ELMO1, Dock180 and Glioma Invasion

描述（由申请人提供）：恶性人类神经胶质瘤的共同特征之一是单个肿瘤细胞分散在整个大脑中的固有能力，使得所有现有疗法都无法改变这些致命肿瘤的生长。 目前，神经胶质瘤细胞的危险侵袭性的机制知之甚少。 胶质瘤细胞恶性生长和潜伏侵袭的获得涉及多种遗传改变，包括表皮生长因子受体（EGFR）和PTEN的突变。 遗传研究表明，EGFR突变体的最常见形式EGFRvIII和PTEN缺失促进脑中胶质瘤细胞的侵袭。 最近，我们确定了一个上调的吞噬和细胞运动1（ELMO 1）在侵袭性胶质瘤细胞系。 ELMO 1及其结合伴侣胞质分裂专用因子1（dedicator of cytokinesis 1，Dock180）作为一种双向鸟嘌呤核苷酸交换因子（ganine nucleotide exchange factor，GEF）激活Rac 1并促进C.线虫、果蝇和哺乳动物细胞。 使用各种模型系统，包括大量的原发性人脑胶质瘤标本的分析，我们报告说，这种二分的Rac1 GEF，ELMO1-Dock180促进胶质瘤细胞迁移和侵袭在体外和离体（脑切片模型）。 我们的初步研究显示，在这些临床胶质瘤标本中，EGFRvIII与ELMO1-Dock180的表达和PTEN在浸润区域的丢失，而不是在中心区域。 通过人胶质瘤细胞系和短期培养的具有内源性EGFRvIII的原代人胶质瘤细胞外源性表达EGFRvIII显示Rac 1活性、ELMO 1和Dock180的酪氨酸磷酸化以及胶质瘤细胞运动性增加。 这种GEF的抑制减弱了EGFRvIII促进的细胞运动性。 此外，在表达EGFRvIII的胶质瘤细胞中敲低PTEN导致内源性ELMO 1和Dock180的诱导酪氨酸磷酸化，并进一步增强细胞运动性。 本研究的目的是探讨EGFRvIII和PTEN通过ELMO1-Dock180-Rac1通路调节胶质瘤细胞侵袭的机制，并建立ELMO1-Dock180作为胶质瘤治疗的治疗靶点。 我们的中心假设是EGFRvIII通过ELMO1-Dock180刺激胶质瘤细胞侵袭，并且表达EGFRvIII的细胞中的PTEN的缺失进一步增强胶质瘤细胞侵袭。 我们将通过三个具体目标来解决我们的假设。 在具体目标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测试我们的假设。 在具体目标2中，我们将通过评估PTEN在EGFRvIII-ELMO 1-Dock180诱导的胶质瘤细胞运动中的作用来检验我们的假设。 在具体目标3中，我们将通过确定ELMO 1、Dock180、PTEN及其效应物的抑制对表达EGFRvIII且具有已知PTEN状态的侵袭性胶质瘤中脑中胶质瘤细胞侵袭的影响来检验我们的假设。 我们有很好的模型系统来测试我们的假设，在胶质瘤细胞侵袭特性方面的丰富经验和所有必要的资源。 这些研究有助于我们进一步了解胶质瘤细胞的内在浸润能力，并为限制胶质瘤细胞在脑内的浸润提供重要的策略。 该项目具有转化意义，为胶质瘤细胞的内在侵袭性提供了新的见解，并探索了影响现有胶质瘤治疗反应的方法。 公共卫生相关性：恶性神经胶质瘤的一个标志是单个肿瘤细胞浸润整个大脑的内在能力，使得这些肿瘤几乎无法通过所有现有疗法治愈，并且也是其复发倾向的基础。 因此，进一步了解胶质瘤侵袭过程中细胞运动异常的机制是必要的，因为它可能导致发展新的策略来治疗恶性弥漫性胶质瘤。 本研究拟探讨由吞噬和细胞运动因子1（ELMO 1）和胞质分裂因子1（Dock180）组成的双向鸟嘌呤核苷酸交换因子（GEF）在胶质瘤细胞侵袭中的作用，并探讨ELMO 1-Dock180介导胶质瘤细胞异常脑浸润的分子机制。 拟议的工作具有转化意义，为胶质瘤细胞的内在侵袭性提供了新的见解，并探索了影响现有胶质瘤治疗反应的方法。

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.