The Role Of TRIP13 In Bladder Cancer

Aims:Bladder cancer(BCa)is one of the most common malignancies in the world and the most expensive malignancy to treat from diagnosis to death.Unfortunately,there have been few advances in its clinical management due to a poor understanding of the correlations between its molecular and clinical features.Thus improved understanding of the molecular mechanisms underlying the tumorigenesis of BCa and its progression is indispensable to identify new targets and develop more effective precious therapy.Thyroid hormone receptor interactor 13(TRIP13)is a crucial regulator of spindle apparatus checkpoint and double-stranded break repair.Recently,its abnormal expression has been found in several human cancers which was associated with disease progression,whereas the role of TRIP13 in the malignant development of bladder cancer(BCa)has not been fully elucidated.Our study aimed to investigate the expression of TRIP13 in BCa and normal bladder tissues and to explore the correlation of TRIP13 expression with clinicopathological parameters and the prognostic effect of TRIP13 expression in BCa.We also evaluated whether the modulation of TRIP13 expression altered proliferation,cell cycle,apoptosis and cell motility.Then we further studied the underlying mechanism by which TRIP13 mediates the tumorigenesis of BCa,which would further provide new novel biomarker and therapeutic target for BCa treatment.Methods:1.The expression level of TRIP13 was measured in a tissue microarray(TMA)consisting of human BCa and paired adjacent normal bladder tissue specimens(n=46 cases).2.We first interrogated the Oncomine database for the expression of TRIP13 in twenty different cancer types.BCa cohorts from the Oncomine and TCGA(The Cancer Genome Atlas)databases were analyzed to further validate the expression of TRIP13 in BCa specimens.3.We evaluated the expression of TRIP13 in 342 paraffin-embedded BCa tissue samples using immunohistochemistry staining.The correlation of TRIP13 expression with clinicopathological parameters in BCa was explored.4.We examined the prognostic effect of TRIP13 expression in the BCa dataset GSE13507 from the Gene Expression Omnibus database.5.In vitro: The mRNA expression of TRIP13 was determined in 6 bladder cancer cells(T24、5637、HT-1197、TCCSUP 、ScaBER and J82)by qRT-PCR.TRIP13-silencing T24 and 5637 cell lines were established using lentiviruses encoding the specific shTRIP13.MTT assay,colony formation assay,flow cytometry analysis,wound healing and transwell migration assay were used to evaluate whether themodulation of TRIP13 expression altered the cells’ proliferation,colony-forming ability,cell cycle and apoptosis,mobility of bladder cancer cells.Western blot was used to evaluate whether TRIP13 was correlated with epithelial-mesenchymal transition in bladder cancer cells.6.In animal model: T24 cells stably transduced with lentiviruses encoding shTRIP13 or shCtrl were subcutaneously inoculated into nude mice to investigate the impact of TRIP13 on the proliferation of bladder cancer cells in vivo.The tumor growth was measured twice a week and then compared between the two groups.Forty days after inoculation,bioluminescent signals of the tumor were measured.Then mice were killed and the tumors were isolated for further analysis.7.To further study the underlying mechanism by which TRIP13 mediates the tumorigenesis of BCa,we performed whole-genome expression microarray in T24/ShCtrl and T24/shTRIP13 cells.Analysis of differentially expressed genes was performed by Ingenuity Pathway Analysis(IPA).To validate the array-based findings in cells,several tumorigenesis relevant genes’ expression were measured.To probe the potential interacting partner of TRIP13,we performed LC-tandem mass spectrometry analysis of Flag-TRIP13-immunoprecipitated complexes which was purified with anti-Flag magnetic beads from T24 cells stably overexpressing Flag-TRIP13,stained and immunoblotted.Based on our mass spectrometry data,we performed co-immunoprecipitation assays to confirm the exact interacting factor of TRIP13.To further determine whether this finding is clinically relevant,we investigated the gene expression data in BCa dataset GSE13507.Results : 1.Immunohistochemistry staining results of tissue microarray indicated that TRIP13 expression was significantly elevated in bladder tumor tissues compared with adjacent normal bladder tissues and TRIP13 expression was high in 10(21.7%)of tumor tissues but only in 1(2.2%)of adjacent normal tissues.2.Analysis of the expression of TRIP13 in twenty different cancer types revealed that TRIP13 was overexpressed in 70 out of 448 analyses(cancer tissue vs.normal tissue;fold change threshold: 2).Analysis of BCa cohorts from the Oncomine and TCGA databases confirmed that TRIP13 expression was escalated in tumor tissues in comparison with normal bladder