Chronic, repeated exposure to gefitinib leads to epithelial-to-mesenchymal transition and drug resistance in A549, a non-small cell lung cancer cell line

2346 Epithelial-to-mesenchymal transition (EMT) is a process for cancer cells to acquire the ability of invasion, metastasis and therapeutic resistance. The loss of E-cadherin, an epithelial marker, has been reported to be related with the reduced sensitivity to EGFR tyrosine kinase inhibitors. To investigate whether this phenomenon would exert a role in acquired resistance to gefitinib, we performed this study. The cell line resistant to gefitinib was created by exposing A549 cells to 10 μM gefitinib for 3 months, 20 μM gefitinib for another 3 months, and finally 30 μM for 6 more months. The daughter cells (A549/GR) were 5 to 10 times more resistant to gefitinib, CL-387, erlotinib, and ZD6474. Phenotypic changes such as spindle-cell shape, increase of intercellular separation and pseudopodia formation suggesting EMT were found on immunofluorescent staining. It was accompanied by the decrease of E-cadherin and increase of vimentin, a mesenchymal marker. Western blots revealed the amount of EGFR was decreased and, in consequence, the activation of Erk was reduced in A549/GR cells. Although EGFR expression was decreased, the level of p-Akt was increased more than parent cells. Moreover, the activated Akt was not reduced by the treatment of gefitinib. In the change of factors determining sensitivity to gefitinib, no EGFR mutations were detected by sequencing and EGFR gene copy number by FISH was not changed in A549/GR cells. In conclusion, chronic, repeated exposure to gefitinib leads to EMT, which may be one of possible mechanisms to acquire the gefitinib resistance during treatment.