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

描述（由申请人提供）：拟议研究的主要目标是定义激活的Ras蛋白诱导人胶质母细胞瘤细胞非凋亡细胞死亡的分子信号通路。众所周知，胶质瘤对细胞凋亡具有抵抗力。因此，本研究的长期目标是发现可在治疗背景下操纵的分子靶点，以激活这类癌症的非凋亡性死亡。这些研究是由于观察到胶质母细胞瘤细胞中活化的H-Ras或K-Ras的异位表达导致细胞质空泡积聚，最终破坏细胞活力。液泡与自噬体不同，可能产生于内核体或溶酶体的晚期区室。活性Ras在9种不同的人类胶质瘤细胞系中产生类似的作用，并且在稳定的胶质母细胞瘤细胞系中重现了这种表型，其中Ras有条件表达。Ras的不同寻常的作用依赖于它的膜结合，但不依赖于它对众所周知的效应器如Raf或PI3K的刺激。初步研究表明，激活的Rac1，而不是RhoA或Cdc42，可以模拟激活的Ras的作用，而显性阴性的Rac1则可以阻断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rac1依赖的效应通路，导致内核体或溶酶体形态发生致命的破坏。为了验证这一假设，研究将集中在四个特定目标上：（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。这将包括识别参与液泡生物发生的膜室和描述液泡运输的改变。（目的2）我们将评估Ras和Rac1表达对小鼠胶质母细胞瘤异种移植物生长和活力的影响，使用已建立的细胞系进行蛋白质的条件表达。（目标3）我们将确定激活的Ras诱导空泡表型所需的特定Ras效应通路，重点关注激活Rac1信号的Ras调节的核苷酸交换因子（例如Tiam1， RasGRF）。这将涉及使用显性阴性突变体，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表征。（目的4）我们将定义胶质母细胞瘤中Rac1信号通路与内切溶酶体运输机制之间的下游分子连接。这些研究将集中在Rac1与Rab gtpase和磷酸肌肽4-磷酸5'-激酶的相互作用上，后者在早期和晚期内吞途径中调节运输。这些研究将有助于更好地理解Ras和Rac1信号通路与内溶酶体功能之间的联系，并将为一种新的、尚不清楚的非凋亡性死亡形式提供新的知识，对脑肿瘤的治疗具有潜在的临床意义。

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.