Understanding and Eliminating Oncogenic EGFR Signaling in Malignant Glioma

描述（由申请人提供）：几十年来，我们已经知道表皮生长因子受体（EGFR）的过表达是多形性胶质母细胞瘤（GBM）病因学的主要组成部分，但对GBM中激活的EGFR发出的信号事件知之甚少。越来越清楚的是，信号通路是复杂和高度动态的，不能在真空中进行研究。这当然有助于定义GBM的可塑性，并解释GBM靶向治疗的失败。因此，我们必须在全球范围内研究EGFR信号传导事件，并在我们可以随意进行遗传操作的相关动物模型中进行研究。基于在人类肿瘤中观察到的最常见的遗传畸变，即EGFR沿着过度表达以及p16 lnk 4a/p19 ARF和PTEN肿瘤抑制基因的功能丧失，我们已经开发了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化蛋白质组学方法研究全球EGFR信号通路将揭示负责肿瘤细胞生长，迁移和耐药性的关键节点信号事件。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 GEMM将揭示GBM病因学的新的和有见地的信息。这将通过实现以下目标来实现：1）使用质谱法确定和研究来自我们的小鼠模型的GBM肿瘤中磷酸酪氨酸和磷酸丝氨酸/苏氨酸信号传导事件的网络动力学。2)研究我们的GBM靶向治疗对全局信号磷酸化网络的影响。3)使用定制的短发夹RNA（shRNA）文库系统地消除GBM肿瘤细胞中EGFR篡夺的信号传导事件，并确定产生的表型（肿瘤细胞生长、侵袭、对靶向、化疗和放疗的抗性）。4)验证人GBM样品中小鼠GBM生物学的那些关键磷酸化事件。5)确定各种纳米技术平台的毒性特征和疗效谱，以在体内将小干扰RNA（siRNA）分子有效递送至GBM肿瘤细胞。该应用将建立在RNA干扰介导的治疗干预的背景下，在临床前环境中，使用各种纳米平台作为递送工具，评估特定基因功能的基础。本文提出的前瞻性研究计划的力量在于我们操纵基因表达和在活体动物中进行遗传实验的能力。总之，这些功能通过提供能够有效分析基因功能的急需的动物系统，有力地补充了癌症基因组图谱提出的回顾性研究。

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.