| Lung cancer is the most common type of cancer and is the leading cause of death in both male and female cancer patients worldwide.Epidemiological data suggest that there are significant gender differences in clinical pathological features,genetic mutations,hormone levels,morbidity,treatment response and cancer outcomes in lung cancer patients These differences indicate that the development of lung cancer may be affected by gender-related hormonal factors,especially by estrogen receptors(ERs).ERs are thought to play an important role in the development and progression of lung cancer,especially in non-small cell lung cancer(NSCLC).Both of the two major subtypes of ERs,ERa and ER?,may be involved in the regulation of NSCLC cell behaviors and tumor progression However,the expression levels of ERs in NSCLC and their relationship with prognosis remain controversial.In addition,current reports on the mechanism of ERs in NSCLC are limited,mainly focused on the crosstalk between ERs and some growth factor pathways such as EGF,FGF and IGF1 pathways.The complexity of biological system and signal transduction system suggests that ERs are more likely to regulate cell behaviors and tumor progression through multiple targets or signaling networks than regulating individual genes or pathways.Therefore,it is necessary to further explore the role of ERs in NSCLC in the context of systems biologyRecently,there has been accumulating evidence supporting that cancers,including NSCLC,is a kind of complex systemic disease involving dysregulation of multi-factors,multi-step,multi-omics and loss of homeostasis at multiple levels.In addition,there are extensive and intricate interactions within and between different levels that further exacerbate the complexity of cancer pathogenesis.Therefore,it is particularly important to apply systematic biology methods to study the mechanism of cancer development and explore more effective treatment strategies.Taking the advantage of high-throughput database,cost-effective chip technologies,bioinformatics methods and high-speed computing resources,Cancer Systems Biology can bring together a variety of advanced research and analytical approaches,to reveal the characteristics of cancer evolution from a holistic perspective.And then powerful models can be built to validate or predict new treatment strategies.More importantly,this kind of systematic study can not only analyze the abnormal phenomena and dynamic evolution of cancer across multi-layers and multi-steps,but also reveal new phenomena and new mechanisms arising from various interactionsTherefore,in this study,we intend to integrate bioinformatics method,experimental approach,computer modeling and immunohistochemical analysis,to explore the role of ERs in the development of NSCLC from the perspective of cancer systems biology.On the base of that,we hope to discover the regulation mechanism of ERs on cancer-related molecular signaling network in NSCLC and provide the possibility of exploring new strategies for NSCLC treatment.The study is divided into the following three parts:in the first part,we have explored the effect of ESR1/2 on NSCLC gene expression profile based on TCGA dataset at the gene level.Bioinformatics analysis suggests that ESR1/2 gene may play a role in NSCLC by regulating some important membrane receptor signals;in the second part,the combination of biological experiments and computer modeling have confirmed that ERs promote the development of NSCLC by modulating the membrane receptor signaling network composed of EGFR,Notchl,GSK3?/?