The Roles Of KDM6A And PDW In Bladder Cancer

Part ? The FOXA1-KDM6A-ARHGDIB axis suppresses cell migration and invasion in human bladder cancerBackgroundBladder cancer(BCa)is the tenth most common malignancy worldwide,with approximately 549,000 new cases diagnosed and 200,000 deaths in 2018.Of all BCa cases,nearly 75%of patients are non-muscle-invasive type(NMIBC;stage Ta or T1)and have a favorable prognosis,with a 5-year overall survival rate reaching 90%.The remaining 25%are muscle-invasive(MIBC:stages T2 to T4)and have a 5-year survival rate decreasing to 60%,even worse for patients with positive lymph nodes or distant metastasis.The main cause for stage-dependent survival reduction is the presence of micrometastases when cystectomy is performed.Therefore,a new diagnostic and therapeutic target for early metastatic BCa is urgently needed.Epigenetics,participating in the regulation of gene expression via modification of DNA or chromatin proteins without DNA sequence alteration,is tightly involved in tumorigenesis,metastasis and recurrence of BCa.Notably,whole-exome sequencing of the genomic DNA identified genetic aberrations of the chromatin remodeling genes were highly prevalent in BCa,suggesting that epigenetic dysregulation is an important feature of BCa.Among all chromatin remodeling genes,KDM6A is the second most common mutant cancer-related gene in BCa.KDM6A,also known as UTX,containing a JmjC domain,is a histone demethylase which specifically demethylates di-and trimethylated histone 3 lysine 27(H3K27me2/3)and antagonizes EZH2-mediated transcriptional repression.Besides,KDM6A could promote H3K27 acetylation in a demethylase-independent pattern by recruiting the histone acetyltransferase p300.It is well established that KDM6A is involved in various biological processes and human diseases.Loss-of-function mutations in KDM6A were associated with a specific hereditary disease-Kabuki syndrome.Remarkably,it was reported that 6 Kabuki patients developed different types of cancer,indicating cancer susceptibility for Kabuki patients.Besides,as a tumor suppressor in most malignancies,inactivating mutations in KDM6A were widely observed in human cancers.In colon cancer,KDM6A inhibits cell migration and invasion by increasing E-cadherin expression via coordinate regulation of H3K27 demethylation and acetylation.In clear cell renal cell carcinoma,low expression of KDM6A was related with reduced overall survival and disease free survival.In T-cell acute lymphoblastic leukemia(T-ALL),KDM6A escapes X chromosome inactivation in female T-ALL lymphoblasts and KDM6A mutations only occurs in male patients,who exhibits sensitivity to EZH2 inhibitor.Interestingly,it is controversial about the role of KDM6A in breast cancer.Notably,KDM6A have the highest mutation rate up to nearly 30%in BCa and KDM6A inactivation enhances PRC2-mediated transcriptional repression and can be targeted by EZH2 inhibitor in BCa.However,the effect of KDM6A on migration and metastasis of BCa and the underlying mechanism are not fully clear.Here we demonstrate that KDM6A suppresses the migration and invasion of BCa cells through transcription activation of ARHGDIB in a demethylase-dependent manner.Additionally,we observe that FOXA1 transcriptionally upregulates KDM6A and acts synergistically as a tumor suppressor in BCa.MethodsRT-qPCR and western blot were performed to detect the expression of KDM6A in BCa cells.Immunohistochemistry(IHC)staining were used to investigate the expression of KDM6A in BCa tissues.Overexpression and knockdown assays were carried out to explore the effects of KDM6A on BCa cells.MTT and colony formation assays were performed to detect cell viability.Wound healing and transwell assays were used to investigate cell mobility.RNA-seq were carried out to explore the downstream mechanism of KDM6A.Dual-luciferase reporter assays and chromatin immunoprecipitation(CHIP)were performed to determine the transcription factor regulating KDM6A.Xenograft tumor models were used to explore the effect of KDM6A on BCa cell invasion and metastasis in vivo.ResultsExpression of KDM6A in BCa cell lines and tissuesThe protein level of KDM6A in SV-HUC-1 cells was much higher than those in BCa cells.Besides,the results of IHC on tissue microarray showed high levels of KDM6A is associated with favorable prognosis.However,no significant correlations were found between KDM6A expression level with tumor size,myometrial invasion,lymph node metastasis and TNM stages.KDM6A influences BCa proliferation in a cell type specific mannerStable KDM6A overexpressed RT4,T24 and 5637 cells were established.The results of MTT and colony formation assays showed that overexpression of KDM6A dramatically inhibited cell proliferation in RT4 cells.However,KDM6A overexpressed T24 and 5637 cells showed no significant difference in cell proliferation.Knockdown of KDM6A did not exhibit any effect on proliferation of T24 and 5637 cells,indicating that the role of KDM6A in cell proliferation varies in different BCa cells.KDM6A inhibits BCa migration and invasion in vitro and in vivoThe results of wound-healing assay and transwell assay showed that overexpression