Role of MIG6 in mutant EGFR-driven lung tumorigenesis

我们在基于质谱的实验中将MIG 6（基因符号ERRFI 1）鉴定为突变EGFR的磷酸化靶点。MIG 6，也称为ERBB受体反馈抑制剂1（ERRFI 1）和受体相关晚期转导子（RALT），是一种支架衔接蛋白，其表达由多种生长因子（包括EGF）和激素及其他应激源快速诱导。MIG 6负调节EGFR、ERBB 2和其他几种受体酪氨酸激酶及其信号通路。我们推测MIG 6的缺失可能与突变型EGFR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型突变EGFR转基因小鼠与Mig 6缺失小鼠杂交。在II型上皮细胞中表达的突变EGFR的多西环素诱导后，肺肿瘤发生确实在Mig 6表达丧失后加速。我们已经完成了野生型、杂合子和基因敲除小鼠中多西环素诱导突变EGFR后的存活曲线。我们令人信服地表明，在Mig 6空背景下，肿瘤发生加速，这表明Mig 6确实是突变EGFR诱导的肺肿瘤发生中的肿瘤抑制因子。我们与Ilona Linnoila博士合作完成了对不同基因型小鼠肺肿瘤的组织学评价。我们还与Mark Simpson博士合作，对这些肿瘤中各种靶点的免疫组织化学染色进行量化。我们已经进行了生化实验，以阐明ERRFI 1的Y394和Y395处的酪氨酸磷酸化增加的功能作用。我们已经证明，突变EGFR和Mig 6的相互作用增加。此外，Y394/395是Mig 6中磷酸化的主要位点。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。厄洛替尼治疗携带EGFR TKI致敏突变体的肺腺癌细胞显著抑制磷酸化，表明MIG 6确实是突变EGFR信号传导的靶点。我们还证明了MIG 6不能促进突变型EGFR降解，这与其对WT EGFR的作用相反。这可能是由于MIG 6的Y394/395的过度磷酸化。这些发现已经发表（Cancer Discov. 5（5）：534-49（2015）。我们继续我们的研究，以了解突变EGFR驱动的肺肿瘤维持中Mig 6抑制的生物学。我们目前正在培育具有突变EGFR、CCSP-rtTA和Nkx2.1-CreERT 2转基因的小鼠，这些转基因在floxed Mig 6的背景下。我们通过强力霉素诱导这些小鼠产生肺腺癌。小鼠肿瘤正在进行连续MRI成像。Mig 6在小鼠肿瘤发展后通过托莫西芬注射被删除，随后的肿瘤生长进展将通过连续MRI扫描监测。在另一组小鼠中，我们也注射腺病毒Cre来敲低Mig 6，而不是含有Nkx2.1-Cre启动子的转基因小鼠。我们已经在10只小鼠中注射了腺病毒Cre，并等待系列MRI来监测肿瘤进展。该项目这一部分的总体目标是研究Mig 6在突变EGFR驱动的肿瘤维持中的作用。我们正在跟踪小鼠肿瘤的产生。这些小鼠正在接受系列MRI以监测肿瘤发生。一旦出现肿瘤，将在特定时间段处死小鼠，并通过组织学监测肿瘤负荷。

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. These findings have been published (Cancer Discov. 5(5):534-49 (2015). We have continued our studies to understand the biology of Mig6 inhibition in mutant EGFR-driven lung tumor maintenance. We are currently breeding mice to have mutant EGFR, CCSP-rtTA, and Nkx2.1-CreERT2 transgenes in the background of floxed Mig6. We have generated lung adenocarcinomas in these mice by doxycycline induction. The mouse tumors are being followed by serial MRI imaging. Mig6 is being deleted by tomoxifen injection after the mouse tumors have developed and the subsequent progression of tumor growth will be monitored by serial MRI scan. In a separate cohort of mice we are also injecting adenovirus-Cre to knockdown Mig6 instead of the Nkx2.1-Cre promoter containing transgenic mice. We have injected adenovirus- Cre in 10 mice so far and awaiting the serial MRI to monitor tumor progression. The overall goal of this part of the project is to examine the role of Mig6 in mutant EGFR-driven tumor maintenance. We are following the mice for generation of tumors. These mice are undergoing serial MRIs to monitor tumorigenesis. Once tumors arise, the mice will be sacrificed at particular time periods and tumor burden monitored from histology.