Regulation of glioblastoma migration by PTEN PDZ-binding domain

描述(由申请人提供)：本提案的长期目标是发现治疗恶性胶质母细胞瘤的新靶点。这项建议探索了一种新的相互作用，在调节胶质母细胞瘤的运动行为中，涉及PTEN的PI3-K信号通路与一组促转移蛋白Ezrin-Radioxin-Moesin(ERM)之间的相互作用。多形性胶质母细胞瘤是恶性程度最高的人脑胶质瘤，具有广泛的局部侵袭特点。确定治疗这种恶性肿瘤的分子靶点将对临床结果产生影响。一些癌基因已被证明在胶质母细胞瘤中过度表达。这些受体包括表皮生长因子受体(EGFR)、血小板衍生生长因子(PDGF)和PDGF受体。在晚期胶质母细胞瘤中，最常见的基因改变是PTEN抑癌基因的缺失。胶质母细胞瘤高侵袭性的分子机制尚未完全阐明。主要的发现是PTEN介导了胶质母细胞瘤中运动性伪足的分解。这与膜-细胞骨架连接蛋白moesin的急剧重新分布以及随之而来的细胞运动性降低有关。PTEN中负责这一活性的区域是磷酸酶结构域和最后四个氨基酸PSD95/DLG/ZO-1(PDZ)结合域。有趣的是，PDZ结合区是胶质母细胞瘤突变失活的特异性靶点。这项应用将开发一种新的脑瘤模型，Pten？PDZ，它包含PTEN PDZ结合区域的缺失，以概括胶质母细胞瘤发展的侵袭期。在特定目标1中，将从野生型和Pten？pdz-/-小鼠中制备原代星形胶质细胞。将描述将PTEN连接到Akt和ERM的信号级联。还将比较ERM、PTEN和Akt在这些星形胶质细胞中的激活状态和亚细胞分布。通过异位表达和siRNA，Akt在调节ERM蛋白中的作用将被确定。在特定目的2中，比较Pten？PDZ+/+和Pten？PDZ-/-小鼠星形胶质细胞的迁移特性。Akt和ERM蛋白在细胞迁移中的作用将通过沉默RNA策略来检测。在特定的目标3中，Pten？PDZ-/-小鼠将与RasB8胶质瘤模型杂交。将分析在PasB8：PTEN？PDZ+/+和RasB8：PTEN？PDZ-/-小鼠中产生的胶质瘤的组织学分级、侵袭特征和相对肿瘤潜伏期。这些研究将为脑肿瘤分子疗法的发展奠定基础。公共卫生相关性：人类多形性胶质母细胞瘤是一种高度恶性的肿瘤，临床预后一般较差。这项建议探索使用一种新的临床前小鼠模型来概括这种恶性疾病的侵袭期。长期目标是利用这种小鼠模型作为药物发现工具，以确定可以阻止人类胶质母细胞瘤扩散的化合物。

DESCRIPTION (provided by applicant): The long-term objective of the present proposal is to uncover novel targets for treating malignant glioblastoma. This proposal explores a novel interaction between the PI3-K signaling cascade involving PTEN, and a group of pro-metastatic proteins, Ezrin-Radixin-Moesin (ERM), in regulating the motile behavior of glioblastoma. Glioblastoma multiforme is the most malignant form of human glioma characterized by extensive local invasion. Identification of molecular targets to treat this malignancy will have an impact on the clinical outcomes. Several oncogenes have been shown to be overexpressed in glioblastoma. These include Epidermal Growth Factor Receptor (EGFR), Platelet-derived Growth Factor (PDGF), and the receptors for PDGF. In late stage glioblastoma, the most frequent genetic alteration is the loss of PTEN tumor suppressor. The molecular mechanisms responsible for the highly invasive nature of glioblastoma have not been fully elucidated. The major finding is that PTEN mediates the disassembly of motile pseudopodia in glioblastoma. This is correlated with a drastic redistribution of a membrane-cytoskeleton linker protein, moesin, and a concomitant reduction in cell motility. The regions within PTEN responsible for this activity are the phosphatase domain and the last four amino acid PSD95/Dlg/ZO-1 (PDZ)-binding domain. Interestingly, PDZ-binding domain is specifically targeted for mutation inactivation in glioblastoma. This application will develop a novel brain tumor model, Pten?PDZ, which harbors a deletion in the PTEN PDZ-binding domain to recapitulate the invasive phase of glioblastoma development. In Specific Aim 1, primary astrocytes will be prepared from both wild-type and Pten?PDZ-/- mice. The signaling cascades linking PTEN to Akt and ERM will be delineated. The activation states and subcellular distribution of ERM, PTEN, and Akt in these astrocytes will also be compared. By using ectopic expression and siRNA, the role of Akt in regulating ERM proteins will be determined. In Specific Aim 2, the migratory properties of astrocytes prepared from Pten?PDZ+/+ and Pten?PDZ-/- mice will be compared. The role of Akt and ERM proteins in cell migration will be examined by silencing RNA strategy. In Specific Aim 3, Pten?PDZ-/- mice will be crossed to a RasB8 glioma model. Gliomas generated in PasB8: Pten?PDZ+/+ and RasB8: Pten?PDZ-/- mice will be analyzed for their histological grades, invasive features and relative tumor latency. These studies will lay the foundation for the development of molecular-based therapeutics for brain tumors. PUBLIC HEALTH RELEVANCE: Human glioblastoma multiforme is a highly malignant tumor and the clinical prognosis is generally poor. This proposal explores the use of a novel preclinical mouse model to recapitulate the invasive phase of this malignant disease. The long-term goal is to use this mouse model as a drug discovery tool to identify compounds that can block the spread of human glioblastoma.