of KDM6A significantly suppressed cancer migration and invasion in both T24 and 5637 cells.Consistently,knockdown of KDM6A demonstrated facilitative effect on cell migration and invasion in both BCa cells.Moreover,KDM6A overexpressed T24 cells developed fewer lung metastases in nude mice than control cells,while KDM6A knockdown resulted in a significant increase in lung metastases.Identification of ARHGDIB as a target of KDM6AAs the results of RNA-seq shown,overexpression of KDM6A led to upregulated expression of 1055 genes and downregulation of 834 genes,while knockdown of KDM6A resulted in upregulation of 1285 genes and downregulation of 715 genes.To validate the data of the transcriptome profiling,we performed RT-qPCR on a separate set of RNA samples and confirmed the majority of target genes determined by RNA-Seq analysis.According to the results of RNA sequencing,ARHGDIB was one of the most significantly altered genes(38.3-fold increased expression in KDM6A group,14.7-fold decreased expression in shKDM6A group).We examine the expression of ARHGDIB in a separate set of samples by RT-qPCR and western blot,and the results showed that overexpression of KDM6A significantly increased both the mRNA and protein expression levels of ARHGDIB in T24 and 5637 cells,while knockdown of KDM6A decreased ARHGDIB levels.Moreover,the IHC results indicated the levels of KDM6A and ARHGDIB had high consistency.ARHGDIB was a downstream effector of KDM6A for suppressing BCa migration and invasionWe downregulated ARHGDIB expression in BCa cells using siRNA.Knockdown of ARHGDIB enhanced migration and invasion of T24 cells.Furthermore,the inhibition of KDM6A on BCa migration and invasion was counteracted by knockdown of ARHGDIB in T24 cells.Kaplan-Meier analysis showed high expression ARHGDIB were respectively associated with prolonged overall survival time,suggesting ARHGDIB is an independent prognostic factor in BCa/KDM6A transcriptionally promotes ARHGDIB expression in a demethylase-dependent mannerConsidering KDM6A as a gene expression activator demethylating H3k27me3,we first examine the H3K27me3 levels in KDM6A overexpression and knockdown BCa cells.The results showed KDM6A overexpression decreased prominently H3K27me3 levels in both T24 and 5637 cells,while KDM6A knockdown exhibited stimulative effect on H3K27me3 expression.Then,we introduced a catalytically dead KDM6A mutant(H1146A,E 1148A)into T24 cells.The results manifested that T24 cells overexpressing mutant KDM6A exhibited comparable ARHGDIB mRNA and protein levels compared with control cells,which were much lower than those of cells overexpressing wildtype KDM6A.Moreover,GSKJ4,a KDM6A demethylase inhibitor,could decrease ARHGDIB expression levels in wildtype T24 cells.Notably,EZH2 knockdown in T24 cells led to increased expression of ARHGDIB.Next,we sought to verify the ARHGDIB regulation mechanism in vivo.After tail vein injection with KDM6A knockdown T24 cells and control cells,the mice were administered EZH2 inhibitor-GSK126 or 20%SBE-?-CD by intraperitoneal injection.The results showed GSK126 counteracted the increased lung metastasis caused by KDM6A knockdown.To further elucidate the epigenetic regulatory mediated by KDM6A,we performed CHIP using wildtype T24 cells and found three common binding sites of KDM6A and H3K27me3 at ARHGDIB promoter.CHIP-qPCR assays showed KDM6A exhibited increased enrichment levels at ARHGDIB promoter region in KDM6A overexpressed T24 cells compared with control cells,while the enrichment levels of H3K27me3 decreased.Thus,these results indicated that KDM6A modulates ARHGDIB expression in a demethylase-dependent manner.FOXA1 transcriptionally enhances KDM6A expression in BCaThe dual-luciferase reporter assay showed that the KDM6A promoter region approximately between-127bp and-71 bp is critical for KDM6A transcription regulation.Next,we analyzed the core promoter region with JASPAR program(http://jaspar.genereg.net/).Four transcription factors(FOXAl,USF1,USF2,TFE3)were predicted to potentially bind with this region.To explore their effects in regulating KDM6A transcription,these four transcription factors were separately overexpressed in T24 cells.The luciferase reporter assay indicated the promoter activity from-127bp to+47bp(construct P5)increased prominently in FOXA1 overexpressed T24 cells.To verify the putative FOXA1 binding site,we introduced two point mutations into construct P5.The results showed that the elevated luciferase activity by FOXA1 overexpression was completely abolished in mutant P5 group.Furthermore,CHIP assay confirmed that FOXA1 could bind directly to the promoter of KDM6A.To explore the effect of FOXA1 on KDM6A expression,we knocked down FOXA1 with siRNAs in T24 cells.RT-qPCR and western blot assays demonstrated that FOXA1 knockdown significantly decreased KDM6A and ARHGDIB expression levels.Similar results were obtained in 5637 cellsFOXA1 suppresses migration and invasion of BCa cells through promotion of KDM6A and ARHGD1B expressionTo determine the effect of FOXA1 on BCa migration and invasion,we knocked down FOXA1 expression in wildtype T24 cells,wound healing and transwell assays were