The role of cooperativity with Src in oncogenic transformation mediated by non-small cell lung cancer-associated EGF receptor mutants

Non-small cell lung cancer (NSCLC)-associated epidermal growth factor receptor (EGFR) mutants are constitutively active and induce ligand-independent transformation in nonmalignant cell lines. We investigated the possibility that the ability of mutant EGFRs to transform cells reflects a constitutive cooperativity with Src using a system in which the overexpression of mutant but not wild-type EGFR induced anchorage-independent cell growth. Src was constitutively activated and showed enhanced interaction with mutant EGFRs, suggesting that constitutive EGFR-Src cooperativity may contribute to mutant EGFR-mediated oncogenesis. Indeed, the mutant EGFR-mediated cell transformation was inhibited by Src- as well as EGFR-directed inhibitors. Importantly, a tyrosine to phenylalanine mutation of the major Src phosphorylation site on EGFR, Y845, reduced the constitutive phosphorylation of NSCLC EGFR mutants as well as of STAT3, Akt, Erk and Src, and reduced the mutant EGFR-Src association as well as proliferation, migration, and anchorage-independent growth. Reduced anchorage-independent growth and migration were also observed when DN-Src was expressed in mutant EGFR-expressing cells. Overall, our findings demonstrate that mutant EGFR-Src interaction and cooperativity play critical roles in constitutive engagement of the downstream signaling pathways that allow NSCLC-associated EGFR mutants to mediate oncogenesis, and support the rationale to target Src-dependent signaling pathways in mutant EGFR-mediated malignancies.