The Study Of CD166 Mediating Radioresistance In Nasopharyngeal Carcinoma Via EGFR-ERK1/2 Signaling Pathway

Background CD166,also called activated leukocyte cell adhesion molecule,is one member of the immunoglobulin superfamily,which has been identified as a positive marker for a variety of cancer stem cells(CSCs).In our previous researches,the expression level of CD166 in serum of radioresistant NPC was significantly higher than that of radiosensitive NPC using secretome proteomics.Silencing CD166 enhanced radiosensitivity of radioresistant cells CNE-2R.In additiion,our previous study also found that CNE-2R cells possessed cancer stem cell-like(CSC-like)characteristics.Studies have demonstrated that EGFR signaling pathway plays an important role in tumor radiosensitivity,which is negatively related to radiosensitivity of NPC.Furthermore,studies have confirmed that CSCs contribute to tumor radioresistance in multiple tumor types.Our previous studies have shown that the upregulated genes of radiation resistance have high connectivity with the EGFR pathway.Pre-experiment revealed that the expression of phosphorylated EGFR(p-EGFR)protein in CNE-2R cells was significantly higher than that in parental radiosensitive CNE-2 cells.In light of these,we hypothesized that CD166 may regulate CSC-like characteristics through EGFR signaling pathway,ultimately reducing the radiosensitivity of CNE-2R cells.Therefore,this study aims to investigate the relationship between CD166 and CSC,and further explore the underlying mechanism.Part 1 The effects of CD166 on the cancer stem cell-like properties in nasopharyngeal cancer cellsObjective To study the effects of CD166 on the cancer stem cell-like properties of nasopharyngeal cancer cells.Methods In this study,stable silencing and overexpression of CD166 in nasopharyngeal carcinoma cells was conducted by lentiviral transfection,q RTPCR and western blot analysis were used to detect the expression level of CD166 and the stem cells related genes(Bmi1,Oct4 and Sox2).Sphere formation assay was used to assess sphere formation ability of silencing CD166 of CNE-2R cells.Nude mice mode was preformed to evaluate tumorigenicity in vivo.Results 1.CD166 silencing cell line(CD166-sh RNA-CNE-2R)and CD166 overexpression cell line(CD166-OE-CNE-2)were successfully constructed by lentiviral vector transfection technology.Fluorescence microscope revealed that the transfection efficiency was above 90% in each group.The results of q RT-PCR showed that CD166 expression was significantly downregulated in CD166-sh RNA groups(P<0.001),whereas it was significantly upregulated in CD166-OE groups compared with the control groups(P<0.001).The results of western blot analysis were consistent with that of the q RT-PCR assay.2.Western blot analysis and q RT-PCR assay demonstrated that the expression level of the stemness-associated genes in proteins and m RNA was downregulated in CD166-sh RNA-CNE-2R cells and up-regulated in CD166-OECNE-2 cells compared with the corresponding control groups(P<0.05).3.Sphere formation assay showed that the number and size of spheres in CD166-sh RNA cells was smaller than that in NC-sh RNA and parent cells(P<0.01).4.Nude mice tumorigenicity assay showed that silencing CD166 inhibited tumorigenesis capacity of CNE-2R cells in vivo.The volume of xenografts in CD166-sh RNA group was smaller than that in NC-sh RNA group(0.018± 0.011cm3 vs 0.333±0.078 cm3),P=0.008.Conclusion CD166 enhanced cancer stem cell-like characteristics,which could be served as a positive tumor stem cells marker for nasopharyngeal carcinoma.Part 2 The study of CD166 regulating cancer stem cell-like characteristics via EGFR-ERK1/2 pathway in nasopharyngeal carcinoma cellsObjective To study whether CD166 regulates cancer stem cell-like traits via EGFRERK1/2 pathway in nasopharyngeal carcinoma cells.Methods In this study,western blot analysis was performed to detect the expression of EGFR-ERK1/2 pathway proteins in CD166-sh RNA-CNE-2R cells.Coimmunoprecipitation(Co-IP)was used to analyze the interaction between CD166 and EGFR.CD166-sh RNA and CD166-OE cell lines were cultured with EGFR pathway agonist and inhibitor,respectively,and then western blot analysis was performed to detect the expression of EGFR-ERK1/2 pathway proteins and CSCsrelated proteins.Results 1.The results of western blot analysis showed that there was no significant difference in the expression of EGFR and ERK1/2 proteins between CNE-2 and CNE-2R cells.However,the expression levels of p-EGFR and p-ERK1/2 were increased in CNE-2R cells.2.Western blot analysis demonstrated that silencing CD166 significantly decreased the expression levels of p-EGFR and p-ERK in CD166-sh RNA cells.However,there was an increase in the expression of p-EGFR,p-ERK1/2 and CSC-associated markers after cells stimulation with EGF.3.The results of Co-IP assay suggested that there was direct interaction between CD166 and EGFR.4.Western blot analysis showed inhibiting EGFR signaling down-regulated the expression of p-EGFR,p-ERK1/2 and CSC-related proteins.Conclusion CD166 promotes cancer stem cell-like characteristics via activating EGFRERK1/2 pathway in the nasopharyngeal carcinoma cell