Glypican-1 in gliomagenesis

描述（由申请人提供）： 中枢神经系统胶质瘤的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诊断后几个月内杀死患者。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明显改变。神经胶质瘤细胞的特征之一是它们的遗传不稳定性，这有助于进展和治疗抗性。开发更有效的胶质瘤治疗策略的主要障碍是对胶质瘤发生、进展和遗传不稳定性的机制相对缺乏了解。我们的发现可能为神经胶质瘤的治疗提供一种新的方法。 我们的实验室发现，磷脂酰肌醇蛋白聚糖-1（GPC 1），一种细胞表面蛋白聚糖，在绝大多数胶质瘤中过表达。GPC 1在体外过表达激活c-Myc并触发Skp 2自诱导环，其特征在于E2 F、Skp 2、细胞周期蛋白E和细胞周期蛋白依赖性激酶2（CDK 2）的协调诱导以及细胞周期蛋白依赖性激酶抑制剂（CKIs）p21和p27的抑制。这些细胞周期调节因子的协调变化导致G1-S-转换和DNA复制。在神经胶质瘤细胞和星形胶质细胞中，GPC 1过表达还诱导DNA再复制，导致DNA损伤。初步数据表明，癌基因c-Myc发挥这些变化的主要上游介质。 我们假设GPC 1是一个有效的细胞周期调节因子，在胶质细胞中过表达时作为癌基因发挥作用。我们建议通过破译GPC 1在胶质瘤细胞和星形胶质细胞中的信号通路，并通过确定GPC 1丢失和过表达对体内胶质瘤发展的影响来验证这一假设。目标1：分析Gpc 1在细胞周期调控中的作用机制：分析不同浓度的Gpc 1对星形胶质细胞和不同胶质瘤细胞DNA复制和细胞周期调控因子的影响。特别关注GPC 1在c-Myc激活中的活性。此外，我们将采用发现策略来识别GPC 1相互作用伙伴。目的2：分析GPC 1转化人星形胶质细胞的能力：永生和原代人星形胶质细胞将被转导以过表达GPC 1。将在体外转化试验和体内肿瘤发生实验中评估细胞的恶性潜能。目标3：确定GPC 1在体内胶质瘤发生和进展中的作用：将使用基于RCAS tv-a病毒受体系统的最新体内模型评估GPC 1在早期胶质瘤发生中的作用。使用功能获得方法，GPC 1将在神经祖细胞和星形胶质细胞中过表达，以确定GPC 1是否足以诱导胶质瘤。使用功能丧失的方法，将GPC 1基因缺陷小鼠与转基因小鼠杂交，转基因小鼠以星形胶质细胞特异性方式表达激活的ras癌基因，并发展具有高转移率和短潜伏期的高级别胶质瘤。这些实验将揭示GPC 1缺陷动物是否受到保护免于胶质瘤形成。此外，我们将询问现有的组织芯片，以确定是否GPC 1过表达是一个独立的预后标志物在人类胶质瘤。 重要性：拟议的研究旨在为胶质瘤发展和进展的生物学以及蛋白多糖GPC 1在这些过程中的特定作用提供新的见解。GPC 1信号通路可能通过靶向GPC 1本身或下游介质提供治疗干预的机会。

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.