The Mechanism And Prognostic Significance Of WDR5 In Cholangiocarcinoma

BackgroundCholangiocarcinoma(CCA)is a type of highly aggressive malignancy originating from the biliary system with a rising incidence worldwide.The prognosis of CCA is extremely poor due to the difficulties of early diagnosis and the resistance to chemotherapy and radiotherapy.According to the anatomical location,CCA is further classified as three subtypes including intrahepatic(iCCA),perihilar(pCCA)and distal(dCCA)CCA.pCCA is the most prevalent type of CCA accounting for about 50%-60%of total CCA cases,while dCCA and iCCA accounted for 20%-30%and 10%,respectively.Surgical resection is the only curative option for CCA patients.Unfortunately,most patients are present in an advanced stage and lose the surgical opportunity.The 5-year overall survival rate(OS)of CCA is very unsatisfactory,remaining less than 30%after radical resection.The dismal outcome of CCA can be partially attributed to the insensitivity to systemic treatment such as chemotherapy.Moreover,there are very few well-accepted target drugs of CCA,and the effects of most current targeted drugs are unsatisfactory.Therefore,new biomarkers and targeted therapies are urgently needed to ameliorate the survival of CCA patients.Unfortunately,the discovery of CCA effective biomarkers to guide precise treatment or predict promising drug targets remains stagnant compared with that of common cancer types such as lung cancer.Many reasons lead to this result including the low radical surgical rate,difficulty of specimen obtainment and cohort establishment.Emerging evidence indicated that each CCA subtype has not only different morbidities,clinical characteristics,treatment strategies and prognoses,but also molecular features and biological behaviors,so these three CCA subtypes should be investigated separately,including the biomarker studies.However,this issue did not get enough attention before that the 7th AJCC/UICC classification just defined CCA into iCCA,pCCA and dCCA at 2007.WDR5 is one of the most famous proteins in WD40 protein family.WDR,peptide chain contains seven WD repeats,which form seven β-folds and constitute the main functional domain of WDR5.This structure is critical for the binding and recognition with other proteins including MLL1,c-Myc.Currently,most of the researches on WDR5 are focused on the biological effects of histone methyltransferase complex MLL1 and SET1 in the field of epigenetics.WDR5 protein has no catalytic activity.It could recognize the WIN sequence of MLL1 and formed the MWRAD(MLL1,WDR5,RBBP5,ASH2L and DYP-30)core catalytic complex,which catalyzes H3K4 trimethylation and thus activates gene transcription and affects epigenetics.Recent studies revealed that WDR5 enhances c-Myc recruitment to the chromosomal locations via direct interaction with c-Myc.WDR5 is responsible for Myc-induced gene transcription by binding a conserved motif of c-Myc,which is identified as"Myc box Ⅲb(MBⅢB)"(residues 258-267 of human c-Myc).This interaction of c-Myc and WDR5 creates an avidity-based chromatin recognition mechanism,making c-Myc able to select its target genes in response to different up-stream stimulators.WDR5 has been proved to be over-expressed in colorectal cancer,gastric cancer,bladder cancer and other malignant tumors and promote the proliferation and invasion of tumor cells.However,there is still a lack of systematic study in cholangiocarcinoma.Objective1.Based on the TCGA database,we analyzed the differential expression of WDR5 in 36 CCA tissues and 9 normal bile duct epithelial tissues and showed the potential value of WDR5 as a biomarker in CCA.2.The mRNA expression of WDR5 in different subtypes of CCA was further determined in fresh tissues by qRT-PCR.3.Immunohistochemical(IHC)staining was performed on the CCA tissue microarray(TMA).IHC score was used to compare the expression of WDR5 in different subtypes of CCA tissues and para-tumor tissues.Furthermore,combined with the postoperative follow-up of CCA patients,the relationship between WDR5 expression and the prognosis of CCA patients was determined.4.The effects of WDR5 on the proliferation,migration,invasion,metastasis