ELMO1, Dock180 and Glioma Invasion

描述(申请人提供)：人类恶性胶质瘤的共同特征之一是单个肿瘤细胞在大脑中扩散的固有能力，导致所有现有治疗方法实际上都失败了，无法改变这些致命肿瘤的生长。目前，胶质瘤细胞危险侵袭的机制尚不清楚。胶质瘤细胞获得恶性生长和潜伏侵袭性涉及多种基因改变，包括表皮生长因子受体(EGFR)和PTEN的突变。遗传学研究表明，最常见的EGFR突变体EGFRvIII和PTEN缺失会促进脑内胶质瘤细胞的侵袭。最近，我们在侵袭性胶质瘤细胞系中发现了吞噬和细胞运动1(ELMO1)的上调。ELMO1和它的结合伙伴，胞质分裂的奉献因子1(Dock180)是一种二段鸟嘌呤核苷酸交换因子，激活了rac1，促进了线虫、果蝇和哺乳动物细胞的迁移。利用各种模型系统，包括对大量原代人脑胶质瘤标本的分析，我们报道了这种二分的rac1 gef，ELMO1-Dock180促进胶质瘤细胞在体外和体外(脑片模型)的迁移和侵袭。我们的初步研究显示，在这些临床脑胶质瘤标本中，ELMO1-Dock180表达EGFRvIII，PTEN在侵袭区丢失，但在中央区不表达。外源性表达EGFRvIII的人胶质瘤细胞系和短期培养的原代人脑胶质瘤细胞经内源性EGFRvIII诱导后，细胞内的rac1活性、ELMO1和Dock180的酪氨酸磷酸化水平及胶质瘤细胞的运动能力均增强。抑制这种全球环境基金减弱了EGFRvIII促进的细胞运动。此外，在表达EGFRvIII的胶质瘤细胞中，PTEN的敲除导致内源性ELMO1和Dock180的酪氨酸磷酸化，并进一步增强细胞的运动性。本研究的目的是研究EGFRvIII和PTEN通过ELMO1-Dock180-rac1通路调控胶质瘤细胞侵袭的机制，并建立ELMO1-Dock180作为治疗胶质瘤的靶点。我们的中心假设是，EGFRvIII通过ELMO1-Dock180刺激胶质瘤细胞的侵袭，表达EGFRvIII的细胞中PTEN的缺失进一步增强了胶质瘤细胞的侵袭。我们将用三个具体目标来解释我们的假设。在特定的目标1中，我们将通过确定ELMO1-Dock180在EGFRvIII刺激的胶质瘤细胞运动中的作用来检验我们的假设。在特定的目标2中，我们将通过评估PTEN在EGFRvIII-ELMO1-Dock180诱导的胶质瘤细胞运动中的作用来检验我们的假设。在特定的目标3中，我们将通过确定ELMO1、Dock180、PTEN及其效应物对已知PTEN状态的表达EGFRvIII的侵袭性胶质瘤脑内胶质瘤细胞侵袭的影响来验证我们的假设。我们有优秀的模型系统来测试我们的假设，在胶质瘤细胞侵袭的表征方面有丰富的经验，以及所有必要的资源。这些研究结果有助于我们进一步了解脑胶质瘤细胞的固有侵袭能力，并为限制脑内胶质瘤细胞的侵袭提供重要的策略。该项目在为胶质瘤细胞的内在侵袭性提供新的见解以及探索影响现有胶质瘤治疗反应的方法方面具有翻译意义。公共卫生相关性：恶性胶质瘤的一个特征是单个肿瘤细胞固有的渗透到整个大脑的能力，使这些肿瘤几乎无法通过现有的所有治疗方法治愈，也是它们极大的复发倾向的基础。因此，进一步了解胶质瘤侵袭过程中异常细胞运动的机制是非常必要的，因为这可能导致开发新的治疗恶性弥漫性胶质瘤的策略。在这项建议中，我们计划确定由吞噬和细胞运动1(ELMO1)和胞质分裂奉献因子1(Dock180)组成的两部分鸟核苷酸交换因子(Global)在胶质瘤细胞侵袭中的新作用，并探讨ELMO1-Dock180介导异常胶质瘤细胞向脑内渗透的分子机制。这项拟议的工作在提供对胶质瘤细胞内在侵袭性的新见解以及探索影响现有胶质瘤治疗反应的方法方面具有翻译意义。

DESCRIPTION (provided by applicant): One of the common features of malignant human gliomas is the intrinsic ability of single tumor cells to disperse throughout the brain, rendering the virtual failure of all existing therapies to alter the growth of these deadly tumors. Currently, the mechanisms underlying the perilous invasiveness of glioma cells are poorly understood. Acquisition of malignant growth and insidious invasiveness by glioma cells involves multiple genetic alterations including mutations of epidermal growth factor receptor (EGFR) and PTEN. Genetic studies showed that the most common form of EGFR mutants, EGFRvIII and PTEN loss promote glioma cell invasion in the brain. Recently, we identified an up-regulation of engulfment and cell motility 1 (ELMO1) in invasive glioma cell lines. ELMO1 and its binding partner, dedicator of cytokinesis 1 (Dock180) function as a bipartite guanine nucleotide exchange factor (GEF) that activates Rac1 and promotes cell migration of C. elegans, Drosophila and mammalian cells. Using various model systems including analysis of a large number of primary human glioma specimens, we reported that this bipartite Rac1 GEF, ELMO1-Dock180 promotes glioma cell migration and invasion in vitro and ex vivo (brain slice model). Our preliminary studies reveal expression of EGFRvIII with ELMO1-Dock180 and loss of PTEN in invasive areas but not in central regions on these clinical glioma specimens. Exogenous expression