Relationship Between EGFR Gene Expression And Radiosensitivity In Egfr-mutant NSCLC Cell Line

Background and ObjectiveNon-small-cell lung cancer(NSCLC)is the leading cause of death from cancer in the world.80% of lung cancer is non-small cell lung cancer.Radiation therapy is used to treat about 70 % of NSCLC patients.However,interindividual differences in radiosensitivity exist in patients with NSCLC,and there is still no biomarkers to predict radiosensitivity.Previous studies showed that in clinic,NSCLC patients with EGFR mutation were more sensitive to radiotherapy than those patients with EGFR wild type.EGFR mutation-positive NSCLC experienced superior PFS and OS in comparison with EGFR wild type NSCLC.Das and our study found that in vitro,NSCLC cells with EGFR mutation were more sensitive to ionizing radiation than the cells with EGFR wild type.Results of these studies strongly suggested that EGFR gene status may be one of the predictors of radiosensitivity in patients with NSCLC.However,it was reported that the radiosensitivity of different EGFR mutant NSCLC cells was different and some EGFR mutant cells were relatively resistant to radiation,suggesting that except EGFR gene status,there is still other factors affecting radiosensitivity.It was shown that in NSCLC with EGFR wild type,the expression of EGFR also play an important role in the radiosensitivity.Whether it happened in NSCLC with EGFR mutation needed to be further studied.The epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI)is the first line treatment for the NSCLC patients with EGFR mutation.However,patients receiving EGFR-TKI therapy inevitably develop acquired resistance within 10-12 months.Previous studies have shown that the acquired resistance of EGFR 21 exon mutant cells to gefitinib can be reversed by radiation-induced MET(mesenchymal to epithelial transition).Whether radiation can reverse the acquired resistance of EGFR-TKI in the NSCLC cells with EGFR 19 exon need further investigated.There are two purposes in this study: Firstly,to explore the relationship between EGFR expression and radiosensitivity in the NSCLC cells with EGFR mutation,to try to find the biomarker for individual treatment of radiotherapy.Secondly,to investigate whether radiation can reverse the acquired resistance of EGFR-TKIs.Methods1.Correlation between EGFR expression and the radiosensitivity of NSCLC cells with EGFR mutation: Human NSCLC cell lines PC-9 and HCC827 with mutations in exon 19 of EGFR,H3255 with mutations in exon 21 of EGFR,H1975 harboring both the L858 R mutation in exon 21 and the T790 M mutation in exon 20 of EGFR,and PC-9/AB and PC-9/ZD cell lines harboring both the mutations in exon 19 and the T790 M mutation in exon 20 of EGFR induced by acquired gefitinib-resistance were used in this study.EGFR gene status was detected by next generation sequencing analysis.Western blotting verified the expression of EGFR.RNA interference was used to silence EGFR gene expression.X-ray induced radioresistance.Radiosensitivity was determined by clone-forming test.2.Reversal of acquired EGFR-TKI resistance in NSCLC cells in vitro by ionizing radiation and its possible mechanism:NSCLC cell line PC-9 and the acquired gefitinib-resistant NSCLC cell line PC-9 / AB were used in this study.PC-9 / AB cells were irradiated to induce the radiation-resistant cell,named as PC-9/ AB IR.EGFR gene status was detected by next generation sequencing analysis.Gefitinib-resistance was determined by CCK8 method.Radiosensitivity was determined by clone-forming test.Western blotting was used to find the expression of E-cadherin and Vimentin.The cell apoptosis and cell cycle distribution were detected by flow cytometry.3.Statistical analysis: Data are expressed as the mean ± standard deviation.SPSS 20.0 software(IBM Corp.,Armonk,NY,USA)was used for statistical analysis.The t-test was used to compare data between two groups.P<0.05 was considered to indicate a statistically significant difference.Results 1.The correlation between the expression of EGFR and the radiosensitivity of NSCLC cells with EGFR mutation.1)Among the 6 NSCLC cell lines,H1975 cell was the most radiosensitive cell line,HCC827 and H3255 cells were moderately sensitive to radiation,and PC-9?PC-9/AB and PC-9/ZD cells were the least radiosensitive cell lines.2)The expression of EGFR in H1975 cell was remarkably lower than that in the other five cell lines.HCC827?H3255 and PC-9/ZD cells expressed the intermediate level of EGFR.PC-9 and PC-9/AB cells expressed the highest level of EGFR.3)The EGFR expression in PC-9/ZD cell was significantly decreased with RNA interference,accompanying the enhancing of its radiosensitivity.4)The X-ray induced radioresistance in PC-9/ AB and PC-9 /ZD cell lines and reduced the EGFR expression at the same time.2.Reversal of gefitinib acquired resistance in non-small cell lung cancer in vitro by ionizing radiation and its primary mechanism.1)PC-9 / AB cells were gefitinib-resistant with T790 M and EMT.The IC50 of gefitinib for PC-9 / AB cells was significantly higher than that for PC-9 cells.The growth rate of PC-9 / AB IR is faster than that of the PC-9 / AB,approching to parent cell PC-9.2)Comparing to PC-9/ AB cells,the PC-9/ AB IR cells were less sensitive to ionizing radiation and more sensitive to gefitinib.The IC50 of gefitinib for PC-9 / AB cells close to parent cell PC-9.3)No changes occurred at the genetic level of the radiation-induced PC-9/ AB IR cells.However,the expression of E-cadherin was significantly upregulated and the expression of vimentin was significantly downregulated in PC-9/AB IR cells.4)The gefitinib-induced apoptosis rate and proportion of cells in G0/G1 stage in PC-9/ AB IR cells was significantly higher than that in PC-9/ AB cells(P<0.05).Conclusion1)The radiosensitivity of NSCLC cells with EGFR gene mutation was closely associated with EGFR expression.The higher expression of EGFR,the lower sensitive to radiation.EGFR expression may be one of the prospective factors for radiosensitivity.2)These results suggested that the gefitinib-induced acquired resistance could be reversed by ionizing radiation in NSCLC cells.The mechanism may be related to reversing the EMT?increasing apoptosis and cell cycle block.