Role of MIG6 in mutant EGFR-driven lung tumorigenesis

在我们基于质谱的实验中，我们发现MIG6（基因符号ERRFI1）是突变型egfr的磷酸化靶点。MIG6，也被称为ERBB受体反馈抑制剂1 （ERRFI1）和受体相关晚期传感器（RALT），是一种支架接头蛋白，其表达可被多种生长因子（包括EGF）、激素和其他应激源快速诱导。MIG6负调控EGFR、ERBB2和其他几种酪氨酸激酶受体及其信号通路。我们假设MIG6的缺失可能与EGFR突变体协同诱导肺肿瘤发生。为了验证这一假设，我们将多西环素诱导的EGFR突变转基因小鼠与Mig6缺失小鼠杂交。强力霉素诱导II型上皮细胞中表达的egfr突变体后，Mig6表达缺失确实加速了肺肿瘤的发生。我们在野生型、杂合型和敲除型小鼠中完成了强力霉素诱导突变型egfr的生存曲线。我们令人信服地表明，在Mig6零背景下，肿瘤发生加速，这表明Mig6确实是突变型egfr诱导的肺肿瘤发生的肿瘤抑制因子。我们与Ilona Linnoila博士合作完成了不同基因型小鼠肺肿瘤的组织学评估。我们还开始与Mark Simpson博士合作，量化这些肿瘤中各种靶点的免疫组织化学染色。我们进行了生化实验来阐明ERRFI1 Y394和Y395酪氨酸磷酸化增加的功能作用。我们已经证明突变egfr和Mig6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egfr的肺腺癌细胞中，这些位点存在组成性磷酸化。厄洛替尼治疗含有tki致敏EGFR突变体的肺腺癌细胞可显著抑制磷酸化，这表明MIG6确实是突变EGFR信号的靶标。我们还证明，与对WT型EGFR的影响相反，MIG6不能促进突变型EGFR降解。这可能是由于MIG6的Y394/395的过度磷酸化。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.