HuR-Mediated Bim Expression Contributes To Intrinsic Resistance Of Sensitive EGFR-Mutant NSCLC To EGFR-TKI

BackgroundLung cancer is one of the main causes of tumor-associated deadly disease.Non-small cell lung cancer(NSCLC)accounts for nearly 80% of all lung cancer types.In 2004,the mutations of epidermal growth factor receptor(EGFR)tyrosine kinase domain were found in NSCLC,and most of the NSCLC patients with EGFR mutations were sensitive to EGFR tyrosine kinase inhibitors(EGFR-TKIs).The median overall survival time for EGFR mutation-positive patients who received EGFR-TKIs treatment was substantially longer than those treated with platinum-based chemotherapy.However,primary resistance to EGFR-TKIs was found in about 20-30% of patients.Moreover,people who are initially effective to EGFR-TKIs eventually develops acquired drug resistance.Presently,many kinds of mechanisms of acquired resistance to EGFR-TKI has been confirmed,such as EGFR T790 M gatekeeper mutation(50%),Met amplification(20-25%),High-level expression of HGF,as well as transformation to small cell lung cancer.However,the mechanisms of primary resistance to EGFR-TKIs are not well understood.The Bim gene encodes a BH3-only protein,which is a proapoptotic member of the BCL2 family of proteins.Studies have found that Bim gene expression status may be related to the effect of patients with EGFR mutations on EGFR-TKIs.Other studies have also shown that the presence of the Bim deletion polymorphism was associated with treatment response to EGFR-TKIs.However,the regulation mechanism of Bim gene expression is unknown.Hu antigen R(HuR)is RNA-binding proteins belonged to ELAV family.HuR can regulate the stability of a variety of molecular mRNA including cyclin A,cyclin B,TNF-?,IL-6,and IL-8.Through binding to adenine-uridine rich elements(AREs)located in the 3' untranslated region(UTR),HuR could regulate the mRNA and protein expression of target genes.We also found that Bim contains adenine-uridine rich elements(AREs)within the 3'-UTR of mRNA.This is the molecular function of RNA binding proteins.ObjectiveTo analyze the expression of HuR and Bim and their significance in NSCLC tumors with sensitive EGFR mutations,and further reveal the potential molecular mechanisms of the resistance to EGFR-TKIs in lung adenocarcinoma cancer cells harboring EGFR mutations HuR-mediated expression regulation of Bim.Methods1.HuR and Bim protein expression levels in 54 EGFR mutation-positive NSCLC tissues which was clinically sensitive to EGFR-TKIs and 27 control NSCLC tissues which was clinically resistant to EGFR-TKIs were detected by immunohistochemistry method(SABC method).The relationship between the expression of HuR and Bim and clinicopathological features were analyzed.2.H1650,HCC827 and PC-9 cells were used in in vitro experiments,and the cell proliferation were detected by CCK-8 after gefitinib treatment.3.The expression of HuR,Bim mRNA and protein in three kinds of cells were detected by Western blotting and quantitative RT-PCR.4.The siRNA targeting HuR was used to interfere with the expression of HuR in HCC827 cells.After treatment with gefitinib,the IC50 and apoptosis in the interference group and control group were detected by CCK-8 kits and Annexin V/PI double staining method.5.Western blotting and RT-PCR were used to detect the expression of mRNA and protein of HuR and Bim in HCC827 interference group and non interfering group.6.A vector ovexpressing HuR GV365-HuR was constructed and transfected to H1650 cells,and the changes of IC50 and the cell apoptosis were measured when treated with gefitinib.7.Western Blotting and RT-PCR were used to detect the changes of mRNA and protein levels of HuR and Bim in H1650 infected group and control group.8.HuR-transfected H1650 cells were subcutaneous inoculation into the nude mice to develop a tumor model.To compare with the growth of transplanted cancer when treated with gefitinib,and analysis the effect of high HuR level on drug sensitivity of EGFR-TKIs in vivo.9.HuR and Bim expression level were measured by western blotting and RT-PCR in the transplanted tumor.10.HuR and Bim expression were measured by immunohistochemical staining in the transplanted tumor.Results1.There is a difference in gender between two