Non-small-cell lung cancer (NSCLC) cells that expressed mutant EGFR are more sensitive to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) than that expressed wild-type EGFR. Recent clinical trials revealed that more than 70% NSCLC patients that expressed mutant EGFR showed sensitivity to EGFR-TKIs, such as gefitinib and erlotinib. To elucidate the mechanism of this hypersensitivity, we explored the difference of EGFR-binding proteins between wild-type EGFR and 15 bp deletion mutant EGFR using respective stable transfectants.Methods
Wild-type EGFR and a 15-bp deletion mutant EGFR plasmids were transfected into HEK293 cells. The stable transfectant cells were established, and were designated 293_pEGFR and 293_pΔ15, respectively. Cell lysate was precleared by centrifugation and the supernatant was mixed with a polyclonal EGFR antibody for 1 h and EGFR was immunoprecipitated by Protein A-Sepharose. After adequate washing, coprecipitated proteins that bound to EGFR were eluted and separated by 2D-PAGE (Immobiline DryStrip (pH 3–10 NL, 7 cm) and 10% SDS–PAGE). Proteins were visualized by silver staining and identified by LC-MS/MS. HSP70 siRNA was transfected into the cells by lipofection method. Sensitivity to gefitinib was measured by the MTT assay. EGFR binding affinity to gefitinib was measured using [14C] gefitinib.Results and discussion
We detected several EGFR-binding proteins. Among these proteins, one candidate (lesser binding to wild-type EGFR than the mutant EGFR) was identified as HSP 70 by LC-MS/MS. The total amount of HSP70 protein was not different between 293_pEGFR and 293_pΔ15 cells. A specific binding of this protein to the mutant EGFR was confirmed by western blotting analysis. This binding was not modulated by EGFR activation. Knockdown of HSP 70 protein by a specific siRNA enhanced gefitinib sensitivity and its binding to the receptor in the mutant EGFR transfectant.Conclusion
These results suggest that HSP 70 may decrease sensitivity to EGFR-TKIs in the cells that expressed 15-bp deletion mutant EGFR. There is a possibility that HSP70 overexpression might be a newly resistant mechanism to EGFR-TKIs in NSCLC patients that expressed mutant EGFR.