Role of MIG6 in mutant EGFR-driven lung tumorigenesis

在我们的基于质谱学的实验中，我们确定MIG6(基因符号ERRFI1)是突变的EGFR的磷酸化靶标。MIG6又称ERBB受体反馈抑制因子1(ERRFI1)和受体相关晚期转导蛋白(RALT)，是一种支架适配蛋白，在多种生长因子(包括EGF)、激素和其他应激源的诱导下迅速表达。MIG6负性调节EGFR、ERBB2和其他几种受体酪氨酸激酶及其信号通路。我们推测MIG6的缺失可能与突变的EGFR协同诱导肺肿瘤的发生。为了验证这一假设，我们将多西环素可诱导突变的EGFR转基因小鼠与Mig6基因缺失的小鼠进行杂交。当多西环素诱导在II型上皮细胞中表达的突变的EGFR时，Mig6的表达缺失确实加速了肺肿瘤的发生。我们已经完成了野生型、杂合型和基因敲除小鼠中多西环素诱导突变的EGFR的存活曲线。我们令人信服地表明，在Mig6缺失的背景下，肿瘤的发生被加速，这表明在突变的EGFR诱导的肺肿瘤发生中，Mig6确实是一种肿瘤抑制因子。我们与伊洛娜·利诺伊拉博士合作，完成了对不同基因类型的小鼠肺肿瘤的组织学评估。我们还与马克·辛普森博士合作，对这些肿瘤中不同靶点的免疫组织化学染色进行了量化。我们已经进行了生化实验，以阐明ERRFI1基因Y394和Y395酪氨酸磷酸化增加的功能作用。我们已经证明了突变的EGFR和Mig6之间的相互作用增加。此外，Y394/395是Mig6的主要磷酸化位点。在携带突变的EGFR的肺腺癌细胞中，这些位点存在结构性的磷酸化。对于携带TKI敏感的EGFR突变体的肺腺癌细胞，厄洛替尼处理可显著抑制其磷酸化，这表明MIG6确实是突变的EGFR信号的靶点。我们还证明了MIG6不能促进突变的EGFR的降解，这与它对WT EGFR的影响相反。这可能是由于MIG6的Y394/395过度磷酸化所致。一份描述这些发现的手稿正在审查中。

We identified MIG6 (gene symbol ERRFI1) as a phosphorylation target of mutant EGFRs in our mass spectrometry-based experiments. MIG6, also known as ERBB receptor feedback inhibitor 1 (ERRFI1) and receptor-associated late transducer (RALT), is a scaffolding adaptor protein whose expression is rapidly induced by a variety of growth factors (including EGF) and by hormones and other stressors. MIG6 negatively regulates EGFR, ERBB2, and several other receptor tyrosine kinases and their signaling pathways. We hypothesized that loss of MIG6 may cooperate with mutant EGFR to induce lung tumorigenesis. To test this hypothesis we crossed doxycycline inducible mutant EGFR transgenic mice with Mig6 null mice. Upon doxycycline induction of mutant EGFRs that are expressed in type II epithelial cells, lung tumorigenesis is indeed accelerated upon loss of Mig6 expression. We have completed the survival curve upon doxycycline induction of mutant EGFRs in wild type, heterozygous and knock-out mice. We convincingly show that tumorigenesis is accelerated in Mig6 null background suggesting that Mig6 is indeed a tumor suppressor in mutant EGFR-induced lung tumorigenesis. We have completed the evaluation of histology of mouse lung tumor in various genotypes in collaboration with Dr. Ilona Linnoila. We have also initiated a collaboration with Dr. Mark Simpson to quantify immunohistochemical staining of various targets in these tumors. We have performed biochemical experiments to elucidate the functional role of increased tyrosine phosphorylation at Y394 and Y395 of ERRFI1. We have demonstrated that there is increased interaction of mutant EGFRs and Mig6. In addition Y394/395 are the predominant sites of phosphorylation in Mig6. There is constitutive phosphorylation of these sites in lung adenocarcinoma cells harboring mutant EGFRs. Phosphorylation is significantly inhibited by erlotinib treatment of lung adenocarcinoma cells that harbor TKI-sensitizing mutants of EGFR, suggesting MIG6 is indeed a target of mutant EGFR signaling. We have also demonstrated that MIG6 cannot promote mutant EGFR degradation contrary to its effects on WT EGFR. This is likely due to hyperphosphorylation of Y394/395 of MIG6. A manuscript is under review describing these findings.