groups of patients.In the resistant group,female patients accounted for 18.5%,female patients in the sensitive group accounted for 57.4%,and the drug resistance group was significantly lower than that in the sensitive group(p=0.01).There was no observed difference between EGFR-sensitive group and EGFR-resistant group with respect to age,performance status,mutation types,history of surgery and chemotherapy and brain metastases.2.There was a significant difference of HuR and Bim expression between two groups.It was showed that 81.5% of EGFR-resistant group had the negative expression of HuR,and 20.4% of EGFR-sensitive group were HuR negative.It was showed that 70.4% of EGFR-resistant group had the negative expression of Bim,and 7.4% of EGFR-sensitive group were Bim negative.There was a significant difference between the two groups(p<0.001),and the negative expression rate of HuR and Bim was significantly higher in the resistant group than that in the sensitive group.3.The expression of cytoplasmic Bim was closely related to HuR expression(p < 0.01),but not correlated with sex,age and mutation types.(p > 0.05).4.Median progression-free survival was 12.8 months in positive Bim expression patients of EGFR-sensitive group,and 9.7 months in negative Bim expression patients.The PFS of positive Bim expression patients were significantly higher than those in negative Bim expression patients by Kaplan-Meier analysis.5.Median progression-free survival was 16.5months in positive HuR expression patients of EGFR-sensitive group,and 9.7 months in negative HuR expression patients.The PFS of positive HuR expression patients were significantly higher than those in negative Bim expression patients by Kaplan-Meier analysis.6.The IC50 values for gefitinib in the PC-9,HCC827 and H1650 cells were 0.05,0.004 and 21.28 ?M,respectively.The IC50 value for gefitinib in the H1650 cells was markedly higher than the other two cells(p < 0.01).7.RT-PCR showed that HuR and Bim mRNA expression significantly lower in H1650 cells than the other two cells.As expected,western blotting showed that HuR and Bim expression also lower in H1650 cells.8.The IC50 value for gefitinib in the siHuR HCC827 cells was markedly increased than HCC827 cells(8.142?M vs 0.004?M).Annexin-V/propidium iodide(PI)staining indicated that,compared with the control group,the apoptosis rate of HCC827 cell interference group was decreased after treatment with gefitinib(p<0.05).9.The results of RT-PCR and western blotting showed that the expression of HuR and Bim mRNA and protein levels in the interference group was lower than that in the control group(p<0.01).10.The vector GV365-HuR targeting HuR was successfully constructed.After transfection,H1650 cells were stably overexpression of HuR,named GV365-HuR-H1650.11.The IC50 value for gefitinib in the GV365-HuR-H1650 cells was markedly decreased than H1650 cells(21.28?M and 0.875?M).This suggests that the GV365-HuR-H1650 cells restored response to gefitinib.Annexin-V/propidium iodide(PI)staining indicated that,compared with the control group,the apoptosis rate of H1650 cell infection group was significantly increased after treatment with gefitinib(p<0.01).12.RT-PCR showed that the expression of HuR and BIM mRNA in H1650 infected group was higher than that in control group(p<0.01).Western blotting showed that the expression of HuR and Bim protein in the infection group was higher than that in the control group.13.Animal experiment is divided into transfection group and the control group.After formation of subcutaneous xenograft in mices,they were tall reated with gefitinib.Tumor growth curves showed that the tumor growth was slow in transfection group.The tumors volumes of transfection group are much smaller and lighter than the control group.14.Immunohistochemistry indicated that Cytoplasm HuR and Bim dying in transfection group were positive,while the control groups were negative.15.RT-PCR and Western blotting showed that expressing level of HuR and Bim in transfection groups was higher than that in control groups.ConclusionsBim plays a major role in primary EGFR-TKI resistance in harboring sensitive EGFR mutations adenocarcinoma patients.HuR through adjust the expressing of the Bim lead to the resistance to EGFR-TKI.