tissues.3.We did not find a correlation of TRIP13 expression with age,sex or tumor size in BCa patients.Notably,expression of TRIP13 was positively correlated with advanced American Joint Committee on Cancer(AJCC)stage,lymph node metastasis and distant metastasis.4.Kaplan–Meier analysis of GSE13507 implied that patients with elevated TRIP13 mRNA expression exhibited significantly reduced overall survival(OS)(68.0 months vs.96.0 months,P<0.01)and disease-specific survival(DSS)(94.0 months vs.123.9 months,P<0.001).Of note,increased TRIP13 expression predicted poor overall survival in patients with NMIBC(non-muscle-invasive bladder cancer,OS: 78.7 months vs.103 months,P<0.05),but not in patients with MIBC(muscle-invasive bladder cancer,P=0.358).However,the prognostic power of TRIP13 was lost in a multivariate analysis,indicating that TRIP13 may not be an independent prognostic factor in BCa.5.TRIP13 expression was assessed by qRT-PCR in T24、5637、HT-1197、TCCSUP、ScaBER and J82.In TRIP13-silencing T24 and 5637 cells,transcript and protein expression of TRIP13 were consistently knocked down.MTT assay results showed that the depletion of TRIP13 statistically attenuated proliferation in both cells relative to control.Colony-forming ability of T24 and 5637 cells were substantially repressed upon the knockdown of TRIP13 by 29.6% and 59.3% respectively.TRIP13 inhibition also resulted in cell cycle arrest at G2/M phase in both cells and induced cell apoptosis.Wound-healing assay and transwell migration assay validated that silencing TRIP13 obviously impeded the motility of T24 and 5637 cells.Western blot results showed that after the depletion of TRIP13,the expression of the epithelial marker E-cadherin increased,whereas the expression of the mesenchymal markers vimentin and fibronectin decreased.6.In the control group,all injected mice grew tumors in 4 weeks after injection while only 9 out of 10 injected mice with TRIP13-silenced cells had tumors.tumors formed by TRIP13-silenced cells grew significantly slower than the control group at different time points.At the end of the experiment,bioluminescent signals in the TRIP13 knockdown group were lower than those in the control group and the net weight of tumors formed by TRIP13-silenced cells was significantly reduced in comparison with controls.7.We performed whole-genome expression microarray in T24/ShCtrl and T24/shTRIP13 cells.Specifically,Afffymetrix Human Gene Expression Array was used and led to the identification of 1175 differentially expressed genes where 588 were up-regulated and 587 were down-regulated.Gene Ontology enrichment analysis revealed that among the enriched biological processes,mesenchyme development was at the top of the list.And the cellular components involved encompassed the endoplasmic reticulum membrane,nuclear membrane,cytoplasmic microtubule,etc.To validate the array-based findings in cells,several tumorigenesis relevant genes’ expressions were measured.Interestingly,mRNA and protein expression of several key regulators in the EGFR signaling pathway including inhibitor of DNA binding 1(ID1),cyclooxygenase-2(Cox-2),Slug and EGFR were consistently decreased.In addition,pathway analysis showed that among the top 15 statistically significant pathways,seven pathways were associated with the EGFR signaling pathway.LC-tandem mass spectrometry results showed that a total of 2476 proteins were identified in association with TRIP13,including several key regulators in the EGFR signaling pathway like EGFR,JAK2,and AKT1.Based on our mass spectrometry data,we performed co-immunoprecipitation assays to confirm the exact interacting factor of TRIP13.The result indicated thatTRIP13 directly bound to EGFR,but not JAK2 or AKT1.Pearson correlation analysis revealed that TRIP13 was positively correlated with EGFR(P<0.001).Kaplan–Meier analysis demonstrated that patients with high expression for both TRIP13 and EGFR had a significantly shorter survival time(OS: 71.7months;DSS: 95.1 months),while patients with low expression for both TRIP13 and EGFR had a significantly longer survival time(OS: 100.3 months;DSS: 125.4 months).Conclusions : TRIP13 was over-expressed in bladder cancer tissues compared to normal bladder tissues and its expression was closely correlated with advanced AJCC stage,lymph node metastasis and distant metastasis,indicating that TRIP13 may serve as a biomarker to predict the progression and prognosis.In addition,high expression of TRIP13 indicated poor prognosis in BCa patients.Our data also demonstrated that the up-regulation of TRIP13 exerted a crucial role in bladder cancer cell proliferation,cell cycle,apoptosis,and mobility,at least partially through interaction with EFGR,consequently modulating the EGFR signaling cascade.Therefore TRIP13 was identified as a promising biomarker,expanding potential therapeutic options for BCa treatment.