Interaction Of Cyclooxygenase-2 Variants And Smoking In Pancreatic Cancer: A Possible Role Of Nucleophosmin

Background & Aim:Pancreatic cancer is a big health problem and challenge for cancer research due to the increasing incidence and extremely poor prognosis of this lethality disease.The mechanism of pancreatic cancer is largely unknown and the only established etiology is tobacco smoking.Over expression of cyclooxygenase-2(COX-2) is implicated in pancreatic cancer development and cigarette smoking can induce the expression of this enzyme.This study examined the interaction of genetic polymorphisms of the COX-2 promoter and smoking in susceptibility to pancreatic cancer and the functional relevance.Methods:Genotypes and haplotypes of COX-2-765G/C,-1195G/A and-1290A/G were analyzed in 393 pancreatic cancer patients and 786 controls.Odds ratio(OR) and 95%confidence interval(CI) were computed by logistic regression.The function of the -765G/C polymorphism was examined by a set of biochemical assays.Dual luciferase reporter assays were performed to compare the COX-2 promoter activity of different alleles by transient transfection of reporter vectors into human pancreatic cancer cell lines PANC-1,AsPC-1 and human colon cancer cell line HCT-116 with or without the stimulation of cigarette smoke condenses.Electrophoretic mobility shift assay(EMSA) was carried out to examine the differential nuclear proteins binding to the-765G/C site and the proteins were purified and identified by a matrix assisted laser desorption ionisation time-of-flight mass spectrometry(MALDI-TOF-MS) and verified by supershift assay.Chromatin immunoprecipitation assay(CHIP),immunofluorescent staining and confocal imaging,western blot and RNA interference were employed to investigate the regulation of COX-2 expression by the verified nuclear protein.Results:The-1195AA or-765GC genotype carriers had a 1.34-fold(95%CI= 1.12-1.60) or 1.63-fold(95%CI=1.25-2.10) excess risk for developing pancreatic cancer.These two variants showed a cooperative effect in context of haplotype,with the ORs for the A_-1195-C_-765-containing haplotypes being significantly greater than those for the G_-1195-G_-765-containing haplotypes.Smoking also increased pancreatic cancer risk, with the OR being 1.48(95%CI=1.26-1.74).The-765C allele and smoking displayed a multiplicative joint effect,with the OR being 3.72(95%CI=1.70-8.14) for heavy smokers carrying the -765GC genotype.Dual luciferase reporter assays showed that cigarette smoke remarkably increased COX-2 promoter activity and this effect was more pronounced for the -765C allele-containing reporter plasmid compared with the -765G allele-containing counterpart.The differential nuclear protein binding to the -765C allele found in EMSA was identified as nucleophosmin(NPM) by MALDI-TOF-MS and verified by antibody against phosphorylated NPM(p-NPM) in supershift assay.Western blot and immunofluorescence staining analysis revealed that cigarette smoke reduced nuclear NPM levels,especially p-NPM levels,which was reversely associated with COX-2 expression.Knock-down of NPM by RNAi leads to decrease in COX-2 protein expression.ChIP assay with antibody against NPM or p-NPM indicated that cigarette smoke reduced nuclear p-NPM levels,which was reversely associated with COX-2 expression by reducing the p-NPM binding to the -765 site of COX-2 promoter, suggesting that the -765G to C change creates a binding site for p-NPM,which acts as a transcriptional inhibitor.Conclusion:Functional COX-2 polymorphisms are associated with susceptibility to pancreatic cancer and tobacco smoke specifically increases -765C promoter activity, which might be mediated by p-NPM.