Effect Of Aspirin On Prognosis Of Nasopharyngeal Carcinoma And Its Mechanism

Nasopharyngeal carcinoma（NPC）is a common malignancy in southern China.The main cause of the treatment failure is local recurrence and distant metastasis.Therefore,exploring how to monitor and prevent distant metastasis of NPC is urgently desired.Previous studies found that platelet activation upregulated expressions of Twist and Snail to promote epithelial-mesenchymal transition（EMT）in tumor cells and the increasement of circulating tumor cells（CTCs）.In addition,prostaglandin E2 produced by COX-2 was demonstrated to activate EGFR to promote EMT and increase the number of CTCs.In previous studies,it was found that aspirin inhibited platelet aggregation and COX-2 activity.Aspirin could improve the prognosis in patients with various human cancers.However,whether aspirin,a widely used COX inhibitor,can improve the prognosis of NPC and whether it has inhibitory effect on CTCs and EMT remained poorly understood.Therefore,aspirin was employed into this study,to investigate its effects on the EMT process and prognosis of NPC and the underlying mechanism.Part 1.Influence of aspirin on the hematology-related indicators and prognosis in patients with nasopharyngeal carcinomaObjective:To explore whether orally-taken aspirin affects hematology-related indicators and prognosis in patients with nasopharyngeal carcinoma.Methods:A total of 420 patients with NPC treated in the ward of the Nasopharyngeal Cancer Research Institute,People’s Hospital of Guangxi Zhuang Autonomous Region were enrolled and divided into an aspirin group and a non-aspirin（control）group.Absolute lymphocyte count（ALC）,platelet count（PLT）,platelet/lymphocyte ratio（PLR）,neutrophil/lymphocyte ratio（NLR）,fibrinogen（FIB）,T-cell subsets and EBV-DNA were analyzed before and after the treatment.Kaplan-Meier survival curves were applied for single-factor survival analysis.Log-rank tests were used to compare survival curves between the two groups.Results:（1）The PLR and FIB level increased with clinical disease stage（both P<0.05）.Baseline ALC and CD4+CD8+double-positive（DP）T cells were higher in the aspirin group than in the control group（P=0.021,P=0.032）.After treatment,the PLR and NLR were lower in the aspirin group than in the control group（both P<0.05）.（2）Higher PLT and FIB levels were found in the patients with recurrence and metastasis than those without recurrence and metastasis.（3）Distant metastasis rates after treatment in the aspirin group and the control group were 14.5%（11/76）and 25.9%（89/344）,respectively（P<0.05）.（4）Five-year overall survival rate（OS）was 82.08%in the aspirin group and67.40%in the control group（P=0.015）.Five-year OS,DFS and DMFS in aspirin group were superior to the control group（all P<0.05）.Multivariate analysis showed that,taking aspirin,the pretreatment PLR and FIB level were independent risk factors for OS in NPC patients.Conclusions:Aspirin could improve the prognosis of the patients with NPC,and its mechanism may be associated with the decreased PLR and distant metastasis.Part 2.Effect of aspirin on circulating tumor cells of nasopharyngeal carcinoma and the correlation with distant metastasisObjective:To investigate the status of circulating tumor cells（CTCs）in patients with nasopharyngeal carcinoma（NPC）and the effect of orally-taken aspirin on CTCs.Methods:CTCs were detected by CanPatrol^TM CTCs detection technology.From October2014 to November 2015,92 patients with NPC were enrolled into this study,and20 healthy subjects were selected as the control group.Of the 92 NPC patients,18 patients were taking aspirin daily and 74 patients were not.The CTCs were detected before and after the treatments,and the correlations between CTCs and clinical pathological characteristics of patients were analyzed.The effect of orally-taken aspirin on the number and classification of CTCs were also analyzed.Results:（1）CTCs were detected in 80 patients with NPC,and the detection rate was86.96%.The detection rate of epithelial,hybrid and mesenchymal CTCs were55.43%,77.173%and 40.22%,respectively.No CTCs were identified in any of the 20 healthy volunteers.