Understanding and Eliminating Oncogenic EGFR Signaling in Malignant Glioma

描述(申请人提供)：几十年来，我们已经知道表皮生长因子受体(EGFR)的过度表达是多形性胶质母细胞瘤(GBM)病因的主要组成部分，但对GBM中由激活的EGFR发出的信号事件知之甚少。越来越清楚的是，信号通路是复杂的、高度动态的，不能在真空中研究。这当然有助于确定基底膜的可塑性，并解释了基底膜靶向治疗的失败。因此，我们必须在全球范围内研究EGFR信号转导事件，并在相关动物模型的背景下进行研究，我们可以随意在基因上操纵这种动物模型。我们基于在人类肿瘤中观察到的最常见的遗传异常，即EGFR的过度表达以及p16lnk4a/p19ARF和PTEN抑癌基因的功能丧失，开发了一种GBM的基因工程小鼠模型(GEMM)。我们假设，在我们的模型中使用磷酸蛋白质组学方法研究全球EGFR信号通路将揭示与肿瘤细胞生长、迁移和治疗耐药性有关的关键结节信号事件。通过使用我们的模型来研究负责这些影响的信号事件，我们的GBM的GEMM将揭示关于GBM病因的新的和有洞察力的信息。这将通过实现以下目标来实现：1)使用质谱仪来确定和研究我们的小鼠模型中GBM肿瘤中磷酸酪氨酸和磷酸丝氨酸/苏氨酸信号事件的网络动力学。2)研究靶向治疗大鼠肾小球系膜细胞对全球信号磷酸化网络的影响。3)使用定制的短发夹状RNA(ShRNA)文库系统地消除我们GBM肿瘤细胞中被EGFR篡夺的信号事件，并确定由此产生的表型(肿瘤细胞生长、侵袭、对靶向治疗、化疗和放射治疗的耐药性)。4)在人的基底膜样本中验证这些关键的小鼠基底膜生物学事件。5)确定不同纳米技术平台高效地将小干扰RNA(SiRNA)分子输送到GBM肿瘤细胞的毒性分布和效能谱。这项应用将为使用各种纳米平台作为输送工具，在临床前环境中评估RNA干扰介导的对GBM的治疗干预的特定基因功能奠定基础。本文提出的前瞻性研究计划的力量在于我们在活体动物身上操纵基因表达和进行遗传学实验的能力。总而言之，这些功能通过提供一个能够有效分析基因功能的急需的动物系统，与癌症基因组图谱提出的回溯性研究相称。

DESCRIPTION (provided by applicant): For decades, we have known that overexpression of the epidermal growth factor receptor (EGFR) is a major component of the etiology of glioblastoma multiforme (GBM), yet little is known regarding the signaling events that emanate from activated EGFR in GBM. It is becoming increasingly clear that signaling pathways are complex and highly dynamic and cannot be studied in a vacuum. This certainly helps define GBM's plasticity and explain failures of targeted therapies for GBMs. It is therefore imperative that we study EGFR signaling events globally and in the context of a relevant animal model that we can genetically manipulate at will. We have developed a genetically engineered mouse model (GEMM) of GBM, based on the most common genetic aberrations observed in human tumors, that is overexpression of EGFR along with loss of function of the p16lnk4a/p19ARF and PTEN tumor suppressor genes. We hypothesize that investigating global EGFR signaling pathways in our model using phosphoproteomic methods will reveal key nodal signaling events that are responsible for tumor cell growth, migration and resistance to therapies. By using our model to study signaling events responsible for these effects, our GEMM of GBM will reveal new and insightful information on the etiology of GBMs. This will be accomplished by fulfilling the following goals: 1) To determine and study network dynamics of phosphotyrosine and phosphoserine/threonine signaling events in GBM tumors from our mouse models using mass spectrometry. 2) To study the effects of targeted therapeutic treatment of our GBMs on global signaling phospho-networks. 3) To systematically eliminate the signaling events usurped by EGFR in our GBM tumor cells using custom-made short hairpin RNA (shRNA) libraries, and determine resulting phenotypes (tumor cell growth, invasion, resistance to targeted, chemo and radiation therapies). 4) To validate those key phosphoevents for mouse GBM biology in human GBM samples. 5) To ascertain toxicity profiles and efficacy spectrum of various nanotechnology platforms for the efficient delivery of small interfering RNA (siRNA) molecules to GBM tumor cells in vivo. This application will establish the groundwork for evaluating specific gene function in the context of RNA interference-mediated therapeutic intervention for GBM in a pre-clinical setting, using various nanoplatforms as delivery tools. The power of the prospective research program proposed herein lies in our ability to manipulate gene expression and perform genetic experiments in live animals. Together, these features commensurably complement the retrospective studies brought forward by The Cancer Genome Atlas by providing a much-needed animal system capable of efficient analysis of gene function.