocyte-specific manner and which develop high-grade gliomas with high penetrance and short latency. These experiments will reveal whether GPC1-deficient animals are protected from gliomagenesis. In addition, we will interrogate an existing tissue microarray to determine whether GPC1 overexpression is an independent prognostic marker in human glioma. Significance: The proposed studies are designed to offer novel insights into the biology of glioma development and progression and into the specific role of the proteoglycan GPC1 in these processes. The GPC1 signaling pathway may offer opportunities for therapeutic intervention by targeting either GPC1 itself or downstream mediators.