and epithelial-mesenchymal transition(EMT)of CCA cells were detected using molecular biological techniques such as qPCR and Western blot and the in vivo and in vitro experiments.Methods1.With the help of GEPIA,a visualized website of TCGA database,WDR5 mRNA expression of 36 CCA tissues and 9 normal bile duct epithelial tissues in TCGA database were calculated and the statistical differences were analyzed by One Way ANOVA.2.We collected 18 pairs of iCCA,16 pairs of pCCA and 14 pairs of dCCA tissues as well as their para-tumor tissues,and further determined the mRNA expression of WDR5 in different subtypes of cholangiocarcinoma and para-tumor tissues by means of qRT-PCR.3.We collected 336 paraffin tissue specimens from patients who underwent radical cholangiocarcinoma surgery in Qilu Hospital of Shandong University from January 2012 to December 2018,including 78 cases of iCCA,141 cases of pCCA,88 cases of dCCA,and 12 cases of iCCA para-tumor tissues,17 cases of pCCA para-tumor tissues.The TMA was made and IHC staining was performed.The expression of WDR5 in TMA was evaluated by Quant Center software.4.The protein expression of WDR5 were compared in different subtypes of cholangiocarcinoma or in tumor tissues and para-tumor tissues.5.We used the ROC curve(receiver operating characteristic curve)to calculate the cut-off values of iCCA,pCCA and dCCA cohorts respectively,and then divided the patients into the group with low WDR5 expression and the group with high WDR5 expression.Postoperative survival of the patients was investigated by telephone follow-up and clinicopathological data including age,gender and TNM stage were gathered.Univariate analysis with Kaplan-Meier method and long-rank test was first performed to analyze the prognostic value of WDR5 and other clinicopathological factors.Multivariate analysis was also performed to identify the independent prognostic factors using Cox regression model.6.WDR5 was knocked down or overexpressed in CCA cells using lentiviral vector.The effects of WDR5 on the proliferation,migration and invasion ability of CCA cells were detected by CCK8,wound-healing assay and transwell assay.The expression of EMT-specific proteins were detected by western blot.7.The mice model was established to investigate the effect of WDR5 on CCA metastasis.Results1.By analyzing the TCGA database,we found that the expression of WDR5 was significantly increased in CCA tissues.2.qRT-PCR results confirmed that the mRNA expression of WDR5 in iCCA,pCCA and dCCA tissues was significantly higher than that in the corresponding para-tumor tissues.3.The score of IHC staining showed that WDR5 was expressed in all sub-types of CCA and the highest expression was found in dCCA.The expression of WDR5 in iCCA and pCCA tissues was significantly higher than that in the corresponding para-tumor tissues.4.The Cut-off value of iCCA,pCCA and dCCA was calculated as 139.7,120.2 and 127.9 respectively by the ROC curve.5.Univariate analysis showed that high expression of WDR5 was significantly correlated with poor outcome of iCCA,pCCA,and dCCA.All factors with p ≤0.10 in univariate analysis were enrolled into multivariate analysis,except TNM stage because of its natural interaction with T,N or M stage.We found that WDR5 was an independent prognostic factor in extrahepatic cholangiocarcinoma(pCCA and dCCA)rather than in iCCA.Patients with pCCA and dCCA had 1.67 and 2.01 fold higher risk of cancer-caused death than those with lower WDR5.6.Overexpression of WDR5 promoted the migration and invasion of CCA cells and induced the EMT,while knockdown of WDR5 resulted in the opposite result.7.WDR5 knockdown inhibited the liver and lung metastasis of CCA cells in the mice model.Conclusion1.The expression of WDR5 in CCA tissues was significantly higher than that in para-tumor tissues.2.High expression of WDR5 was significantly correlated with poor prognosis of all subtypes of CCA.WDR5 was an independent risk factor which resulted in the unsatisfactory prognosis of extrahepatic cholangiocarcinoma(pCCA and dCCA)patients.3.WDR5 promoted the migration,invasion and metastasis of CCA cells and induce EMT,but it had no effect on the proliferation.