-Catenin pathways;in the third part,results of immunohistochemistry echo the combined cancer-promoting effects of ERs and EGFR,Notch1 pathways,that is ER?,ER?,EGFR and Notch 1 synergistically promote the poor prognosis of the late-stage NSCLCPart ? ESR1/2 affected some important membrane receptor signaling pathways in NSCLCObjective:To investigate the effect of ESR1/2 on NSCLC gene expression profile based on TCGA NSCLC gene expression dataMethods:The TCGA NSCLC gene expression dataset was obtained from The UCSC Xena online database.The R package "limma" was used for gene differential expression analysis to assess whether the variance in ESR1/2 expression levels affects the expression of other genes in NSCLC tumor tissue samples.Differential expression genes(DEGs)screened based on different levels of ESR1 and ERS2 were used as target gene sets,which were uploaded to the online DAVID Functional Annotation Tool.GO function and KEGG pathway enrichment analysis were performed to analyze the potential impact of ESR1/2 on key molecular events and pathways in NSCLCResults:Differential expression analysis showed that there were 1237 DEGs between the low-and high-ESR1 group and 102 DEGs between the low-and high-ESR2 group,respectively.The results of GO function and KEGG pathway enrichment showed that the 769 up-regulated DEGs in the high-ESR1 group were mainly enriched in the terms of signal transduction,immune and inflammatory responses,cell adhesion,receptor binding and activation.The 468 down-regulated DEGs in the high-ESR1 group were mainly enriched in the terms of transcriptional regulation,oxidation-reduction process,calcium ion binding,CYP450-related metabolism.For ESR2,no significant term was found for enrichment analysis of these 7 downgraded DEGs.The 95 up-regulated DEGs in the high-ESR2 group were mainly enriched in the terms of cell adhesion,morphogenesis,epithelial cell differentiation,calcium ion binding and structural molecular activity.For cell component of GO analysis,both of DEGs in ESR1 and ESR2 group were mainly enriched in plasma membrane and extracellular space.In addition,DEGs enriched in the above terms of GO and KEGG pathways were also involved in many membrane receptor signaling pathways,such as the growth factor signaling pathway,Wnt/GSK/?-Catenin pathway and Notch pathway.Conclusion:ESR1/2 might affect many important molecular events and pathways,especially membrane-associated cell communication,including receptor activation,signal transduction,cell adhesion,immune response.ESR1/2 might influence the development of NSCLC by regulating important membrane receptor signaling pathways.Therefore,in the next part,we will validate the effect of ERs on EGFR,Notchl,GSK3?/?-Catenin pathway and on the progression of NSCLC.Part ? ERs promoted NSCLC progression by modulating the membrane receptor signaling networkObjective:To investigate the effects of ERs on the behaviors of NSCLC cells,and the effects of ERs on the EGFR,Notchl,GSK3?/?-Catenin pathways and on the signaling network.Methods:(1)After treated with different concentrations of 17?-E2(0,10,20 nM)for 72 h,CCK-8 was used to detect the proliferation of NSCLC cells;Western blot was used to detect the expression of ER? and ER? in NSCLC cells.(2)si-ER?/si-ER? was transfected into PC9/G and H1299 cells,Western blot was used to detect the effect of ERs silencing on the expression of apoptosis-related proteins,migration and invasive-related proteins.After transfection with si-ER?/si-ER? or treated with Fulvestrant,cells were incubated with Gefitinib or blank control(DMSO)for 48 h,Flow cytometry was used to detect the effect of ERs silencing on apoptosis rate of PC9/G and H1299 cells,Transwell was used to detect the effect of ERs on cell migration and invasion.(3)After transfection,PC9/G and H1299 cells were incubated for 48 h,the effect of ERs silencing on the expression of key proteins in Notchl and GSK3?/?-Catenin pathway was detected by Western blot.(4)After incubation with different concentrations of 17?-E2(10,20,50 nM)and Fulvestrant(0.25,0.5,1?M)for 12 h,Western blot was used to detect the expression of key proteins of Notchl and GSK3?/?-Catenin pathways in PC9/G cells.(5)pEGFR,pAkt and pERK expression in PC9/G cells was detected by Western blot after 0,2,5,10,30,60,120 and 180 min of stimulation with 100 ng/mL EGF.NICD,Hes1 and PTEN expression in H1299 cells was detected by Western blot after 0,2,5,10,30,60 and 120 min of stimulation with 100 ng/mL Dll1.Expression levels of key members in ERs,Notch1,EGFR and GSK3?