Mutant Epidermal Growth Factor Receptor Signaling in Lung Cancer

描述（由申请人提供）：此提案描述了我在癌症生物学学术生涯和作为内科科学家的职业生涯发展的培训计划。在这个奖项期间，我将发展我的独立研究计划。我还将利用这段时间来积累我的科学知识和指导技能。哈罗德·瓦姆斯博士是癌症生物学领域的领军人物，他培养了许多成功的科学家，他将是我的导师。纪念斯隆-凯特琳癌症中心（MSKCC）将提供机构支持，包括充足的资源，职业发展活动和科学家和临床医生之间的互动机会，以帮助我实现我的目标。这项建议的重点是肺癌，死亡率最高的癌症。表皮生长因子受体（EGFR）基因突变与肺癌患者对酪氨酸激酶抑制剂（TKIs）的敏感性相关，如吉非替尼（易瑞沙）或厄洛替尼（特罗凯）。在过去的三年里，我在Varmus博士的实验室进行博士后研究，发现了肺癌中EGFR突变体的许多靶点。在本提案中，我建议进一步扩展这项工作，以了解传递突变egfr信号以促进细胞存活和增殖的信号通路以及被TKIs抑制以促进敏感性的信号通路。具体目标1：我建议研究人类肺癌细胞系和这些细胞系变体中突变EGFR下游蛋白的磷酸化状态，这些细胞系在实验室培养和操作后对TKIs产生了抗性。我们还建议在我们实验室产生的转基因小鼠中研究信号转导，这些小鼠在肺上皮中有条件地表达突变的egfr，并产生类似于人类的肺肿瘤。我们将利用技术富集磷酸化蛋白，利用灵敏质谱法检测这些蛋白，并量化这些蛋白在各种条件下的磷酸化程度，包括配体刺激和TKI抑制。特异性目标2：我建议通过在肺癌组织微阵列（tma）中进行免疫组织化学来证实特异性目标1中鉴定的突变EGFR靶点的磷酸化存在。我还将使用较新的专业质谱技术（精确包涵体质量筛选，AIMS和多重反应监测，MRM）来特异性确认人类肺肿瘤组织中靶点的特异性磷酸化。具体目标3：我建议进行功能研究，包括通过RNAi技术敲除蛋白表达的实验，以测定细胞在体外和体内的存活和增殖。我们还建议研究突变EGFR和Mig-6表达缺失的合作关系，Mig-6是一种在表达突变EGFR的细胞中酪氨酸上被过度磷酸化的蛋白。

DESCRIPTION (provided by applicant): This proposal describes a training program for the development of my academic career in cancer biology and professional career as a physician scientist. Throughout the period of this award, I will develop my independent research program. I will also use this time to build on my scientific knowledge and mentoring skills. Dr. Harold Varmus, a leader in the field of cancer biology who has trained numerous successful scientists, will be my mentor. Memorial Sloan-Kettering Cancer Center (MSKCC) will provide institutional support, including ample resources, career development activities and opportunities for interactions between scientists and clinicians to help me achieve my goals. This proposal focuses on lung cancer, the cancer with the highest mortality. Mutations in the epidermal growth factor receptor (EGFR) gene have been associated with sensitivity of lung cancer patients to tyrosine kinase inhibitors (TKIs), such as gefitinib (Iressa(R)) or erlotinib (Tarceva). My postdoctoral research in the laboratory of Dr. Varmus over the last three years has led to the identification of numerous targets of mutant EGFR in lung cancer. In this proposal, I propose to extend this work further to understand the signaling pathways that relay the signals from mutant EGFRs to promote cell survival and proliferation and the pathways that are inhibited by TKIs to promote sensitivity. Specific Aim 1:I propose to study the phosphorylation status of proteins downstream of mutated EGFR in human lung cancer cell lines and in variants of these cell lines, which have developed resistance to TKIs upon culture and manipulation in the laboratory. We also propose to study signaling in transgenic mice generated in our laboratory that conditionally express the mutant EGFRs in lung epithelium and generate lung tumors similar to the ones in humans. We will use techniques to enrich phosphorylated proteins, use sensitive mass spectrometry to detect these proteins, and quantify the degree of phosphorylation in these proteins under a variety of conditions, including ligand stimulation and TKI inhibition. Specific Aim 2:I propose to confirm the presence of phosphorylation of targets of mutant EGFR identified in Specific aim 1 by performing immunohistochemistry in lung cancer tissue microarrays (TMAs). I will also use newer specialized mass spectrometry techniques (accurate inclusion mass screening, AIMS and multiple reaction monitoring, MRM) to specifically confirm site-specific phosphorylation of the targets in human lung tumor tissue. Specific Aim 3: I propose to perform functional studies, including experiments to knock-down protein expression by RNAi technology in order to assay survival and proliferation of cells in vitro and in vivo. We also propose to study the cooperation of mutant EGFR and loss of expression of Mig-6, a protein identified to be hyper-phosphorylated on tyrosines in cells expressing mutant EGFR. RELEVANCE: Lung cancer is the leading cause of death from cancer. EGFR mutations account for around 10% of all patients of lung cancer in the US and are associated with sensitivity to tyrosine kinase inhibitors. This proposal is aimed at elucidating the signaling pathways that relay the signals from mutant EGFRs to promote cancer cell survival and proliferation and validate those targets of mutant EGFR in lung cancer patients.