Distinctive activation patterns in constitutively active and gefitinib-sensitive EGFR mutants

Abstract

Mutations in the kinase domain of epidermal growth factor receptor (EGFR) are associated with clinical responsiveness to gefitinib in patients with non-small-cell lung cancers (NSCLC). Recently, we have identified many novel EGFR mutations in NSCLC tissues. In this study, we found that gefitinib could suppress the tyrosine phosphorylation of most EGFR mutants better than the wild-type receptor. However, gefitinib had quite variable growth-suppressive effects on different EGFR mutantexpressing cells. All tested EGFR mutants have high basal phosphorylation at multiple tyrosine residues. Upon EGF stimulation, the mutated EGFRs did not have apparently stronger phosphorylation at tyrosines 845, 992, 1068, and 1173 than the wild-type receptor. However, stronger phosphorylation at tyrosine 1045 was observed in the S768I, L861Q, E709G, and G719S mutants. The E746-A750 deletion mutant was less responsive to EGF than the wild-type and other mutant receptors. The S768I, L861Q, E709G, and G719S mutants were refractory to EGFinduced ubiquitination and had more sustained tyrosine phosphorylation. E709G and G719S also lacked EGFinduced receptor downregulation. Our results indicate that, in addition to sensitivity to gefitinib, EGFR mutations also caused various changes in EGFR's regulatory mechanisms, which may contribute to the constitutive activation of EGFR mutants and oncogenesis in NSCLC.

EGF-independent activation of cell-surface EGF receptors harboring mutations found in gefitinib-sensitive lung cancer

“…Autophosphorylation specificity is not altered in gefitinib-sensitizing mutants By contrast with two other recent reports Chen et al, 2006), we could not detect differences in phosphorylation site utilization in mutated forms of EGFR. As shown in Figure 3a, the relative degree of phosphorylation at tyrosines 845, 992, 1045 and 1068 was indistinguishable in WT and mutated EGFR, with-and without EGF.…”

“…The G695S and L834R mutants appeared least heavily phosphorylated (but nonetheless elevated over WT), while basal activity appeared similar for the L837Q, DL723-P729insS and DS728-I735 mutants. These findings concur with other studies published while this paper was in preparation Jiang et al, 2005;Chen et al, 2006), and contrast with initial reports that these mutations do not increase ligand-independent receptor activity (Lynch et al, 2004;Pao et al, 2004).…”

“…Single amino-acid substitutions in the P-loop (G695S) or A-loop (L834R and L837Q) do not appear to impair maximal EGF response, whereas the two deletion mutants (DL723-P729insS and DS728-I735) showed substantially reduced maximal levels of EGF-induced autophosphorylation. Reduced maximal activity has also been observed in other recent studies of deletion mutants (Pao et al, 2004;Amann et al, 2005;Chen et al, 2006).…”

Overview of molecular testing in non-small-cell lung cancer: mutational analysis, gene copy number, protein expression and other biomarkers of EGFR for the prediction of response to tyrosine kinase inhibitors

“…Among all mutations, four predominantly result in TKI drug sensitivity by in vitro and in vivo studies. These include point mutations in exons 18 (G719A/C) and 21 (L858R and L861Q) and in-frame deletions in exon 19, which eliminate four amino acids (LREA) downstream of the lysine residue at position 745; other mutations appear to be associated with variable or less sensitivity (Eberhard et al, 2005;Chen et al, 2006;Sharma et al, 2007;see Gazdar, 2009). …”

Autophosphorylation of the carboxyl‐terminal domain is not required for oncogenic transformation by lung‐cancer derived EGFR mutants

“…Furthermore, accumulating data suggest that the downstream signaling pathways activated by oncogenic mutant EGFR differ from those activated by ligand stimulated wild‐type EGFR 17, 18, 19. Notably, C‐terminal deletion EGFR mutants, lacking some or all of the autophosphorylation sites that have been identified in GBM and lung adenocarcinoma, are able to induce cellular transformation 8, 9, 20.…”