/?-Catenin pathways were detected by Western blot after 0,2,5,10,30,60,120 and 180 min of stimulation with 20 nM 17?-E2.(6)Based on the time-dependent expression data of EGFR,Notchl and GSK3?/?-Catenin pathway proteins,the kinetic model of membrane receptor signal network was constructed by ordinary differential equations;the Matlab toolbox PottersWheel was used for model construction,parameter estimation and model simulation.Results:1.ERs promoted NSCLC cell proliferation,migration,invasion and apoptosis escape.(1)After E2 treatment,the proliferation ability of PC9/G,PC9 and H1299 cells was significantly increased(p<0.05).However,the proliferation of A549,H1975 and HCC827 cells was not influenced by E2;ER? and ER? were significantly high-expressed in PC9/G,PC9 and H1299 cells,while the expression levels in A549,H1975 and HCC827 cells were relatively low.(2)ERa and ER? silencing increased the apoptosis rate of PC9/G and H1299 cells(p<0.05);compared with Gefitinib alone,the combination of si-ERs and Gefitinib increased the apoptotic rate of PC9/G and H1299 cells(p<0.05).The expression of Survivin and Bcl-2 was down-regulated and the expression of Cleaved Caspase 3 was up-regulated after silencing ER? and ER? in PC9/G and H1299 cells(p<0.05).The expression of Bim in PC9/G cells was up-regulated(p<0.05),which in H1299 cells was not affected after ER?and ER? silencing.(3)Cell migration and invasion were reduced after silencing ER? and ER? in PC9/G and H1299 cells(p<0.05).si-ERs also increased the inhibitory effect of Gefitinib on cell migration and invasion in PC9/G and H1299 cells(p<0.05).In PC9/G cells,ERs silencing down-regulated the expression of Fibronetin,N-Cadherin,ZEB1,Vimentin and Snail(p<0.05),and restored the expression of E-Cadherin(p<0.05).ERs silencing also reduced the expression of Fibronetin,N-Cadherin,ZEB1,Vimentin and Snail in H1299 cells(p<0.05),but no recovery of E-Cadherin expression was observed.2.ERs regulated the expression and activation of key proteins of EGFR,Notchl and GSK3?/?-Catenin pathway(1)After ERs silencing,the expression of Notchl,NICD,Hesl and ?-Catenin were down-regulated in PC9/G and H1299 cells(p<0.05).ERs silencing also down-regulated GSK3? expression and up-regulated the phosphorylation of GSK3? in H1299 cells(p<0.05).The expression levels of GSK3? and pGSK3? in PC9/G cells were not affected by si-ERs.(2)In PC9/G cells,17?-E2(10,20 nM)up-regulated the expression of ER? and ER?(p<0.05).17?-E2 stimulation also affected the expression of key proteins in Notchl pathway.10 or 20 nM 17?-E2 up-regulated the expression of Notch1,NICD and Hesl(p<0.05);50 nM E2 only up-regulated Hesl expression(p<0.05),while down-regulating the expression of Notchl(p<0.05),and had no effect on NICD expression.17?-E2 up-regulated the expression of ?-Catenin(p<0.05),but did not affect the expression of GSK3?.Fulvestrant had the opposite effect to 17?-E2.After treated with Fulvestrant,the expression of ER? and ER? was significantly inhibited(p<0.05).At the same time,the expression of Notch1,NICD,Hes1 and ?-Catenin was also inhibited(p<0.05).However,the expression of GSK3? is not affected by Fulvestrant in PC9/G cells.(3)In PC9/G cells,after stimulated with 100 ng/mL EGF,all of EGFR,Akt and ERK were phosphorylated,and the expression of pEGFR and pERK rapidly reached the peak level,while the expression of pAkt did later.After stimulated H1299 cells with 100 ng/mL Dll1,NICD and Hesl were transiently activated and then returned to baseline levels;PTEN,as a downstream target of Hesl,was slightly down-regulate with a slow rate.20 nM E2 promoted the expression of ER? and ER?,increased the expression and activation of Notchl,enhanced the activation of EGFR pathway,but did not affect the expression of EGFR,increased the expression of ?