| Introdution:Lung cancer is one of the most common malignancies and a major cause of cancerrelated deaths worldwide,approximately 85% of all lung cancers are non-small-cell lung cancers(NSCLCs).NSCLC patients with EGFR sensitizing mutation are highly responsive to first-generation EGFR-TKIs,such as gefitinib and erlotinib,~20%-30% of NSCLC patients show primary resistance to EGFR-TKIs,although they harbor activating EGFR mutation.Clinical application of EGFR-TKIs drug was obviously limited by primary resistance.It is of important scientific significance and clinical value to i mprove curative effects,alleviate side effects and improve prognosis if we have develop potently therapeutic strategies to overcome this resistance.Previously,we have found that metformin could recover sensitivity of acquired resistant cells to EGFR-TKIs in vitro and vivo through suppressing IL-6/STAT3 pathway.IL-6/STAT3 pathway is the key mechanism of metformin overcome acquired resistance to EGFR-TKIs in lung cancer cells.But we examined the protein of p-STAT3 between primary resistant cells and sensitized cells and found no significant difference,IL-6/STAT3 pathway might not be an important role in primary resistance to EGFR-TKIs.It has been reported that tumor tissue from NSCLC patients with de novo EGFR-TKIs resistance showed increased expression of IGF-1R.We also observed increased protein level of p-IGF-1R in primary resistant H1975 cells(L858R/T790M)compared with sensitized cells,while inhibiting the IGF-1R signaling could restore EGFR-TKIs sensitivity.Thus,primary resistance to EGFR-TKIs was mediated by the activation of IGF-1R pathway.Metformin has been a classical anti-diabetic drug with relatively low cost and high safety profile.It increasingly report that metformin exerts antineoplastic effects in many types of malignancies in vitro and in vivo.And metformin could enhance cancer cell killing effects via suppressing IGF-1R signaling activation.Metformin combined with EGFR-TKIs is a promising therapeutic strategy to overcome primary resistance through inhibiting IGF-1R signaling.In this study,we further investigated the effect of metformin combined with gefitinib on primary resistant lung cancer cells and clarify the underlying mechanism.Objectives: EGFR-TKIs have shown dramatic effects against sensitizing EGFR mutations NSCLC patients.But approximately 20%-30% of NSCLC patients with activating EGFR mutation showing intrinsic resistance to EGFR-TKIs.Studies have shown that this primary mechanism of drug resistance of EGFR-TKIs is associated with IGF-1R signals and EMT progressing.In this study,we aim to further explore the action of inhibiting IGF-1R and EMT in primary EGFR-TKIs resistant NSCLC cell line with de novo T790M(H1975),and the effect of metformin on EGFR-TKIs primary resistant cells through inhibition of IGF-1R signaling and EMT reversal.Methods: H1975 cell lines were treated with metformin and/or gefitinib to examine cell growth inhibition and potential mechanism of action in vitro by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium(MTT),ki67 incorporation assay,clone formation assay,invasion assay,flow cytometry analysis,immunofluorescence staining,small interfering RNA technology and Western blot analysis.Results: EGFR-TKIs primary resistant H1975 cells displayed an EMT phenotype and showed overexpression of p-IGF-1R as compared to EGFR-TKI acquired resistance cells(PC-9GR,H1650-M3)and EGFR-TKI sensitivity cells(PC-9,HCC827).Inhibition of IGF-1R activity by AG-1024(a small molecule IGF-1R inhibitor),as well as downregulation of IGF-1R by siRNA,significantly reversed EMT-related morphologies and enhanced the ability of gefitinib to suppress proliferation and induce apoptosis in H1975 cells via the inhibition of AKT activation and subsequently upregulation of BIM.Interestingly,the observation showed metformin combined with gefitinib treatment had similar tumor growth suppression effects in comparison with the addition of AG-1024 to therapy with gefitinib and knockdown of IGF-1R in H1975 cells.Notably,IGF-1R silencing mediated by RNAi attenuated anticancer effects of metformin without obviously resensitization H1975 cells to gefitinib.Conclusions: Our findings demonstrated that IGF-1R signal and EMT plays an important role in primary drug resistance against EGFR-TKIs.Metformin combined with EGFR-TKIs would be a promising strategy to sensitize primary resistant NSCLC cells via suppressing IGF-1R pathway and reversing EMT. |
