Abstract 699: HSP70 specifically binds to 15 bp deletion mutant EGFR and modulates sensitivity to EGFR-TKI

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL [Purpose] Non small 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. Cells were incubated in a medium with/without 10 ng/ml of TGFα for 1 h and lysed. Cell lysate was precleared by centrifugation at 15,000 rpm for 15 min. 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 (pH3-10 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 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 and293\_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 in the mutant EGFR transfectant. 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. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 699. doi:10.1158/1538-7445.AM2011-699