Blocking ROR1 Through SiRNA Enhances The Roles Of Erlotinib In Lung Adenocarcinoma Cell Lines

Objective: Lung cancer is a kind of cancer with high incidence.The effect of epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs)has brought good news to patients,however after a period of drug treatment,patients will develop acquired resistance to EGFR-TKIs.Transmembrane receptor tyrosine kinase protein ROR1 is highly expressed in various cancers,such as lung adenocarcinoma,breast cancer and ovarian cancer and also involved in growth,proliferation,migration and invasion of cancer cells.The main purpose of this study is to investigate whether blocking ROR1 can enhance the cytotoxic effect of erlotinib in lung adenocarcinoma cell lines,then to explore the possible molecular mechanisms,and to provide a pontential strategy to overcome EGFR-TKIs resistance in lung adenocarcinoma.Method: An acquired erlotinib-resistant cell line named as PC-9erlo was established by exposing an erlotinib-sensitive cell line PC-9 to a certain concentration of erlotinib for 8 months.The expression level of ROR1 was blocked down in XLA-07,NCI-H1975 and PC-erlo cell lines by ROR1-specific small interfering RNA(si ROR1),and the blocking efficacy was detected by flow cytometry.Once ROR1 expression was blocked,NCI-H1975,PC-9erlo and XLA-07 cells were treated with different concentrations of erlotinib,and the levels of cell proliferation and apoptosis were tested by MTS method and flow cytometry,respectively.Key molecules in AKT/m TOR signaling pathway in different treated groups were detected by western blot and Bio-Plex Pro assays.Results:1.The maternal cell line named as PC-9 acquired resistance to erlotinib after treated with a certain concentration of erlotinib for 8 months,IC50 value of erlotinib in the newly established cell line named as PC-9erlo increased,and its sensitivity to erlotinib decreased.2.ROR1-specific small interfering RNA inhibited the expression of ROR1 in each lung adenocarcinoma cell lines,and the average inhibition rate was greater than 75%.3.After blocked ROR1 in NCI-H1975 and PC-9erlo cell lines,the cytotoxicity effect of erlotinib on lung adenocarcinoma cell lines was enhanced.4.Compared with cells treated with erlotinib alone,the apoptosis-inducing role of blocking ROR1 along with erlotinib in NCI-H1975,PC-9erlo and XLA-07 cell lines were significantly increased.5.After blocked with si ROR1 and treated with erlotinib,the phosphorylation level of each key molecule in the AKT/m TOR signaling pathway was decreased in NCI-H1975 cells and the expression level of Bcl-2,which was positively correlated with cell survival,was also significantly decreased and the difference was statistically significant(p<0.01,p<0.001).Conclusion: Blocking ROR1 enhanced the roles of erlotinib through AKT/m TOR signaling pathway.