Abstract
BackgroundEpidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and altered EGFR signaling contributes to human cancer. EGFR kinase domain mutants found in non-small cell lung cancer (NSCLC) are constitutively active, a trait critical for cell transformation through activation of downstream pathways. Endocytic trafficking of EGFR is a major regulatory mechanism as ligand-induced lysosomal degradation results in termination of signaling. While numerous studies have examined mutant EGFR signaling, the endocytic traffic of mutant EGFR within the NSCLC milieu remains less clear.ResultsThis study shows that mutant EGFRs in NSCLC cell lines are constitutively endocytosed as shown by their colocalization with the early/recycling endosomal marker transferrin and the late endosomal/lysosomal marker LAMP1. Notably, mutant EGFRs, but not the wild-type EGFR, show a perinuclear accumulation and colocalization with recycling endosomal markers such as Rab11 and EHD1 upon treatment of cells with endocytic recycling inhibitor monensin, suggesting that mutant EGFRs preferentially traffic through the endocytic recycling compartments. Importantly, monensin treatment enhanced the mutant EGFR association and colocalization with Src, indicating that aberrant transit through the endocytic recycling compartment promotes mutant EGFR-Src association.ConclusionThe findings presented in this study show that mutant EGFRs undergo aberrant traffic into the endocytic recycling compartment which allows mutant EGFRs to engage in a preferential interaction with Src, a critical partner for EGFR-mediated oncogenesis.
Highlights
Epidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and altered EGFR signaling contributes to human cancer
non-small cell lung cancer (NSCLC)-associated oncogenic EGFR mutants are constitutively endocytosed To examine the cell surface versus intracellular localization of EGFR, we carried out confocal immunofluorescence imaging analyses of a normal bronchial epithelial cell line HBE135 expressing wtEGFR and NSCLC cell lines expressing wtEGFR (H1666) or mutant EGFRs (H1650 and HCC827 expressing EGFR Δ746-750, HCC4006 expressing EGFR Δ747-749/A750P, and H1975 expressing EGFR L858R/T790 M) [4,33]
Mutant EGFRs ectopically expressed in human bronchial epithelial cell (HBEC) cell lines as EGFR-GFP chimeras showed similar results, indicating that the constitutive intracellular localization of mutant EGF receptors observed in Figure 1 was not due to cell type specificity (Additional File 1A)
Summary
Epidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and altered EGFR signaling contributes to human cancer. EGFR kinase domain mutants found in non-small cell lung cancer (NSCLC) are constitutively active, a trait critical for cell transformation through activation of downstream pathways. Missense mutations or small in-frame deletions within the kinase domain, which render EGFR constitutively active, are observed in a subset of patients with nonsmall cell lung cancer (NSCLC) [4,5,6]. As mutational activation of EGFR imparts a higher sensitivity to inhibition by EGFR-selective tyrosine kinase inhibitors (TKIs), there is considerable interest in understanding biological mechanisms whereby mutant EGFRs mediate aberrant oncogenic signaling in cancer cells. While the normal EGFR signaling cascade is initiated by ligand-dependent dimerization and subsequent transphosphorylation of tyrosine residues within the cytoplasmic tail of the receptor, constitutively active mutant EGFRs associated with human cancer are thought to engage downstream signaling pathways in a constitutive fashion. Other studies have indicated a relatively selective activation of Src downstream of mutant EGFRs [7,8,9]
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