of EGFRvIII by human glioma cell lines and short-term cultured primary human glioma cells with endogenous EGFRvIII displayed increases in Rac1 activity, tyrosine phosphorylation of ELMO1 and Dock180, and glioma cell motility. Inhibition of this GEF attenuated EGFRvIII-promoted cell motility. Additionally, knockdown of PTEN in EGFRvIII-expressing glioma cells resulted in induced tyrosine phosphorylation of endogenous ELMO1 and Dock180 and further enhanced cell motility. The goal of this proposal is to investigate the mechanisms by which EGFRvIII and PTEN modulate glioma cell invasion through the ELMO1-Dock180-Rac1 pathway and establish ELMO1-Dock180 as a therapeutic target for glioma treatment. Our central hypothesis is that EGFRvIII stimulates glioma cell invasion through ELMO1-Dock180 and loss of PTEN in EGFRvIII-expressing cells further enhances glioma cell invasion. We will address our hypotheses with three Specific Aims. In Specific Aim 1, we will test our hypothesis by determining the role of ELMO1-Dock180 in EGFRvIII-stimulated glioma cell motility. In Specific Aim 2, we will test our hypothesis by evaluating the effect of PTEN in EGFRvIII-ELMO1-Dock180-induced glioma cell motility. In Specific Aim 3, we will test our hypothesis by determining the impact of inhibition of ELMO1, Dock180, PTEN and their effectors on glioma cell invasion in the brain in invasive gliomas expressing EGFRvIII with known PTEN status. We have excellent model systems in place to test our hypotheses, extensive experience in characterization of glioma cell invasion and all of the necessary resources. The proposed studies could advance our understanding of the inherent ability of glioma cell infiltration and provide important strategies to limit the infiltration of glioma cells in the brain. This project has translational significance in providing new insights into intrinsic invasiveness of glioma cells and exploring approaches that impact the response to existing glioma therapies. PUBLIC HEALTH RELEVANCE: A hallmark of malignant gliomas is the intrinsic ability of single tumor cells to infiltrate throughout the brain, rendering these tumors virtually incurable by all existing therapies and also underlies their great propensity for recurrence. Therefore, further understanding of mechanisms underlying the aberrant cell motility during glioma invasion is imperative because it could lead to developing new strategies to treat malignant diffuse gliomas. In this proposal, we plan to determine the novel role of a bipartite guanine nucleotide exchange factor (GEF) composed of engulfment and cell motility 1 (ELMO1) and dedicator of cytokinesis 1 (Dock180) in glioma cell invasion and investigate the molecular mechanisms by which ELMO1-Dock180 mediate aberrant glioma cell infiltration into the brain. The proposed work has translational significance in providing new insights into intrinsic invasiveness of glioma cells and exploring approaches that impact the response to existing glioma therapies.