Metformin Restores Crizotinib Sensitivity In Crizotinib-Resistant Human Lung Cancer Cells Through Inhibition Of IGF1-R Signaling Pathway

Introduction: In the gene of anaplastic lymphoma kinase(ALK),genomic alterations widely exist in numerous malignancies [1].First discovered by Soda et al.in 2007 [2],ALK rearrangements are either translocations or inversions of the receptor gene of ALK-tyrosine kinase(-TK)with other partners of fusion.With pro-oncogenic effects,a constitutively activated receptor TK constitutes the product of the ALK rearrangement [3].As consequence,with ALK rearrangements tumors are addicted to signaling of ALK.Therefore,small molecules ALK-TK inhibitors(-TKIs)greatly hinder the tumors.Crizotinib is a multitargeted TKI with activity against ALK,MET[4],and ROS1[5].It was found that the active substance can outperform the most effective chemotherapy agents in NSCLC patients with rearrangements of ALK[6] [7].Recently,the study(PROFILE 1014)demonstrated that in the first-line treatment,ORR and PFS were greatly improved by crizotinib for ALK-positive NSCLC patients,with a reasonable security profile.Hence,crizotinib is established as a criterion of care for ALK-positive patients who have not received treatment before[8].Nonetheless,acquired resistance eventually develops in most ALK-positive NSCLC patients.Therefore,in order to enhance the survival rate of NSCLC patients,new strategies of treatment are of great importance to overcome the therapeutic resistance to crizotinib.Under acquired crizotinib resistance,molecular mechanisms have not been completely comprehended,mainly due to the small number of included patients and experimental results [9].Gatekeeper mutations,such as L1196 M,C1156Y,F1174 L and L1152 R hinder drug binding and are frequently detected in these crizotinib-resistant samples[10] [11].In addition,the signaling pathway of IGF-1R is included in resistance of crizotinib [12].IGF-1 addition caused resistance to the inhibitory effects of crizotinib for growth.Chronic ALK inhibition was related to the enhanced signaling of IGF-1R.Furthermore,the addition of an IGF-1R inhibitor can sensitize the resistant tumor cells to the effects of ALK blockade[11].Therefore,the signaling axis of IGF-1R is a therapeutic target in ALK+ lung cancer.As an insulin-sensitizing agent,metformin has been widely applied in the treatment of type II diabetes for more than 40 years.Since clinical evidence was published that the mortality and cancer risk were decreased in diabetics who were treated with metformin,lots of interest has been paid to metformin as a latent anticancer agent[13].Previously,it has been reported that metformin combined with erlotinib or gefitinib had a synergistic inhibitory influence on the migration,invasion and proliferation of cell lines resistant to EGFR-TKIs[14].Nonetheless,it remains unknown whether metformin might overcome crizotinib resistance.Interestingly,it was found that metformin has an inhibitory impact on the signaling of IGF-1R [15] [16],which encouraged us to research whether crizotinib sensitivity in crizotinib-resistant cells can be restored by metformin via inhibiting the signaling pathway of IGF-1R.Herein,we demonstrated that metformin combined with crizotinib showed a synergistic inhibitory effect on the invasion,migration and proliferation of both crizotinib-sensitive cells,and crizotinib-resistant cells.The ability to reduce IGF-1R signaling activation led to the effect of metformin against crizotinib resistance.Therefore,the experimental and rationale evidence was offered for the combined usage of crizotinib and metformin to overcome crizotinib resistance in patients with ALK+ NSCLC.Aim: Despite the impressive efficacy of crizotinib to treat ALK-positive lung cancer,patients develop therapeutic resistance invariably.Studies have demonstrated that the suppression of the signaling pathway of IGF-1R might abrogate this mechanism of drug resistance to crizotinib.Experimental design: The effects of metformin to overcome crizotinib resistance was examined in vitro by using 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium(MTT),ki67 incorporation assay,flow cytometry analysis,invasion assay,colony-forming assay,and Western blot analysis.Results: The present study demonstrated that metformin,which has been widely applied as an antidiabetic agent,can effectively increase the sensitivity of both crizotinib-sensitive and crizotinib-resistant ALK+ NSCLC cells to crizotinib,as evidenced by decreased invasion and proliferation and enhanced apoptosis.Metformin could reduce IGF-1R signaling activation in crizotinib-resistant cells.Moreover,the addition of IGF-1 to crizotinib-sensitive H2228 cells led to crizotinib resistance,which could be reversed by metformin.Conclusion: Metformin could be applied in combination with crizotinib in ALK+ NSCLC patients to overcome crizotinib resistance and prolong surviv al.