Study The Role Of Abnormal Expression Of Wnt/β-catenin Signaling Pathway Key Molecules In The Nasopharyngeal Carcinogenesis Using High-throughput Tissue Microarray

ObjectivesTo investigate the role of abnormally regulated Wnt/β-catenin signaling pathway in the development and progression of nasopharyngeal carcinogenesis and screen the molecular markers of NPC,by detecting the protein expression of key molecules of the Wnt/β-catenin signaling pathway,such as Wnt5a,β-catenin,c-myc,CyclinD1,E-cadherin,COX-2 and Epstein-Barr virus(EBV) latent membrane protein 1(LMP-1) in nasopharyngeal carcinogenesis tissue with different pathological morphology,Methods①Immunohistochemical S-P method was used to detect the protein expression of Wnt5a,β-catenin,c-myc,CyclinD1,E-cadherin,COX-2 and LMP-1 in high-throughout nasopharyngeal cancer tissue microarrays previously made with different stages in nasopharyngeal carcinogenesis, different histological type,different pathological grades and different clinical stage.②The relation between protein expressions and clinicopathological features in nasopharyngeal carcinoma were analyzed by chi-square test. Spearman's correlation analysis was used to investigate the relationship between the seven protein expressions.Comprehensive evaluation for the effectiveness of the seven protein mentioned above as diagnostic molecular markers for nasopharyngeal carcinoma were done with sensitivity,specificity,positive predictive values,negative predictive values and agreement rate.Calculations were performed using SPSS16.0 statistical software for Windows. Results①We found that positive expression of Wnt5a,c-myc,CyclinD1, COX-2 and LMP-1 proteins were significantly increased in NPC compared with atypically hyperplastic nasopharyngeal epithelium (AHNPE)(P＜0.05),hyperplastic nasopharyngeal epithelium(HNPE) (P＜0.01) and histologically normal nasopharyngeal epithelium(NPE) (P＜0.01);Wnt5a,c-myc,CyclinD1,COX-2 and LMP-1 proteins in AHNPE were also significantly higher than those in the HNPE(P＜0.05) and NPE(P＜0.01);The membranous expression ofβ-catenin in NPC reduced or expression deletion and showed high abnormal protein expression in the of cytoplasm and nucleus,the abnormal positive expression of cytoplasm and nucleus protein in the NPC was significantly higher than that in AHNPE(P＜0.05),HNPE(P＜0.01) and NPE(P＜0.01); E-cadherin shows significantly positive expression in NPE than that it in the AHNPE and NPC(P＜0.05;P＜0.01).②The positive expression of Wnt5a,c-myc and CyclinD1 protein in clinical T3 and T4-staged NPC was significantly higher than that in clinical T1 and T2-staged NPC(P＜0.01,respectively),the positive expression rate of Wnt5a,c-myc and CyclinD1 protein was also significantly higher in NPC with lymph node metastasis than that in NPC without lymph node metastasis(P＜0.01,respectively);the abnormal expression ofβ-catenin protein in clinical T3 and T4-staged NPC was evidently higher than that in T1 and T2-staged NPC(P＜0.05) and the abnormal expression ofβ-catenin protein was also significantly higher in NPC with lymph node metastasis than that in NPC without lymph node metastasis(P＜0.01);the positive expression of E-cadherin protein in clinical T3 and T4-staged NPC was evidently lower than that in T1 and T2-staged NPC(P＜0.05),the positive expression of E-cadherin protein in NPC with lymph node metastasis was evidently lower than that in NPC without lymph node metastasis(P＜0.01);there were no significant correlations between the positive expression of COX-2 protein and pathological type,clinical stage and cervical lymph node metastasis of nasopharyngeal carcinoma(P＞0.05);the expression of LMP-1 protein in clinical T3 and T4-staged NPC was evidently higher than that in T1 and T2-staged NPC(P＜0.05) and the signals of LMP-1 protein expression was significantly increased in non keratinizing carcinoma compared with undifferentiated carcinoma and keratinizing carcinoma(P＜0.05;P＜0.05), there was no significant correlation between the positive expression of LMP-1 and lymph node metastasis of NPC(P＞0.05).③The expression of Wnt5a protein had significantly positive correlations withβ-catenin protein(r=0.354,P＜0.05);the expression ofβ-catenin protein had respectively positive correlations with COX-2, c-myc and CyclinD1 protein expression in NPC(r=0.382,r=0.432, r=0.425,P＜0.01,respectively) and had negative correlation with E-cadherin protein expression in NPC(r=-0.215,P＜0.05).We also found that LMP-1 had respectively positive correlations withβ-catenin, CyclinD1 and c-myc protein expression in NPC(r=0.418,r=0.427, r=0.486,P＜0.01,respectively).④Sensitivity,specificity,positive predictive values,negative predictive values and agreement rate,which were hired to evaluate practical values of molecular marker to distinguish NPC from the non-cancerous NPE.Results indicated that combined molecular phenotype of expression of LMP-1,Wnt5a,β-catenin and c-myc proteine was the best molecular marker to distinguish NPC from the non-cancerous NPE. Conclusions①The activation of proto-oncogene c-myc,CyclinD1 and COX-2, and inaction of E-cadherin,which were mediated by abnormal expression of wnt5a andβ-catenin protein which are key molecules of the Wnt/β-catenin signaling pathway,play important role in the carcinogenesis,invasion,metastasis and development of nasopharyngeal carcinoma.②The high protein expression key molecules of the Wnt/β-catenin signaling pathway,such as wnt5a andβ-catenin,c-myc,CyclinD1, decreased expression of E-cadherin protein were related to malignant biological behavior of N-PC.Wnt5a,β-catenin,c-myc,CyclinD1 and E-cadherin can be used for molecular markers in judging clinical progress, metastasis and prognosis prediction of NPC.③The EBV-encoded latent membrane protein 1(LMP-1) can indirectly caused abnormal expression ofβ-catenin and then activated the downstream effect genes of Wnt/β-catenin signaling pathway such as c-myc,CyclinD1 and COX-2,participating in the development and progression of nasopharyngeal carcinoma;LMP-1 may be interaction enhancement and synergistic effect with c-myc and CyclinD1 in the development and progression of nasopharyngeal carcinogenesis, promoting together or successively causing malignant transformation of the nasopharyngeal epithelial cell and the development and progression of nasopharyngeal carcinoma.④Combined molecular phenotype of expression of LMP-1,Wnt5a,β-catenin and c-myc proteine was the best molecular marker to distinguish NPC from the non-cancerous NPE.