Synergistic effect of afatinib with su11274 in non-small cell lung cancer cells resistant to gefitinib or erlotinib

Abstract

Epidermal growth factor receptor (EGFR) and c-MET receptors are expressed on many non-small cell lung cancer (NSCLC) cells. Current single agent therapeutic targeting of a mutant EGFR has a high efficacy in the clinic, but is not curative. Here, we investigated the combination of targeting EGFR and c-MET pathways in NSCLC cells resistant to receptor tyrosine kinase inhibitors (TKIs), using RNA interference and inhibition by TKIs. Different NSCLC cell lines with various genomic characteristics (H358, H1650 and H1975) were transfected with EGFR-specific-siRNA, T790M-specific-siRNA, c-MET siRNA or the combination. Subsequently EGFR TKIs (gefitinib, erlotinib or afatinib) or monoclonal antibody cetuximab were combined respectively with the c-MET-specific TKI su11274 in NSCLC cell lines. The cell proliferation, viability, caspase-3/7 activity and apoptotic morphology were monitored by spectrophotometry, fluorimetry and fluorescence microscopy. The combined effect of EGFR TKIs, or cetuximab and su11274, was evaluated using a combination index. The results showed that the cell lines that were relatively resistant to EGFR TKIs, especially the H1975 cell line containing the resistance T790M mutation, were found to be more sensitive to EGFR-specific-siRNA. The combination of EGFR siRNA plus c-MET siRNA enhanced cell growth inhibition, apoptosis induction and inhibition of downstream signaling in EGFR TKI resistant H358, H1650 and H1975 cells, despite the absence of activity of the c-MET siRNA alone. EGFR TKIs or cetuximab plus su11274 were also consistently superior to either agent alone. The strongest biological effect was observed when afatinib, an irreversible pan-HER blocker was combined with su11274, which achieved a synergistic effect in the T790M mutant H1975 cells. In a conclusion, our findings offer preclinical proof of principle for combined inhibition as a promising treatment strategy for NSCLC, especially for patients in whom current EGFR-targeted treatments fail due to the presence of the T790M-EGFR-mutation or high c-MET expression.

Figure 1. Effects of EGFR-specific-siRNA on cell growth and apoptosis.

Lung cancer cell lines were treated with EGFR-specific-siRNA 1247. Live cells and apoptotic cells were detected with Hoechst 33342 and PI double fluorescent staining. The number of apoptotic cells was normalized to the number of live cells in the same well. Hoechst 33342 and PI double fluorescent staining ×200.

Figure 2. Functional effect of combination of EGFR-specific-siRNA with c-MET siRNAs.

Panel A: Cell proliferation following transfection with different siRNAs. Cell lines H358 (white), H1650 (grey) and H1975 (black) were transfected with EGFR-specific-siRNA 1247, T790M specific siRNA, a c-MET siRNA pool, or combinations of these, and the proliferation was assayed 72 h post transfection using a colorimetric MTS assay. Panel B: Caspase−3/7 activity. * P<0.05 and ** P<0.01 compared to both single treatment.

Figure 3. Protein level of combination of EGFR-specific-siRNA with c-MET siRNA.

Panel A: Immunoblot analysis of H358 and H1650 cells transfected with wild type EGFR siRNA, a c-MET siRNA pool or a combination of these. Panel B: Immunoblot analysis of H1975 cells, as in panel C. Here, the T790M-specific-siRNA was also included. Antibodies included phosphorylated (p-) EGFR, EGFR, p-c-MET, c-MET, p-AKT, p-ERK1/2, p-STAT5, p-STAT3, and β-actin.

Figure 4. Effect of su11274 in lung cancer cells containing TKI resistance mutations.

H358, H1650 and H1975 cells were incubated with a concentration range of su11274, and the cells were incubated for 72 h. Panel A: proliferation. Panel B: caspase−3/7 activity. Panel C: apoptosis. Panel D: dose-effect curves.

Figure 5. Combination of EGFR-specific TKIs or cetuximab and the c-MET inhibitor su11274.

Panel A: proliferation of cells following single or combined TKI treatment. Cell lines H358 (white), H1650 (grey) and H1975 (black) were cultured in RPMI containing 10% FBS and 1 µM su11274, gefitinib, erlotinib, afatinib or cetuximab, and their combinations. The proliferation was assayed 72 h post treatment using a colorimetric MTS assay. Panel B: caspase −3/7 induction by single or combined TKI treatment. * P<0.05 and ** P<0.01 compared to both single treatment.

Figure 6. Combination Index of EGFR-specific TKIs or cetuximab and the c-MET inhibitor su11274.

combination index (CI) was calculated in H1975. CIs >1, indicating additive effect (Panel A: gefinib+su11274; Panel B: erlotinib+su11274; Panel C: cetuximab+su11274).

Figure 7. Combination effect of afatinib and su11274 in H1975 cells.

Combination Index (CI) of afatinib and su11274. Here, CI of afatinib + su11274<1, indicating synergistic effect.

Figure 8. Protein level of combination of EGFR TKIs or cetuximab and the c-MET inhibitor su11274.

Immuno blotting of cell lysates of H358, H1650 and H1975 cells treated with single or combined TKIs/cetuximab. Antibodies included phosphorylated (p-) EGFR, EGFR, p-c-MET, c-MET, p-AKT, p-ERK1/2, p-STAT5, p-STAT3, and β-actin.