Study Of Estrogen Receptor ?1 And 5 Up-regulate IGF1 Pathway Though Genomic Signaling By Estrogen Stimulation In Non Small Cell Lung Cancer Development

Objective:A body of preclinical evidence now demonstrates that estrogen is a driver of Non-Small Cell Cancer(NSCLC).This study was performed to elucidate the expression and functions of ER?1,2 and 5,the major molecules of estrogen signaling in NSCLC tissues.Methods:Samples 113 patients with primary NSCLC(diagnosed between October 2008 and February 2014)who underwent surgery in the Department of Thoracic Surgery(affiliated to Tongji Hospital of Huazhong University of Science and Technology Tongji Medical College)were enrolled into the study.IHC staining was performed to detect nuclear and cytoplasmic staining of ER?1,2,5,Insulin Growth Factor 1 Receptor(IGF1R)and Ki67.Association between expression of ER?1,2,5 and IGF1R and Ki67,and clinicopathological characteristics include gender,age,smoking index,histological type,TNM stage,degree of differentiation and Overall Survival(OS)after surgery were analysed.The univariate survival analysis was performed using Kaplan-Meier method,survival curves were compared with the log-rank test.Additionally,univariate and multivariate COX regression analyses were carried out.Results:Overexpression of c/n ER?1,2,5 was detected by immunohistochemical analysis in the NSCLC samples at different levels.High expression of Ki67 was detected in tissues with overexpression of cER?1 and cER?5.cER?5 expression was significantly associated with elder than 55 years old patients,and cER?1 strong expression(+++)was higher in IV stage patients.cER(31 expression was significantly associated with cER(35,IGF1R and Ki67 expression and cER?5 xpression was associated with IGF1R and Ki67 expression.Strong expression of cER?1 and cERp5was associated with worse overall survival.Multivariate analysis showed that cER?1 and cER?5 were independent prognostic markers of decreased overall survival.Conclusion:Overall,these findings support ER?1 and ER?5 play important roles in tumorigenesis and development of NSCLC.Potential accociation exists between estrogen signaling and IGF1 signaling.ER?1 and ER?5 may play different roles on the growth and progression of NSCLC and may be considered as a new prognosis predict marker and therapeutic target in NSCLC.Objective:The roles of ER?3,ER?2,ER?5 in proliferation and metastasis of NSCLC are not completely appreciated in tumour development.And the relation between ER?1 ER?2,ER?5 and IGF1 signal is still unknown.The aim of the present study is to determine the function and possible mechanism of ER?3,ER?2,ER(35 in NSCLC development.Methods:A549 and H1793 cells were transfected siRNA-ER?1,siRNA-ER?2,siRNA-ER?5,pcDNA3.1-ER?1,pcDNA3.1-ER?2,pcDNA3.1-ER?5,lentivirus-ER?1,lentivirus-ER?2,lentivirus-ER?5.Immunofluorescence was used to detected location and stain of ER?1,ER?2,ER?5 and IGF1R.Proliferation was analysed by CCK8 and Edu assays,invasion and migration was analysed by Transwell assays after transfection of siRNA-ER?1,siRNA-ER(32,siRNA-ER?5,pcDNA3.1-ER?1 pcDNA3.1-ER?2 and pcDNA3.1-ER(35.CCK8 and wound healing assays was to investigated growth and migration after transfection of pcDNA3.1-ER?1 +siRNA-IGF1R,pcDNA3.1-ER?2+siRNA-IGF1R,pcDNA3.1-ER?5+siRNA-IGF1R.A549 cells transfected lentivirus-ER?3,lentivirus-ER?2,lentivirus-ER?5,were subcutaneously injected in BALB/c mice to built xenograft tumor model.Tumor weight and volumes were calculated,and IGF1R expression in tumors were detected.Results:Green staining of ER?3,ER?2,ER?5 located in both nucleus and cytoplasm,stain of ERP2,ER?5 deepened after 17?