（2）CTCs were found to be associated with N stage,and the number of CTCs in the N₂/N₃ patients were significantly more than that in the N₁ patients.The total number of CTCs and the number of epithelial CTCs were positively correlated with PLT（r=0.205;r=0.244）.（3）The total number of CTCs,hybrid CTCs,mesenchymal CTCs,and non-epithelial CTCs were significantly decreased in the aspirin group after treatment（all P<0.05）,and the ratio of non-epithelial CTCs was also significantly decreased（P<0.05）.（4）As compared with the patients took aspirin and/or received targeted therapy,the mesenchymal CTCs ratio was higher in the patients who did not took aspirin and receive targeted therapy（P<0.05）.（5）At the end of treatment,the incidence of distant metastasis in hybrid CTCs positive group and non-epithelial positive CTCs group were significantly higher than that in the negative group（all P<0.05）.The DMFS rate of hybrid CTCs negative（CTC≤1）group was higher than that of the positive group,and the difference was close to the assumed statistical significance（P=0.051）.Conclusion:Non-epithelial CTCs are associated with distant metastasis in NPC.Orally-taken aspirin might reduce the risk of distant metastases by inhibiting EMT and decreasing the number of CTCs.Part 3.Expression of COX-2,EGFR,E-cadherin and Vimentin in nasopharyngeal carcinoma tissues and their relationship with circulating tumor cellsObjective:To investigate the expression of COX-2,EGFR,E-cadherin and Vimentin in nasopharyngeal carcinoma tissues and their relationship with circulating tumor cells.Methods:The expression of COX-2,EGFR,E-cadherin and Vimentin were detected using immunohistochemical method.Results:（1）The positive expression rates of COX-2,EGFR,E-cadherin and Vimentin in nasopharyngeal carcinoma were 85.3%,92.6%,100.0%and 20.6%respectively.（2）Vimentin expression was associated with lymph node staging（P<0.05）.Cox-2 was positively correlated with EGFR expression（Rs=0.515,P<0.05）.Co-expression of COX-2/EGFR was positively correlated with Vimentin expression（Rs=0.277,P<0.05）（3）The patients with high expression of Vimentin had higher rates of non-epithelial CTCs than those with low expression of Vimentin.（4）The decrease of the CTCs number caused by orally-taken aspirin were more significant in the high-COX-2 group than in the low-COX-2 group.（5）The DMFS rate of Vimentin-positive group was lower than that in the negative group,and the difference was statistically significant（P<0.05）.Conclusion:Aspirin might reduce the occurrence of distant metastases by inhibiting the expression of COX-2 and reducing the ratio of mesenchymal CTCs mediated by Vimentin.Part 4.Effects of COX-2 on nasopharyngeal carcinoma cell migration Objective:To observe the effects of COX-2 on the migration of human nasopharyngeal carcinoma（NPC）cell line HK1 in vitro and preliminarily explore the potential mechanism.Methods:The short tandem repeat（STR）loci was performed in HK1 cells.Targeted knockdown of COX-2 expression in HK1 cells using retrovirus-mediated shRNA interference.COX-2 expression in these cells were detected by Western blot.The effect of COX-2 on the migratory ability of HK1 cells were determined by wound healing and transwell assays.Results:（1）The cell lines used,HK1,were identified as single cell lines of nasopharyngeal carcinoma without any cell-cell cross contamination.（2）After infection with retrovirus-PTGS2-sh RNA,the expression of COX-2protein in HK1 cells was significantly decreased.（3）After the COX-2 expression in HK1 cells was reduced,the migratory ability was verified to be decreased significantly in PTGS2-shRNA HK1 cells.Conclusion:COX-2 might serve as a functional protein that closely corelate with the migratory ability in nasopharyngeal carcinoma cells.