Epidermal growth factor receptor mutations in non-small cell lung cancer influence downstream Akt, MAPK and Stat3 signaling

Abstract

Purpose: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC) has been linked to activating mutations in the EGFR gene. So far these mutations have been extensively characterized in established cell lines. The aim of this study was to determine the effects of EGFR mutations on downstream signaling in human tumor specimens.

Methods: We have looked for mutations of the EGFR gene in specimens of 67 patients with NSCLC and correlated these with EGFR phosphorylation and the activity of its three main downstream signaling cascades Akt, MAPK and Stat3 by immunohistochemistry.

Results: We show that the phosphorylation of tyrosine residues 922 and 1173, but not 1068, are primarily affected by the activating EGFR mutations. Akt activity was significantly higher in patients with EGFR mutations but we found no difference in Stat3 or MAPK phosphorylation. Our results suggest that EGFR mutations not only increase receptor activity, but also alter responses of downstream signaling cascades in human NSCLCs and that these finding differ from results obtained in cell lines.

Fig. 1

An example of the four-tier scoring system used for evaluating the staining intensity in EGFR and phosphotyrosine-EGFR IHC, here demonstrated with an antibody targeting the EGFR independent of phosphorylation. 0 no or undefined background staining, 1+ weak and discontinuous membrane staining, 2+ distinct membrane staining of moderate intensity, 3+ strong and complete plasma membrane staining. Samples with 1+ or 2+ staining intensity were considered positive. The black bar represents 400 μm

Fig. 2

Examples of mutations found in the kinase domain of the EGFR. We found E709G, G719C and G719A substitutions in Exon 18. The deletions in Exon 19 ranged from delE746-A750 to delE746-S752. Both the L858R and G857R substitution were found in Exon 21

Fig. 3

Percent of NSCLC tumors staining positive in EGFR, P-Tyr-992, P-Tyr-1068 and P-Tyr-1173 immunohistochemistry with regard to their mutation status. There was no difference between EGFR-WT and EGFR-M in EGFR and P-Tyr-1068 IHC, but a drastic difference in P-Tyr-992 and P-Tyr-1173 staining

Fig. 4

Percent of tumors with positive IHC staining in Stat3, Akt and MAPK; the three main downstream signaling cascades of the EGFR. Both P-Tyr-705-Stat3 and phospho-p44/p42 MAPK showed no difference between NSCLC tumors with mutations in the EGFR and those without. Staining of phospho-Akt, however, showed a significant difference between EGFR mutant and wild type tumors

Fig. 5

Effect of EGFR specific tyrosine residue phosphorylation on downstream signaling cascades. a Phosphorylation of Tyr-992 correlates to Akt and MAPK activation. b Stat3, Akt and MAPK are not affected by Phospho-Tyr-1068. c Tyr-1173 does not directly correspond to Akt, MAPK or Stat3 phosphorylation