The Role And Mechanism Of IR Signaling Pathway In The Resistance Of Esophageal Squamous Cell Carcinoma To IGF-1R Inhibitor AXL1717

BackgroundInsulin like growth factor（IGF）signaling system consists of two ligands（IGF-1 and IGF-2）,two receptors（IGF-1R and IGF-2R）and six IGF binding proteins.A large number of studies have confirmed that the IGF signaling system plays an important role in promoting tumor growth and survival.IGF-1R is overexpressed in many tumors,including esophageal cancer.However,the anti-IGF-1R monoclonal antibodies in recent clinical trials showed limited efficacy.The poor efficacy is firstly due to the lack of biomarkers to predicte the efficacy,therefore,patients who may benefit from the anti-IGF-1R agents can not be screened out.Secondly,it may be due to the compensatory effect caused by the existence of insulin receptor（IR）bypass pathways.IR has two isoforms:IR-A and IR-B.IR-A was highly expressed in a variety of tumors and mainly transmited cell proliferation and survival signals,while IR-B mainly bound to insulin to regulate glucose metabolism.When the IGF-1R pathway is inhibited,tumor cells may undergo signal transduction through the IR-A pathway.As a result,the resistance mechanism of tumor cells to IGF-1R inhibitors may be related to the compensatory effect of the IGF-1R/IR-A alternative pathway.ObjectiveTo clarify the role and mechanisms of IR signal pathway in the resistance of IGF-1R inhibitor AXL1717 in esophageal squamous cancer cells（ESCC）,so as to provide necessary preclinical experimental data and basis for the treatment of esophageal squamous cancer with IGF-1R inhibitors,and explore a new scheme for the targeted therapy of esophageal squamous cancer.Methods1.MTT assays were used to measure the effects of IGF-1R specific inhibitor AXL1717on cell viability of 8 esophageal squamous cancer cell lines,and the most and least sensitive cell lines to AXL1717 were selected.2.The expression levels of IGF-1R,IR,phospho-IGF-1R and phospho-IR on the esophageal squamous carcinoma cells were detected by Western blot assay,and the correla-tion analysis was performed between the IC50 values of AXL1717 and the expression levels of IGF-1R,IR,phospho-IGF-1R or phospho-IR by Graph Pad Prism 5 software.3.RT-PCR was used to detect IR-A and IR-B gene expression levels in eight esophageal squamous cancer cell lines,after which the correlation analysis was performed between the ratio of IR-A:IR-B and the IC50 values of AXL1717.4.PI staining was used to detect the effect of AXL1717 on the cell cycle progression of esophageal squamous cell carcinoma.5.IR-interfering sh RNA（sh IR#D）was transfected into AXL1717-insensitive esophageal squamous cancer cells EC9706 and TE-7 by using lipofectine 3000.The effects of AXL1717 on cell viability and cell apoptosis were evaluated in EC9706 and TE-7 cells before and after knockdown of IR by MTT assays and Annexin V-FITC/PI staining.6.The IR expression plasmid（h IR-GFP）was transfected into the AXL1717-sensitive esophageal squamous cancer cells EC109 and KYSE450 by using lipofectine 3000.The effects of AXL1717 on cell viability and cell apoptosis were evaluated in EC109 and KYSE450 cells before and after overexpression of IR by MTT assays and Annexin V-FITC/PI staining.7.Western blot analysis was used to detect the effects of AXL1717 on the phosphorylation and total protein levels of IGF-1R and IR as well as their main downstreatm signaling molecules IRS-1,AKT and ERK before and after IR knockdown or overexpression.Results1.IGF-1R specific inhibitor AXL1717 showed potent proliferation inhibitory effect on8 ESCC cell lines with IC₅₀ values of 48.32μmol/L,0.01μmol/L,0.47μmol/L,0.80μmol/L,0.41μmol/L,0.07μmol/L,0.87μmol/L,7.81μmol/L,respectively.2.Correlation analysis revealed that the IC50 values of AXL1717 are not correlated with the phosphorylation of IGF-1R and IR,nor with the total level of IGF-1R and IR,but closely related to the ratio of IR-A/IR-B.3.IR expression plasmid h IR-GFP was successfully transfected into AXL1717-sensitive ESCC cells KYSE450 and EC109,and IR-specific sh RNA（sh IR#D）was successfully transfected into AXL1717-insensitive ESCC cells EC9706 and TE-7.4.Cell viability assays showed that the proliferation inhibitory effects of AXL1717against KYSE450 and EC109 cells were significantly reduced after transfection of IR expression plasmid h IR-GFP.On the contrary,the inhibitory effect of AXL1717 on the proliferation of EC9706 and TE-7 cells was dramatically enhanced after transfection of IR-specific sh RNA.5.The results of cell cycle assay showed that AXL1717 induced esophageal squamous cell carcinoma cells to block in S phase and G2/M phase.6.The results of cell apoptosis showed that AXL1717 induced apoptosis of esophageal squamous cell carcinoma cells EC9706,EC109,KYSE450 and TE-7 in a dosage dependent manner.In addition,the effect of apoptosis-induction after overexpre-ssion of IR plasmid in KYSE450 cells was significantly decreased（P<0.001 vs transfe-ction of vector）.While in EC9706 cells,the apoptosis inducing ability of AXL1717 was significantly increased after IR knockdown in（P<0.001 vs transfection of mock）.7.Western blot analysis showed that the phosphorylation levels of IGF-1R,IR and downstream insulin receptor substrate-1（IRS-1）,AKT and ERK in KYSE450 cells decreased in a concentration-dependent manner after AXL1717 treatment of KYSE450 and EC109 cells,while the phosphorylation levels of IGF-1R and IR did not change significantly in EC109 cells,but the phosphorylation levels of IRS-1 and ERK decreased,indicating that IGF-1R inhibitor AXL1717 could significantly inhibit the activation of IGF-1R and IR signaling pathways.8.Western blot analysis showed that after AXL1717 treatment of TE-7 and EC9706cells,the phosphorylation levels of IGF-1R and downstream AKT and ERK in TE-7 cells decreased,but the activation levels of IR and important junction molecule IRS-1 increased.Furthermore,the phosphorylation levels of IGF-1R,IR and IRS-1 in EC9706 cells increased significantly,which may be one of the reasons for the drug resistance of EC9706 cells to AXL1717.9.Results from Western blot analysis also revealed that the phosphorylation of IGF-1R and IR in KYSE450 cells treated with AXL1717 after IR overexpression increased,and the activation IRS-1 and AKT were also observed,which indicated that IR overexpression may counteract the inhibitory effect of AXL1717,an IGF-1R inhibitor,on the activation of IGF-1R and IR signaling pathways.When EC9706 cells were transfected with IR sh RNA plasmid,AXL1717 treatment at the concentration of 0.3μmol/L could reverse the increase of IGF-1R and IR phosphorylation,but the activation of IRS-1,AKT and ERK was not inhibited.Conclusions1.IGF-1R specific inhibitor AXL1717 demonstrated potent growth inhibitory effects and cell apoptosis induction on 8 ESCC cells.And the sensitivity of ESCC cells to AXL1717was closly related with the cell IR-A/IR-B ratio.2.IR signaling plays importeant role in the resistance of ESCC cells to AXL1717.After overexpression or knockdown of IR,the activity of growth inhibition and cell apoptosis induction was greatly reduced or enhanced.The machanisms of AXL1717 resistance may due to the signal transduction through IR pathway,which counteracts the inhibitory activity of AXL1717 on IGF-1R and its downstream signaling molecules.