Mechanisms of Ras-Induced Non-Apoptotic Cell Death in Glioblastoma

Ras 诱导胶质母细胞瘤非凋亡细胞死亡的机制

• 批准号: 7315564
• 负责人: WILLIAM A MALTESE
• 金额: $ 28.18万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7315564
• 关键词: 1-Phosphatidylinositol 3-Kinase; Affinity; Animals; Apoptosis; Apoptotic; Autophagocytosis; Autophagosome; Binding; Biogenesis; Brain Neoplasms; Cell Death; Cell Line; Cell Proliferation; Cell Survival; Cells; Cellular Morphology; Cessation of life; Complex; Diagnostic Neoplasm Staging; Disruption; Dominant-Negative Mutation; Ectopic Expression; Endosomes; Event; Exhibits; Gene Silencing; Glioblastoma; Glioma; Goals; Growth; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; HRAS gene; Hot Spot; Human; Incidence; Knowledge; Lead; Lysosomes; Malignant Neoplasms; Mediating; Membrane; Modeling; Molecular; Molecular Target; Morphogenesis; Mus; Mutation; Neuroblastoma; Nucleotides; Organelles; Pathway interactions; Phenotype; Phosphatidylinositols; Phosphotransferases; Positioning Attribute; Predisposition; Premalignant; Process; Proteomics; RAS genes; RNA Interference; Range; Research; Research Design; Research Personnel; Resistance; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Structure; Testing; Tetanus Helper Peptide; Therapeutic; Therapeutic Agents; Tumor stage; Vacuole; Virus; Xenograft Model; Xenograft procedure; cancer type; clinically significant; established cell line; inorganic phosphate; late endosome; novel; osteosarcoma; programs; promoter; protein expression; rab GTP-Binding Proteins; raf Kinases; ras Proteins; repaired; trafficking; tumor

DESCRIPTION (provided by applicant): The main goal of the proposed studies is to define the molecular signaling pathways whereby activated Ras proteins induce a novel form of non-apoptotic cell death in human glioblastoma cells. Gliomas are notoriously resistant to apoptotic death. Therefore, the long-term goal of this research is to uncover molecular targets that can be manipulated in a therapeutic context to activate non-apoptotic death in this type of cancer. These studies were prompted by the observation that ectopic expression of activated H-Ras or K-Ras in glioblastoma cells causes accumulation of cytoplasmic vacuoles that ultimately disrupt cell viability. The vacuoles are distinct from autophagosomes and may arise from late endosome or lysosome compartments. Active Ras produces similar effects in nine different human glioma cell lines, and the phenotype is recapitulated in stable glioblastoma cell lines where Ras is conditionally expressed. The unusual effects of Ras depend on its membrane association, but are independent from its stimulation of well- known effectors like Raf or PI3K. Preliminary studies indicate that activated Rac1, but not RhoA or Cdc42, can mimic the effects of activated Ras, whereas dominant-negative Rac1 blocks the Ras-induced phenotype. These findings lead to the central hypothesis that Ras activates Rac1-dependent effector pathways in glioblastoma to cause lethal disruptions of endosome or lysosome morphogenesis. To test this hypothesis, studies will focus on four specific aims: (Aim 1) We will define the defective organelles and trafficking events that contribute to non-apoptotic death in human glioblastoma cells. This will include identification of membrane compartments involved in the biogenesis of the vacuoles and delineation of alterations in vesicular trafficking. (Aim 2) We will evaluate the effects of Ras and Rac1 expression on the growth and viability of glioblastoma xenografts in mice, using established cell lines for conditional expression of the proteins. (Aim 3) We will identify the specific Ras effector pathway(s) required for activated Ras to elicit the vacuolar phenotype, focusing on Ras-regulated nucleotide exchange factors (e.g., Tiam1, RasGRF) that activate Rac1 signaling. This will involve the use of dominant-negative mutants, RNAi-mediated gene silencing, and affinity isolation and proteomic characterization of Ras binding partners. (Aim 4) We will define the downstream molecular connections between Rac1 signaling pathway(s) and the endo-lysosomal trafficking machinery in glioblastoma. These studies will focus on Rac1 interactions with Rab GTPases and phosphoinositide 4-phosphate 5'-kinases, which regulate trafficking in early and late endocytic pathways. These studies will contribute to a better understanding of connections between Ras and Rac1 signaling pathways and endo-lysosomal function, and they will provide new knowledge about a novel and poorly understood form of non-apoptotic death with potential clinical significance for treatment of brain tumors.

描述(申请人提供)：拟议研究的主要目标是确定激活的RAS蛋白在人胶质母细胞瘤细胞中诱导一种新的非凋亡性细胞死亡的分子信号通路。众所周知，胶质瘤对细胞凋亡性死亡具有抵抗力。因此，这项研究的长期目标是发现可以在治疗背景下操纵的分子靶点，以激活这种类型的癌症的非凋亡性死亡。这些研究是由于观察到在胶质母细胞瘤细胞中激活的H-RAS或K-RAS的异位表达导致细胞质空泡的积累，最终破坏细胞的活力。液泡不同于自噬小体，可能来自晚期的内小体或溶酶体室。活性RAS在9个不同的人脑胶质瘤细胞系中产生类似的作用，在稳定的胶质母细胞瘤细胞系中有条件地表达RAS的表型重现。RAS的特殊作用依赖于它的膜结合，但不依赖于它对著名的效应分子如Raf或PI3K的刺激。初步研究表明，被激活的rac1，而不是RhoA或Cdc42，可以模拟激活的RAS的作用，而显性负向的rac1则阻断RAS诱导的表型。这些发现导致了一个中心假设，即RAS激活胶质母细胞瘤中依赖于rac1的效应通路，导致内小体或溶酶体形态发生的致命中断。为了验证这一假设，研究将集中在四个特定的目标上：(目标1)我们将定义导致人类胶质母细胞瘤细胞非凋亡性死亡的缺陷细胞器和运输事件。这将包括识别与液泡的生物发生有关的膜室，并描述泡运输中的变化。(目的2)我们将利用已建立的条件表达RAS和rac1蛋白的细胞系，评估RAS和rac1的表达对小鼠胶质母细胞瘤移植瘤生长和存活的影响。(目的3)我们将确定激活RAS所需的特定RAS效应通路(S)，以诱导空泡表型，重点是RAS调节的核苷酸交换因子(例如，TIAM1，RASGRF)激活RAC1信号。这将涉及到显性负性突变体的使用，RNAi介导的基因沉默，以及RAS结合伙伴的亲和分离和蛋白质组学特征。(目的4)我们将确定在胶质母细胞瘤中RAC1信号通路(S)和内切酶转运机制之间的下游分子联系。这些研究将集中在rac1与Rab GTP酶和磷酸肌醇4-磷酸5‘-激酶的相互作用，这两个酶调节早期和晚期内吞途径的运输。这些研究将有助于更好地了解RAS和rac1信号通路与内溶酶体功能之间的联系，并将为了解一种新的、鲜为人知的非凋亡性死亡形式提供新的知识，这些死亡形式对脑肿瘤的治疗具有潜在的临床意义。