Molecular dissection of signaling and regulatory mechanisms of human cancer-associated epidermal growth factor receptor mutants

Epidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and aberrant EGFR signaling through overexpression and/or mutation occurs in many types of cancer. Not surprisingly, human cancer-associated mutants of EGFR are directly linked to enhanced tumorigenicity. Non-small cell lung cancer (NSCLC)-associated EGFR kinase domain mutants as well as a class III variant EGFRvIII are constitutively active and induce ligand-independent transformation in nonmalignant cell lines. However, how these naturally-occurring EGFR mutants exert their oncogenic effects is not well understood, which prompted current investigations of their signaling and regulatory mechanisms. First, the possibility that the ability of NSCLC-associated mutant EGFRs to transform cells reflects a constitutive cooperativity with Src, a well known oncogenic partner of EGFR, was examined. Through the use of kinase inhibitors and genetic alterations, it was demonstrated 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 cell transformation, proliferation and migration. Next, it was observed that mutant EGFRs in NSCLC cell lines undergo constitutive internalization and endocytic recycling. In the presence of a recycling inhibitor monensin, mutant EGFRs but not wild-type EGFR accumulated in perinuclear vesicles and showed colocalization with various recycling endosomal markers. Importantly, inhibition of endocytic recycling delayed the ligand-induced mutant EGFR degradation and enhanced the mutant EGFR association and colocalization with Src. Together, these findings indicate that mutant EGFRs undergo altered endocytic trafficking via the recycling pathway and that this route allows mutant EGFR to engage in preferential interaction with Src. Defects in endocytosis as well as ligand-induced downregulation of EGFR were also observed in cells overexpressing EGFRvIII, where EGFRvIII remained constitutively at the cell surface. Lastly, three-dimensional culture on reconstituted basement membrane was utilized to study EGFR and its mutants in in vivo-related setting. Immortalized normal bronchial epithelial cells formed polarized acini while the expression of mutant EGFRs resulted in the loss of cell polarity. Overall, our studies demonstrate that the oncogenic mutant EGFRs exhibit altered endocytic trafficking which regulate signaling properties, and these findings reveal a novel aspect of how cancer-associated EGFR mutants promote oncogenesis.