The Correlation And Preliminary Mechanism Study Between Cyclooygenase-2 And CD8~+T Cell Infiltration In Nasopharyngeal Carcinoma

PurposeIn nasopharyngeal carcinoma(NPC),the infiltration of CD8~+Tumor infiltrating lymphocytes(TILs)correlates positively with the prognosis,while the cyclooxygenase-2(COX-2)is opposite,which is overexpressed and relates to the poor prognosis.In recent years,studies have found that COX-2 is not only closely associated with the occurrence and development of tumors,but also participates in the formation of suppressive immune microenvironment,thereby mediating immune escape.The current studies have discovered COX-2 negatively correlated to the infiltration of CD8~+TILs in other solid tumors,yet this correlation isn’t reported in NPC.Therefore,this study will be to explore the influence of the COX-2 expression on CD8~+TILs infiltration and the mechanism in NPC.Materials and MethodThe expression of COX-2 in NPC and head and neck squamous carcinoma(HNSCC)was analyzed on Ocomine Data Set and TIMER software.The correlation of COX-2 and the infiltration of immune cells in HNSCC was also explored on TIMER.The COX-2 expression and CD8~+TILs infiltration in 236 NPC patients diagnosed in 2011 without metastasis were detected by using immunohistochemistry and their correlation were analyzed.The prognostic values were calculated by using Kaplan–Meier method and Cox multivariate regression analysis.The COX-2 overexpression and knock-down/interference cells were constructed by using lentiviral infections or RNA interference in the NPC cell line CNE-2(of low COX-2 expression)and C666-1(of COX-2 high expression).The microarray was applied to detect the differential secretions of chemokines in the cell cultural supernatant,and ELISA and q PCR were employed to verify the difference.RNA-seq was adopted to explore the alterations of signal pathways,and the molecules changes were validated by q PCR.ResultsThe Oncomine Data Set shows the COX-2 is elevated in NPC,compared with normal controls(P<0.001).Searching on the TIMER finds that the expression of COX-2 is higher than normal tissues in HNSCC(P<0.001)and negatively correlates with CD8~+TILs infiltration(r=-0.139,P=0.0024).The elevated COX-2 is detected in NPC tissues via IHC,and of negative correlation with the tissue infiltration of CD8~+TILs(r=-0.233,P=0.003).Through the Kaplan-Meier analysis,the overall survival(OS)(73.9%vs.82.9%,P=0.12)and distant metastasis-free survival(DMFS)(73.8%vs.86.9%,P=0.014)for patients with high COX-2expression are worse than those with low COX-2 expression,but RFS(89.0%vs.91.9%,P=0.49)is not different.Otherwise,the OS(84.4%vs.72.1%,P=0.028),DMFS(87.6%vs.74.1%,P=0.025)and RFS(96.3%vs.84.3%,P=0.0031)for patients with high CD8~+TILs infiltration are better than those with low CD8~+TILs infiltration.The Cox multivariate regression analysis validates both COX-2(HR=2.041,95%CI:1.073-3.883,P=0.03)and CD8~+TILs(HR=0.518,95%CI:0.271-0.992,P=0.047)are independent prognostic factors for DMFS,and CD8~+TILs(HR=0.222,95%CI:0.074-0.884,P=0.007)is also the independent prognostic factors for RFS in NPC.The microarray finds that the secretions of CXCL9 and CXCL10 are both down-regulated in the cell cultural supernatant of CNE-2-COX-2oe(of COX-2overexpression)and C666-1-COX-2-RNAi(of COX-2 knock-down).These alterations are coincident with the results of ELISA and q PCR.The changes of CXCL9/10 following the opposite regulation of COX-2 expression are not consistent,especially the changes are alien to the expectation in C666-1 of down-regulated COX-2.The RNA-seq reveals that the CXCL10 is apparently down regulated when the COX-2 is interfered in C666-1 and the typeⅠinterferon signaling pathway and cellular response to typeⅠinterferon pathways are down-regulation via GO enrichment analysis.The q PCR shows the down-regulated alterations of IFNα1/α2,which promote the secretion of CXCL9/10.ConclusionThe COX-2 is over-expressed in NPC and negatively correlates with the infiltration of CD8~+TILs.Both COX-2 and CD8~+TILs are independent prognostic factors for DMFS,but only CD8~+TILs is the independent prognostic factor for RFS in NPC.The up-regulated COX-2 in CNE-2 of negative EBV suppresses the secretion of CXCL9/10,while the COX-2 may improve CXCL9/10 secretion in C666-1 with naturally positive EBV through the impact of typeⅠinterferon pathway.