EGFR Mutant Lung Adenocarcinoma Showed Reduced Radiosensitivity after Acquired Resistance to EGFR TKIs

Abstract

<h3>Purpose/Objective(s)</h3> Though earlier application of local radiotherapy for the primary lesion may improve the survival of patients, the timing of radiotherapy intervention for advanced EGFR mutant lung adenocarcinoma is still controversial. This study aimed to detect the difference of radiosensitivity before and after resistance and explore the related mechanism by constructing EGFR mutant lung adenocarcinoma cell lines with acquired resistance to gefitinib in vitro, so as to provide theoretical basis for determining the optimal intervention timing of radiotherapy. <h3>Materials/Methods</h3> Two lung adenocarcinoma cell lines harboring EGFR mutation-PC9 and HCC827, were used for establishment of acquired resistant cells to gefitinib. The radiosensitivity was detected using a cell counting kit. Flow cytometry was introduced to explore the cell cycle distribution and Western blot was applied to further test cell cycle and DNA repair related proteins. Soft agar assay and Western blot were performed to determine the cancer stem cell (CSC) characteristics. RT-PCR detected the expression of CSC markers. Finally, the relevant signal pathways were explored. <h3>Results</h3> The cell counting kit showed that the inhibitory activity of cells with irradiation after gefitinib resistance is weaker than that before resistance. Flow cytometry revealed that the G2/M phase proportion of PC9 and HCC827 were significantly higher than that of PC9/GR and HCC827/GR under the same irradiation dose, which is consistent with the results of western blot that the increased expression of G2/M regulatory protein, including p21, Cyclin B1, CyclinA2, pCDC2 and Myt1 were more significant increased in parental cells than resistant cells. The phosphorylated level of RAD17, ATM and CHK2 which were DNA damage repair protein were more upregulated in PC9GR and HCC827GR cells. Furthermore, GR had strong capability of sphere-forming and EMT phenotype. RT-PCR showed that the expression of many genes regulating CSC characteristics increased obviously after gefitinib resistance, including SOX2, OCT4, Nanog, Notch1, Notch3, ALDH1A1, ALDH1A3 and BMI. But the up-regulated genes in the two drug-resistant cells were not completely same. Western blot and RT-PCR showed that BMP/SMAD signal pathway was more activated in both resistant cells than that in parental cells. <h3>Conclusion</h3> Lung adenocarcinoma with EGFR mutations showed reduced radiosensitivity after acquired resistance to EGFR TKIs through obtaining CSC characteristics, which may be mediated by BMP/SMAD signal pathway.