The Tumor-promoting Role Of CDCA5 In Bladder Cancer And Its Mechanism

Bladder cancer(BCa)is the most common urinary malignancy arising from the urinary system in China,which the annual incidence rate and mortality rate are 80.5/100,000 and 32.9/100,000,respectively.Despite the great improvement in the treatment of bladder cancer,its mortality rate still ranks 13th among all malignant tumors.According to the depth of tumor invasion,about 3/4 of BCa are nonmuscle-invasive bladder cancer(NMIBC).One fourth of BCa have already developed into muscle-invasive bladder cancer(MIBC)when first diagnosed.Its progression and metastasis are the two leading causes of BCa-related death.So far,the diagnosis of bladder cancer still lacks of specific molecular markers,and the molecular mechanism of its occurrence and development has not been conclusively determined.Hence,it is necessary to study the mechanism of the development of BCa and determine novel biomarkers for early diagnosus if BCa to improve the prognosis of these patients.Cell division cycle associated 5(CDCA5),a substrate of the anaphase-promoting complex,which could regulate cell cycle.It is reported that CDCA5 is upregulated in several types of cancer.However,the function of CDCA5 in BC remains unclear.At present,MIBC patients with localized disease show a 5-year survival rate of 60%,however,only 10%of MIBC patients with distant metastasis survive past five years.Now the TNM system is most often used to predict the survival of bladder cancer patients,However,even in patients with the same stage of MIBC there might be significant differences in prognosis and survival.Hence,in clinical,it is in great urgent of more convinent and more precise prognostic tools to stratify risk and predictive biomarkers that facilitate selection of patients likely to respond to treatments such as bladder preservation,NAC,radical cystectomy,and adjuvant systemic therapies are essential to advance the field and personalize treatment.Part 1:The expression of CDCA5 in bladder cancer tissuesand cell linesObjectives:Through comparing the different expression of CDCA5 in bladder cancer tissues and their adjacent tissues by qRT-PCR,comparing the expression of CDCA5 in different bladder cancer cell lines by qRT-PCR and Western Blot,we try to illuminate the mechanism of CDCA5 in the development of bladder cancer.We also analysis the expression of CDCA5 in bladder cancer tissuses from TCGA database and Oncomine database,to explore the relation between CDCA5 and tumor stage and patient outcome Methods:1)Quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to detect CDCA5 mRNA expression in BCa tissues and their adjacent normal tissues.And immunohistochemical staining(IHC)was also performed in these BCa tissues to detect the CDCA5 protein expression.qRT-PCR and Western Blot were performed to detect the CDCA5 mRNA expression and protein levels in bladder cancer cell lines(RT4,T24,UMUC3,TCCSUP and 5637)and immortalized uroepithelium cell line(SVHUC)2)According to the information from Oncomine and TCGA database,we explored CDCA5 expression in bladder cancer tissues,and the correlation between CDCA5 expression and prognosis of BCa patientResults:1)The result of qRT-PCR unraveled that mRNA expression of CDCA5 in 20 pairs of BCa tissues is higher compared with their matched adjacent normal tissues,and immunohistochemical staining(IHC)results showed that the protein level of CDCA5 was higher in tumor samples than adjacent normal tissues samely.In addition,qRT-PCR and Western Blot results revealed that expression of CDCA5 was higher in bladder cancer cell lines than immortalized uroepithelium cell line2)Oncomine database and TCGA database revealed that CDCA5 expression is higher in tumor samples than normal samples,and Kaplan-Meier analysis result showed that CDCA5 expression levels correlated with overall survival rates(P=0.0384),patients with high expression had shorter survival time than patients with low CDCA5 expression.Conclusion:1)The mRNA and protein levels of CDCA5 in BCa tissues and BCa cell lines are significantly higher than the matched adjacent normal tissues and normal bladder cells,respectively.2)Tumor T stage is positively related with CDCA5 expression,high CDCA5 expression in BCa patients was associated with poor survival rates,indicating that high CDCA5 expression might be a poor prognostic factor in BCa.Part 2:The function role of CDCA5 in bladder cancer and its mechanismObjectives:We knock-down and overexpress the expression of CDCA5 in different bladder cancer cell lines respectively,to observe the change in cell apoptosis and cell cycle.Then we further studied the molecular pathway of CDCA5 in bladder cancer.Methods:1)We built up stable transfected CDCA5 knockdown(T24,5637)and overexpress(UMUC3)cell lines by short hair RNA(shRNA)transfection and lentivirus overexpression.Cell Counting Kit-8(CCK-8)assay,clone formation assay,flow cytometry analysis of cell apoptosis and cell cycle assays were performed to study the function of CDCA5 in vitro,how it affects BCa cell proliferation,cell cycle and cell apoptosis.2)Western Blot was performed to explore the corresponding mechanism of CDCA5 in BCa cell cycle.3)Western Blot was performed to explore the corresponding mechanism of CDCA5 in BCa proliferation,and looking for potential therapeutic target for BCa.4)Subcutaneous tumor growth experiment was performed to study the function of CDCA5 in the tumor growth