line CNE-2R.Part 3 Effects of silencing CD166 on the cancer stem cell-like characteristics and radioresistance of xenografts in nude miceObjective This study aimed to explore the effect of silencing CD166 on the CSC-like characteristics and radioresistance of xenografts in nude mice.Methods The xenograft tumor model was constructed in nude mice to verify the effect of CD166 on radiosensitivity of nasopharyngeal carcinoma in vivo.Western blot analysis and immunohistochemical assay were performed to detect the expression levels of CD166,CSC-related proteins,p-EGFR and p-ERK1/2.Results 1.The results of xenograft model revealed that silencing CD166 could suppress the growth of xenografts compared with the controls group under nonirradiation.The growth of xenografts in all groups was suppressed after 8Gy Xray exposure.Particularly,the inhibitory effect in CD166-sh RNA group was more obvious(P<0.05).EGF stimulation attenuated the inhibitory effects of CD166-silencing on xenograft growth.2.Western blot analysis demonstrated that downregulation of CD166 decreased the expression of p-EGFR,p-ERK1/2,and CSC-related proteins in xenograft tissues.3.Immunohistochemistry assay unveiled that the expression of p-EGFR,pERK1/2 and CSC-related proteins were decreased in CD166-sh RNA xenograft tissues compared with the control and parental groups.Conclusion CD166 silencing decreased the radioresistance of nasopharyngeal carcinoma and cancer stem cell-like characteristics in vivo.Part 4 Prognostic value of EGFR/p-EGFR in nasopharyngeal carcinoma patients: a meta-analysisObjective To evaluate the prognostic impact of the EGFR pathway molecules and assess their clinical usefulness.A meta-analysis was performed to investigate the correlation between EGFR/p-EGFR expression and prognosis in patients with nasopharyngeal carcinoma.Methods Literatures which were published before November 5,2020 were systematically searched in relevant database,including Pub Med,Web of Science,Embase,China National Knowledge Infrastructure(CNKI),and Wanfang databases.Studies were reviewed according to the inclusion and exclusion criteria,which was made based on the research object,research type,expression of EGFR and prognosis justification.The literature screening,data extraction,and quality assessment were conducted by two independent reviewers.STATA 13 statistical software was used to analyze the pooled hazard ratio(HR)and 95% confidence interval(CI).Heterogeneity of studies was examined by I2.The potential publication bias was assessed using both Egger’s and Begg’s tests.Results A total of twenty literatures with 1545 patients were included for the metaanalysis.Fourteen studies were from the China,three from Republic of Korea,one from Japanese,one from Singapore and one from Sweden.EGFR was used as a prognostic indicator in 19 studies,of which overall survival(OS)was evaluated in seventeen studies,disease-free survival(DFS)was evaluated in eight studies,progression-free survival(PFS)was evaluated in two studies,and distant metastasis-free survival(DMFS)was evaluated in five studies.Whereas p-EGFR was used as a prognostic indicator in three studies,of which OS was used in all studies,DMFS was used in two studies,and PFS was used in only one study.The meta-analysis results suggested that high expression of EGFR was significantly associated with poor OS(HR=1.70,95%CI: 1.24-3.15,P=0.001)and DFS(HR=2.58,95%CI: 1.87-3.56,P=0.000).However,it was not significantly associated with PFS(HR= 1.85,95%CI: 0.90-3.82,P=0.09)and DMFS(HR=1.39,95%CI: 0.73-2.67,P=0.319).Subgroup analyses were based on the source of country(Asia or non-Asia),sample size(N>100 or ≤100),immunohistochemistry(IHC)cut-off value(10% or 25%),histological differentiation(differentiated vs undifferentiated)and TNM stage(I-II vs III-IV).The subgroup analysis indicated that patients with EGFR high-expression in studies of higher TNM stage(III-IV)ratio had significantly poor OS(HR=2.27,95%CI:1.09-4.73,P=0.03),but heterogeneity was existed in studies(I2?=?95.1%,P= 0.000).The prognostic value of EGFR was not significantly associated with the country,sample size,IHC cutoff value,and histological differentiation.Sensitivity analyses revealed that EGFR expression did not significantly affect OS by an individual study solely,indicating there was inherent heterogeneity in OS cohorts.There was no significant heterogeneity among eight studies in the DFS cohorts(I2= 0%,P=0.606).There was significant heterogeneity between EGFR expression and DMFS(I2=82.8%,P=0.000).Sub-group analysis in differentiated carcinoma demonstrated a smaller heterogeneity(I2=33.2%).In addition,p-EGFR highexpression had no significant correlation with OS(HR=1.00,95%CI: 0.88-1.14,P= 0.982)and DMFS(HR=1.21,95%CI: 0.96-1.52,P=0.112).The heterogeneity among p-EGFR and OS studies was small(I2=21%,P=0.26).There was no significant heterogeneity in the DMFS cohorts(I2= 0 %,P=0.497).Conclusion In conclusion,our meta-analysis showed that EGFR high-expression was significantly associated with poor OS and DFS,which may serve as a prognostic predictor for nasopharyngeal cancer.However,p-EGFR expression is not significantly associated with poor prognosis in NPC.