Mig6 Puts the Brakes on Mutant EGFR-Driven Lung Cancer

A model of the regulation of wild type (WT) and mutant (Mut) epidermal growth-factor receptor (EGFR) by Mig6. (A) Mig6 binds to and inhibits the kinase activity of activated WT EGFR, promoting EGFR trafficking to the degradation pathway. Phosphorylation of Mig6 by EGFR enhances the interaction of the two proteins but reduces Mig6's inhibition of EGFR. (B) Mut EGFR is constitutively active, which results in constitutive phosphorylation of Mig6, increased interaction between Mig6 and Mut EGFR, and decreased d

A model of the regulation of wild type (WT) and mutant (Mut) epidermal growth-factor receptor (EGFR) by Mig6. (A) Mig6 binds to and inhibits the kinase activity of activated WT EGFR, promoting EGFR trafficking to the degradation pathway. Phosphorylation of Mig6 by EGFR enhances the interaction of the two proteins but reduces Mig6's inhibition of EGFR. (B) Mut EGFR is constitutively active, which results in constitutive phosphorylation of Mig6, increased interaction between Mig6 and Mut EGFR, and decreased degradation of Mut EGFR.

Lung cancer is the most common cause of cancer-related death worldwide. These cancers are often induced by mutations in the epidermal growth factor receptor (EGFR), resulting in constitutive activation of the protein’s tyrosine kinase domain. Lung cancers expressing these EGFR mutants are initially sensitive to tyrosine kinase inhibitors (TKIs), such as erlotinib, but often become resistant by developing compensatory mutations in EGFR or other growth-promoting pathways. To better understand how mutant EGFR initiates and maintains tumor growth in the hopes of identifying novel targets for drug development, Udayan Guha, M.D., Ph.D., of CCR’s Thoracic and Gastrointestinal Oncology Branch, and his colleagues examined the landscape of proteins phosphorylated in EGFR wild type and mutant cells. One protein hyper-phosphorylated in mutant EGFR cells was Mig6, a putative tumor suppressor.

To tease apart the role of Mig6 in EGFR-mediated lung cancer, the researchers generated Mig6 knockout mice with inducible expression of EGFR mutants. After mutant EGFR induction, mice with homozygous or heterozygous loss of Mig6 (Mig6-/- or Mig6+/-, respectively) developed tumors more rapidly than mice expressing normal levels of Mig6 (Mig6+/+). Histopathological analysis of the lungs revealed frequent adenocarcinomas and gross airway hyperplasia in Mig6-/- and Mig6+/- mice while Mig6+/+ mice had mostly non-invasive lung adenomas and rare adenocarcinomas. Lack of Mig6 also significantly reduced the survival time of the mice after the induction of mutant EGFR.

When the investigators examined the lungs of the various transgenic lines using immunohistochemistry, they found an increase in the proliferation markers Ki67 and phosphorylated ERK but no reduction in apoptosis or senescence markers. Surprisingly, mutant EGFR expression was reduced in late-stage tumors from mice lacking Mig6. To confirm this result, the scientists immunoprecipitated EGFR from the tumors of mice with or without Mig6 and examined total and phosphorylated EGFR levels. Although total mutant EGFR was lower in tumors lacking Mig6, the ratio of phosphorylated to total protein was higher than in Mig6+/+ tumors, suggesting the higher proportion of phosphorylated EGFR is sufficient for tumor maintenance. The reduction in mutant EGFR did not appear to depend on changes in gene transcription as RNA levels were only slightly reduced in the Mig6-/- tumors.

The researchers next wanted to examine the sites at which Mig6 is phosphorylated and how modification affects its activity. Using mass spectrometry, they determined EGFR target phosphorylation sites by comparing samples from serum starved, EGF stimulated, and erlotinib-pretreated EGF stimulated lung cancer cell lysates. Mig6 tyrosine residues 394 and 395 were identified as targets of mutant EGFR kinase activity and were verified in lysates from mutant EGFR-induced tumors. Intriguingly, these tyrosines are two of six that fall within the previously identified EGFR binding domain of Mig6. To determine if tyrosines 394 and 395 are important for EGFR binding, the investigators mutated the residues to phenylalanine to mimic unphosphorylated tyrosine. They expressed the Mig6 double mutant with wild type or mutant EGFR and performed EGFR immunoprecipitation assays following EGF treatment. Mig6 bound both wild type and mutant EGFR, and EGF enhanced Mig6’s interaction with wild type EGFR. In the absence of EGF the association of Mig6 with mutant EGFR was stronger than with wild type EGFR. Mutant Mig6, however, showed reduced binding to both versions of EGFR. Phosphorylation of tyrosines 394 and 395 were sensitive to erlotinib treatment in cells expressing wild type EGFR but not those with mutant EGFR. Thus, these residues are bone fide targets of EGFR and are important for the interaction between Mig6 and EGFR.

Normally, EGF treatment induces the internalization and turnover of EGFR, but EGFR mutants are not properly endocytosed or degraded. To see whether Mig6 plays a role in the EGF-mediated degradation of mutant EGFR, the researchers expressed wild type or double mutant Mig6 with wild type or mutant EGFR and then treated the cells with cycloheximide, to inhibit protein synthesis, and EGF. As expected, wild type EGFR was degraded after EGF exposure whereas mutant EGFR protein was retained. Breakdown of the wild type EGFR depended on lysosomal activity since treatment with chloroquine significantly delayed the process. Interestingly, neither wild type nor double mutant Mig6 had any effect on the degradation of mutant EGFR. In contrast, wild type Mig6 delayed the destruction of wild type EGFR, suggesting this interaction may stabilize EGFR.

Taken together, these results show that phosphorylation of Mig6 at tyrosines 394 and 395 by EGFR enhances the association of Mig6 and EGFR and directs wild type, but not mutant, EGFR to efficient degradation in the lysosome. Phosphorylation of these residues reduces Mig6’s ability to inhibit mutant EGFR, but this residual inhibitory activity is sufficient to delay the initiation and progression of lung tumors caused by mutant EGFR in mouse models. Clinical studies should be undertaken to determine whether Mig6 levels play a role in the initiation, progression, or TKI response of EGFR-driven lung tumors in patients.

Summary Posted: 04/2015

Reference

Maity TK, Venugopalan A, Linnoila I, Cultraro CM, Giannakou A, Nemati R, Zhang X, Webster JD, Ritt D, Ghosal S, Hoschuetzky H, Simpson RM, Biswas R, Politi K, Morrison DK, Varmus HE, and Guha U. Loss of Mig6 accelerates initiation and progression of mutant epidermal growth factor receptor-driven lung adenocarcinoma. Cancer Discovery. March 3, 2015 PubMed Link