EHD2通过调控EGFR及其突变体内吞及循环参与肺腺癌恶性转化的研究

Lung carcer is a common malignant tumor. Lung adenocarcinoma show higher incidence throughout the world. Almost 40% of lung cancers are adenocarcinomas. Epidermal growth factor receptor (EGFR) is a prototype of receptor tyrosine kinases (RTKs) which control critical cellular responses to extra-cellular growth factors during development and tissue homeostasis. Importantly, overexpression of EGFR is frequently observed in lung adenocarcinoma, and recent studies have identified activating mutations in EGFR as direct determinants of oncogenic transformation in lung adenocarcinoma. Two types of mutations account for approximately 90% of mutated cases: a specific point mutation of L858R and short in-frame deletion of E746-A750. Mutant EGFR probably show different endocytosis or endosomal recycling pathway. Internalization of receptors is an essential process required by all mammalian cells, and is vital for the regulation of surface receptors. EHD2 is a new cellular endocytosis regulatory protein, which regulates endocytosis and transport of receptor. In lung adenocarcinoma tissue, we found that the expression of EHD2 were statistically signi?cantly correlated with the expression of EGFR. Three years survival was also potentially effected by EHD2.We also found that EHD2 inhabited the internalization of EGFR, enhanced the endocytic recycle of EGFR and enhanced the downstream signaling. It is unclear that how EHD2 regulated the endocytosis and recycling pathway of mutant EGFR. Based on the regulation of EGFR and EGFR mutants, we want to explore the mechanism of EHD2 in the cancerogenesis of lung adenocarcinoma.

肺腺癌已趋于成为发病率最高的肺癌组织亚型，约占全部肺癌的40%。EGFR扩增和突变是肺腺癌常见的癌变机制，最普遍的突变形式是发生于酪氨酸激酶区的Del E746-A750和L858R突变，二者共占肺腺癌全部突变的约90%。这两种突变体的内吞和胞内转运可能并不与野生型EGFR相同，具体机制尚不明了。EHD2是一类新型膜转运调控蛋白，调控受体内吞和胞内转运。我们发现肺腺癌组织样本中EHD2和EGFR表达水平呈正相关，呈现出独立影响生存的趋势。文献报道，EHD2具有抑制受体内吞，并可能有促进受体循环的作用。我们还发现EHD2负调控EGFR的内吞，抑制其降解，进而增强EGFR下游信号通路，然而EHD2对EGFR突变体的内吞和循环调控作用尚不清楚。在本课题从研究和比较肺腺癌细胞中EHD2对EGFR及其两种主要突变体的内吞及循环调控入手，尝试阐明EHD2参与肺腺癌恶性转化的机制，为临床干预提供新靶点。

EHD家族蛋白分别调节受体內吞及胞内转运的不同环节，它们多种的生物学功能逐渐被发现。在本课题中，我们对EHD2在肺癌发生发展过程中的作用进行了探索，现小结如下。.第一部分：EHD2与EGFR的关系.我们基于前期在hela细胞模型中的发现，EHD2可调控EGFR下游信号通路。但在肺癌中，我们观察到了EHD2和EGFR的共定位，应用免疫共沉淀证实了二者的相互作用，但我们未发现EHD2对EGFR下游信号通路的影响。本课题设计观察EHD2对EGFR及其突变体的胞内转运过程的影响，但因难以建立细胞模型及其它技术困难而放弃。.第二部分：EHD2和肺腺癌转移 .临床数据分析显示EHD2高表达和低表达，及肿瘤最大径是肺腺癌转移的风险因素。在A549细胞模型中，EHD2降表达后，MTT实验显示细胞增殖能力加强，Softagar实验显示细胞克隆形成能力加强。细胞二维克隆呈现离散趋势，E-cadherin表达降低，Vimentin表达升高。应用phalloidin染色观察细胞骨架显示EHD2降表达后细胞骨架改变，伪足增多。Transwells实验显示细胞迁移和侵袭能力增强。GST Pulldown实验显示A549细胞EHD2降表达后，Cdc42活化水平升高。经尾静脉注射裸鼠肺转移瘤实验中，A549细胞EHD2降表达后，肺表面转移成瘤明显增多。因此EHD2通过调节Cdc42活化水平影响肺腺癌转移。.第三部分：EHD2与肺鳞癌增殖.临床数据分析显示EHD2高表达组5年生存率优于EHD2低表达组。在肺鳞癌H520细胞系中，EHD2降表达后MTT实验显示细胞增殖能力加强；soft agar实验显示细胞克隆形成能力增强；裸鼠皮下种植瘤实验显示，种植瘤生长速度加快。应用GST Pulldown实验证实EHD2降表达后Rac1活化水平升高。Rac1是调控细胞增殖的信号通路之一。我们应用Rac1 T17N逆转了EHD2降表达引起的细胞增殖能力加强，提示Rac1在EHD2调控的细胞增殖信号通路中发挥重要作用。Rac1通过JNK通路调节细胞增殖。JNK抑制剂抑制了EHD2降表达引起的细胞增殖效应。在H520细胞系中，Western blot显示EHD2降表达提升了JNK磷酸化水平。综合上述数据，我们发现EHD2通过Rac1/JNK信号通路调节肺鳞癌细胞增殖，影响肺鳞癌生存预后。