-Catenin,but did not affect the expression of GSK3?.3.Construction and simulation of the membrane receptor signal network model(1)Based on the time-dependent expression data of pathway proteins,an ordinary differential equation model network composed of ERs,EGFR,Notch1 and GSK3?/?-Catenin pathways was constructed.The signal network consisted of 66 reactions,43 nodes and 98 parameters.When EGF,17?-E2 and Dll1 were all at low levels(All-Low group),the outputs of network were maintained at low levels.When EGFR pathway was activated(High-EGF group),Akt and ERK were highly phosphorylated,but ?-Catenin and Hesl were not significantly affected.When Notchl pathway was activated(High-Dll1 group),rapid and transient activation of Hesl appeared,and the pAkt level was slightly elevated.17?-E2(High-E2 group)could directly activate EGFR,then caused high activation of Akt and ERK.Moreover,the expression of ?-Catenin and Hesl was elevated by increased ERs expression and activation.If all pathways were activated(All-High group),the four output indicators would reach the highest levels.(2)Model simulations showed that even if EGFR pathway was inhibited,hyperactivation of ERs would reactive the EGFR pathway,leading to EGFR TKI resistance.The experimental results confirmed that Gefitinib combined with Fulvestrant cooperated in NSCLC cells exhibited higher tumor inhibition effect;for Gefitinib-resistant PC9/G and H1299 cells,ERs silencing could also enhance the tumor suppressive effect of Gefitinib.Conclusion:(1)The proliferative effect of estrogen in NSCLC was ERs-dependent;(2)ERs inhibited NSCLC cell apoptosis by regulating the balance between pro-and anti-apoptotic proteins;(3)ERs promoted NSCLC cell migration and invasion by regulating the EMT process;(4)ERs enhanced the expression and activation of Notch1 pathway-associated proteins,and increased ?-Catenin accumulation by GSK3? or other alternative pathways.(5)17?-E2 could enhance the expression and/or activation of ERs,Notchl pathway-associated proteins and ?-Catenin,and Fulvestrant had the opposite effect to 17?-E2.(6)ERs played a role in NSCLC by adding redundant pathways into the signaling network.When EGFR and Notchl pathways were inhibited,ERs could reactivate the signaling network and mediate high-level outputs,thereby promoting NSCLC progression.(7)Redundancy of ERs signal aggravated EGFR TKI resistance,and ERs inhibition was a potential strategy to overcome NSCLC EGFR TKI resistance.Part ? ERs,EGFR and Notchl promoted poor prognosis of the late-stage NSCLCObjective:To investigate the relationship between the expression levels of ER?,ER?,EGFR,Notchl and the prognosis of NSCLCMethods:Immunohistochemistry was used to detect the expression of ER?,ER?,EGFR and Notchl in human NSCLC tissue microarray.Kaplan-Meier method was used to analyze the relationship between ER?,ER?,EGFR,Notchl expression and the overall survival time of NSCLC patients.Results:(1)The positive expression rates of ER?,ER? and EGFR in NSCLC tumor and their adjacent tissues were significantly different(p=0.0019,p<0.0001,p=0.0009).The IHC scores of ERa,ER?,EGFR and Notchl in tumor tissues were higher than those in their adjacent tissues(ERa:p<0.0001;ER?:p<0.0001;EGFR:p=0.0053;Notchl:p=0.0052).(2)The expression levels of ER?,ER?,EGFR and Notchl were not associated with survival time in early-stage NSCLC patients,but high expression of ER?,ER? and EGFR predicted worse 10-year survival for the late-stage NSCLC patients(p=0.0487,p=0.0419,p=0.0422).(3)For the late-stage NSCLC group,the more receptors that were highly expressed,the worse the patients' survival were(p=0.0042).(4)In the subgroup with at least two high-expression receptors,patients with high-expression Notchl displayed worse survival outcomes(p=0.0404).Conclusion:All of ER?,ER?,EGFR and Notchl were overexpressed in NSCLC tumor tissues.ER?,ER?,EGFR and Notchl were poor prognostic factors for the late-stage NSCLC,and that these receptors had a synergistic effect on poor prognosis of advanced NSCLC patients. |
PDF Full Text Request