-estradiol stimulation.Red staining of IGF1R mainly located in cytoplasm of A549 and H1793 cells.Administration with siRNA-ER?1 and siRNA-ER?5 in A549 and H1793 cells decreased cell growth,invasion and migration while pcDNA3.1-ER?1 and pcDNA3.1-ER?5 treatment accelerated proliferation and metastasis.However,treated with pcDNA3.1-ER?1+siRNA-IGF1R,pcDNA3.1-ER?2+siRNA-IGF1R,pcDNA3.1-ER?5+siRNA-IGF1R,cell growth and migration were reversed in A549 and H1793 cells.In xenograft mice,gourp of lentivirus-ER?1,lentivirus-ER?5 demonstrated a dramatic decrease in tumour weight and volumes.In addition,transfection of lentivirus-ER?1,lentivirus-ER?2,lentivirus-ER?5 weaken expression of IGF1R compared to control group.Conclusion:Therefore,our study strongly highlights the biological significance of the ER?1 and ER?5 identifies IGF1 as a key regulator in the NSCLC proliferation and metastasis.ER?1 and ER?5 promote proliferation and metastasis in NSCLC through IGF1 signal pathway.ER?1 and ER?5 may emerges as a new therapeutic target for Endocrine therapy of NSCLC.Further study will focus on the mechanism of IGF1 signal regulation by ER?1 and ER?5.Objective:Activation of ER?1 and ER?5 promote proliferation and metastasis of NSCLC through IGF1 signal pathway.While the exact mechanism of this phenomenon is still unknown.This study is aim to investigate the mechanism how ER?1,ER?2,ER?5 regulate the IGF 1 signal.Methods:A549 and H1793 cells were transfected of siRNA-ER?1,siRNA-ER?2,siRNA-ER?5.Total RNA was isolated from cells and real-time PCR was used to analysed relative expression of IGF1mRNA in each group.We identified six potential EREL(Estrogen Response Elements Like)sites in the promoter of IGF 1 gene,and chromatin immunoprecipitation analysis was applied to investigate if ER?1 ER?2,ER?5 protein bind on the IGF1 promoter derectly.Dual luciferatse reporter analysis was used to detect if ER?1,ER?2,ER?5 could activate IGF1 transcripts via these EREL sites respectively.Results:Relative expression of IGF1 mRNA decreased in siRNA-ER?1 and siRNA-ER?5 group in A549 and H1793 cells,with estradiol-dependent.Six potential EREL sites were predicted in the promoter of IGF1 gene,named site 1,ATGACATCATAACCC,site 2,AGGGGACAGTGACAG,site 3,ATGAGACAGTGCCCT,site 4,GTGTCTTCATGCCCA,site 5,TTGTCACCATGCCCA,site 6,TAACTTTGCCAG.Chromatin immunoprecipitation analysis found ER?1,ER?2,ERP5 did not bind to Non-ERE site.However,ER?1 bind to site 2,site 5 and site 6,ER?2 bind to site 1,site 3,site 4,site 5,and site 6,and ER?5 bind to site 2,site 3 and site 4.Dual luciferatse reporter analysis showed ER?1,2,5 activated transcription of IGF1 gene.The activated sites of ERELs is site 2,site 5 and site 6 to ER?1,site 1,site 3 to ER?2 and site 2,site 3 and site 4 to ER?5,respectively.Conclusion:Our data highlight the importance of ER?1,ER?2,ER(35 in mediatingtranscription of IGF1 by binding to EREL sites respectively.Taken together,our studyindicating the exact mechanism that ER?1,ER?2,ER?5 bind to IGF1 promoter with specific sites.Our study strongly highlights ER? isoforms as new molecular classifications o f NSCLC from gender perspectives.ER? isoforms could be new molecular classifications of diagnosis and new molecular classifications of prognosis predicters and therapeutic targets in NSCLC.