in vivo.5)By analyzing DNA methylation data from TCGA database,we further explored the change of DNA methylation level in CDCA5 promoter region.Results:1)CCK-8 and clone formation experiments showed that knockdown of CDCA5 inhibited the viability and proliferation of cancer cells,while overexpression of CDCA5 promoted cell viability and proliferation.2)Cell functional experiments showed that CDCA5 knockdown inhibited the proliferation of bladder cancer cells by inducing G2/M arrest and cell apoptosis;Conversely,overexpression of CDCA5 promoted the G2/M progression and inhibited cell apoptosis.3)Western Blot results showed that CDCA5 promoted cell proliferation by upregulating two key cell cycle factors,cell division cycle protein 2(CDC2)and cyclin B1,and by activating the Akt pathway.Furthermore,CDCA5 regulate cancer cell apoptosis through the mitochondrial apoptosis pathway4)An Akt activator,SC79,could rescue the partial effects of CDCA5 knockdown5)Subcutaneous tumor formation experiments in nude mice showed that the CDCA5 high expression group developed a larger tumor mass than the low expression group according to the average tumor volume and weight.And IHC results showed that CDCA5 high expression group expressed more positive staining for Ki67 and cyclin B1 and less positive staining for cleaved caspase-3,indicating that CDCA5 promotes proliferation and suppresses the apoptosis of BCa cells.However,the CDAC5 knockdown group expressed decreased levels of p-Akt6)By analyzing the CpGs methylation data in the promoter of CDCA5 from TCGA database,we found that the DNA methylation level in bladder cancer tissue is lower than adjacent normal tissue,which reveals that the abnormal expression of CDCA5 in bladder cancer may caused by the aberrant DNA methylation in the promoter regionConclusion:1)CDCA5 can enhance the viability and proliferation capacity of bladder cancer cells,and inhibit tumors apoptosis.In vivo experiments showed that CDCA5 can promote the growth and proliferation of subcutaneous tumors in nude mice2)At the cellular level,CDCA5 induced G2/M phase progression by regulating cell cycle related proteins such as cell division cycle protein 2(CDC2)and cyclin B1 Furthermore,CDCA5 regulated cancer cell apoptosis by regulating the mitochondrial apoptosis pathway related proteins(Caspase3,Caspase9,Bax,Bcl2,Bcl-XL,et.al)3)CDCA5 promoted BCa cell proliferation by activating Akt pathway4)Subcutaneous tumor formation experiment in nude mice revealed that knockdown of CDCD5 in tumor cells inhibited the growth of tumor5)The abnormal expression of CDCA5 in bladder may correlated with the aberrant DNA methylation levels in its promoter regionPart 3:A DNA methylation signature based prognostic model for MIBCObjectives:Using the DNA methylation sequence data(Methylation 450K)from The Cancer Genome Atlas(TCGA),we aim to find the differentially methylated regions between tumor samples and adjacent normal samples,then by using statistics methods and R combine with patients’ clinical data to further build a DNA methylation based signature as a prognostic factor for overall survival(OS)in patients with MIBC.Methods:Using The Cancer Genome Atlas(TCGA),we described a DNA methylation based signature as a prognostic factor for overall survival(OS)in patients with MIBC.By using a least absolute shrinkage and selection operator(LASSO)model,differentially methylated regions were first identified using multiple criteria followed by survival and LASSO analyses to identify DNA methylation probes related to OS and build a classifier to stratify patients with MIBC.This model can calculate each patient’s Risk score(RS),then further stratify each patient as high risk or low risk,which was validated in a held-out testing set from the TCGA MIBC cohort.Finally,receiver operating characteristic(ROC)analysis was used to compare the prognostic accuracy of the models built with RS and other often used clinical features.In addition,we further divided low CDCA5 expression patients and high CDCA5 expression patients into high risk group and low risk group,we compared the overall survival between the subgroups.Results:A DNA methylation signature was generated using DNA methylation array from 413 MIBC patients.We found that our seven-probe classifier,risk score(RS),stratifies patients into high-and low-risk groups with significantly different overall survival(Training set:HR=2.660,95%CI=1.718-4.118,P=0.000011;Testing set:HR=1.966,95%CI=1.099-3.514,P=0.02),and with higher predictive value for overall survival(OS)in the training(n=276)(AUC at 3 years,0.73;AUC at 5 years,0.75)and testing(n=137)(AUC at 3 years,0.65;AUC at 5 years,0.65)sets compared to model composed of only clinicalopathologic features(T stage,N stage,tumor stage).Futhermore,this RS model could further subdivide T2 and T3-T4 patients,NO and N1-N3 patients,stage Ⅰ-Ⅱ and stage Ⅲ-Ⅳ patients into high risk group and low risk group,where there is significant difference(P<0.05)between high risk group and low risk group.By using RS model,CDCA5 low expression group and high expression group can be subdivided into low risk subgroup and high risk subgroup,where the difference between high risk and low risk subgroups is significant(P<0.05).Conclusion:The DNA methylation-based RS can be a useful tool to improve predictive accuracy of bladder cancer patients’ overall survival,which could help clinical doctors to make the best therapy choice for the treatment of MIBC.