HER2 overexpression reverses the relative resistance of EGFR-mutant H1975 cell line to gefitinib

Abstract

Gefitinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) that has been demonstrated to be clinically useful for the treatment of patients with non-small cell lung cancer (NSCLC). However, ~50% of patients do not respond to EGFR TKI treatment through the emergence of mutations, such as T790M. Therefore, it is important to determine which patients are eligible for treatment with gefitinib. As a preferred dimerization partner for EGFR, the role of EGFR 2 (HER2) in mediating sensitivity to gefitinib is poorly understood. In the present study, full-length human HER2 cDNA was introduced to the NSCLC cell lines H1975 and H1299, which have a low endogenous expression level of HER2. In addition, it was observed in the present study that the H1975 cell line harbored the L858R and T790M mutations in the EGFR kinase domain. Western blot analysis and MTT assay were used to evaluate the TKI sensitivity of HER2 expression status, and the activation of HER3 and HER2 downstream effectors. The results indicated that the sensitivity of H1975 cells to gefitinib was restored by the overexpression of HER2, which stimulated HER2-driven signaling cascades accompanied by the activation of protein kinase B. By contrast, ectopic HER2 overexpression in H1299 cells did not significantly alter the sensitivity to gefitinib treatment. In conclusion, the current study results suggested that the relatively resistance of the H1975 cell line to gefitinib could be reversed by the overexpression of HER2. Therefore, the expression of HER2 could also be considered when evaluate the patients' potential response to gefitinib, particularly in the subgroup of lung cancer patients who harbor an EGFR mutation.

Keywords: gefitinib; human epidermal growth factor receptor 2; lung cancer; mutation; tyrosine kinase inhibitor.

Figure 1.

Correlation of gefitinib sensitivity with expression levels of EGFR and HER2. (A) Cellular protein levels of EGFR and HER2 were determined by western blotting using 30 µg total cell lysates of each cell type. MTT assays were performed for cells incubated for 48 h with various concentrations of gefitinib. All experiments were performed in duplicate and averaged. (B) Protein expression of HER2 in H1975 and H1299 cell lines, and in their HER2 transfectants. Comparison of HER2 protein expression status in the H1975 and H1299 cells transfected with HER2 or mock vector. EGFR, epidermal growth factor receptor; HER, human EGRF.

Figure 2.

Overexpression of the HER2 enhances the sensitivity of the H1975 cell line to gefitinib. Dose-response curves of (A) H1975 and (B) H1299 cells transfected with HER2 or mock vector following gefitinib exposure. Sensitivity to gefitinib was determined by MTT assay in the absence or presence of an increasing gradient of gefitinib (2.5, 5, 10, 20 and 40 µM). Results are presented as the mean ± standard deviation of triplicate experiments and are shown as a percentage of untreated control cells. HER2, human epidermal growth factor receptor 2.

Figure 3.

Effect of gefitinib on P-Akt (Ser473), P-Erk1/2 (Thr202/Tyr204) and P-HER3 levels. (A) Dose-dependent and (B) time-dependent inhibition of Akt and Erk1/2 phosphorylation in H1975 and H1299 cell lines are shown by western blot analysis. Cells were treated for 1 or 24 h with the indicated concentrations of gefitinib. Two independent experiments provided similar results. (C) Effect of gefitinib on P-HER3 in H1975 cell lines in the presence of 10% serum. P, phosphorylated; Akt, protein kinase B; Erk, extracellular signal-regulated kinase; HER, human epidermal growth factor receptor.