By Pass Signaling Pathways Activation Mediated Resistance To Alectinib In EML4-ALK Fusion Gene Positive Lung Cancer Cell Line H3122

Objective: Anaplastic lymphoma kinase tyrosine kinase inhibitor(ALK-TKI)resistance significantly limits its use in clinical practice,activation of bypass signaling pathways is one of the mechanisms of drug resistance in non-small cell lung cancer(NSCLC)targeted therapy.We aim to assess alectinib resistance in EML4-ALK positive lung cancer cell line H3122 following hepatocyte growth factor(HGF)、 epidermal growth factor(EGF)and transforming growth factor-α(TGF-α).And then explore its potential mechanisms.Methods: 1.To detect the sensitivity of EML4-ALK positive lung adenocarcinoma cells H3122 to alectinib To detect the sensitivity of ALK positive lung adenocarcinoma cells H3122 to alectinib,H3122 cell line contain EML4-ALK fusion gene variant 1.Based on CCK-8 manual,the cells survival of H3122 under different concentrations of alectinib was measured,and detect ALK positive lung adenocarcinoma cells’ sensitivity to alectinib induced by HGF,EGF,and TGF-α.EML4-ALK positive NSCLC H3122 cells were treated with increasing concentrations of alectinib and induced by HGF,EGF,and TGF-α,and cultured 72 h.Detect the changes of H3122’s sensitivity to alectinib in the presense of HGF,EGF,and TGF-α.2.Cell appotosis rate was measured using flow cytometry,H3122 cells were treated with 0.05μmol/L alectinib alone or conbined with 50 ng/mL HGF,100 ng/mL EGF,100 ng/mL TGF-αfor 48 h.3.EML4-ALK positive cell line H3122 was treated with increasing concentrations of alectinib or(and)induced by HGF,EGF,and TGF-α.The protein expression and phosphorylation status of ALK,MET,EGFR and its downstream molecules(AKT,ERK,p-AKT,p-ERK)were examined by Western blotting.Results: 1.ALK-positive lung adenocarcinoma cells H3122 were highly sensitive to alectinib.Alectinib inhibited the growth of H3122 cells in a dose-dependent manner after administrated for 72 h,the IC50 value of H3122 was 0.042μmol/L.2.Under the exposure of HGF,EGF and TGF-α,the sensitivity of H3122 to alectinib was significantly decreased,the curves of concentration-survival of H3122 cell line shifted to the right after induced by HGF,EGF,and TGF-α.HGF,EGF and TGF-α can alleviate the inhibitory effect of alectinib on the proliferation of lung cancer cells.3.Alectinib induces apoptosis in H3122 cells(P<0.05),the apoptosis rate of H3122 cell line was(20.12±1.36)% after treated with alectinib for 48 h,while the apoptosis rate of alectinib combined with HGF,EGF and TGF-α was(7.85±1.03)%,(5.6±0.79)% and(4.58±1)%,respectilvey,significantly lower than alectinib single drug treatment(P <0.05).4.Protein detection suggested that H3122 cells can be measured ALK and downstream signaling pathways AKT,ERK and its phosphorylated protein.Alectinib enable to inhibit p-ALK and its downstream signaling pathways while HGF significantly up-regulated the expression of p-MET and its downstream p-AKT,p-ERK.Besides,EGF,TGF-α remarkablely up-regulated the expression of p-EGFR and its downstream p-AKT,p-ERK.Alectinib inhibited p-ALK,but cannot inhibited the expression of p-AKT,p-ERK induced by HGF,EGF,TGF-α.Conclusion: Bypass signaling pathways can be actived by HGF,EGF and TGF-α in EML4-ALK positive lung cancer cells H3122,which may be linked to alectinib resistance.