performed.The results showed that transient knockdown of FOXA1 enhanced cell migration and invasion in T24 cells.To explore whether KDM6A is essential for FOXA1 regulating cell mobility,we transfected FOXA1 siRNA into KDM6A overexpressed T24 cells and control cells.The increased migration and invasion ability caused by FOXA1 knockdown was nearly neutralized in KDM6A overexpressed cells.Moreover,overexpression of ARHGDIB could also abate the enhanced effect of FOXA1 knockdown on T24 migration.ConclusionsHere we demonstrated that KDM6A inhibited migration and invasion of BCa cells in vitro and in vivo,revealing that KDM6A is a key role in regulating BCa mobility.By RNA-sequencing we identified ARHGDIB as a novel downstream gene which is responsible for the anti-tumor effect of KDM6A in BCa.CHIP assays showed that KDM6A promoted ARHGDIB expression through demethylating H3K27me3 at its promoter region.Furthermore,We found FOXA1,acting as a transcription factor,upregulated KDM6A expression and acted synergistically as a tumor suppressor in BCa.These findings reveal that The FOXA1-KDM6A-ARHGDIB signaling axis plays important role in BCa metastasis,allowing for the identification of more aggressive BCa and providing a better definition of treatment strategies.Part II Evaluation of platelet distribution width(PDW)as a diagnostic and prognostic biomarker in bladder neoplasmBackgroundBladder cancer is the tenth most common cancer and fourteenth leading cause of cancer-related death worldwide.Most of the bladder cancer cases are urothelial type and pathologically divided into non-muscle-invasive bladder cancer(NMIBC;stage Ta or T1),and muscle-invasive bladder cancer(MIBC;stages T2 to T4),accounting for 75%and 25%of the newly diagnosed bladder cancer patients separately.Despite progression in surgery,there is still a 60%-70%recurrence rate in NMIBC patients and 20%of NMIBC will undergo progression to MIBC disease.Due to high recurrence rate,frequent cystoscopic surveillance and reoperation after recurrence is required,making it the most expensive malignancy to treat.Ultrasonography,cystoscopy,and cytology are routinely used diagnostic methods of bladder cancer currently,but they are limited due to complicated procedures,expensive for long-term and frequent surveillance.The inverted urothelial papilloma of bladder is generally categorized histologically and clinically as a benign tumor,accounting for 1-2%of all bladder neoplasms.Simple and non-invasive methods to distinguish urothelial papilloma from bladder cancer,and indicators of tumor malignancy of bladder cancer are still warranted.Platelets play a pivotal role in cancer progression and metastasis.The parameters of platelets are known as platelet indices,which mainly include platelet count(PLT),mean platelet volume(MPV),platelet distribution width(PDW),and plateletcrit(PCT)The MPV reflects the average platelet volume and is considered as a surrogate marker of platelet activation.PDW represents variation in platelet size,and PCT is an indicator of the platelet mass in a unit of volume,which is determined by platelet count and MPV.Several studies have identified aberrations in platelet indices in various cancer types including gastric cancer,lung cancer,colon cancer and breast cancer.Thus,platelet indices hold great importance as diagnostic values in cancer management.However,whether platelet indices were associated with bladder cancer and their diagnostic values in discriminating malignancy and tumor stages have not been put forwarded.MethodsPatients' characteristicsA total of 210 bladder cancer patients were enrolled in this study.A total of 76 patients with urothelial papilloma,and 132 healthy sex-and age-matched healthy volunteers were enrolled as controls.All patients were diagnosed and received surgical intervention as primary treatment at the Department of Urology,Qilu Hospital,Shandong University,between January 2016 and June 2018.The clinicopathological data of all bladder cancer patients,including age,sex,smoking history,blood examination results,and tumor characteristics were obtained.Statistical analysisThe levels of platelet count,MPV,PDW and PCT of bladder cancer patients,urothelial papilloma patients and healthy controls were analyzed using Kruskal Wallis test with multiple comparisons as determined by Nemenyi test.The continuous variables were compared using Student's t-test or Mann-Whitney U,and categorical variables were compared using the Chi-square test.Receiver-operating characteristic(ROC)curves were used to evaluate the diagnostic role of PDW.The diagnostic accuracy of the ROC curve was determined by the area under the curve(AUC).The Youden index was used to determine the optimal cut-off value of PDW.ResultsEvaluation of platelet indices in bladder neoplasm patientsThe levels of MPV and PCT in bladder cancer patients(MPV:P<0.001;PCT:P<0.001)and urothelial papilloma patients(MPV:P<0.001;PCT:P<0.01)were considerably higher than that in healthy controls,whereas the PDW in bladder cancer patients and urothelial papilloma patients was lower when compared to healthy controls(P<0.001).Moreover,the PDW levels were lower in bladder cancer patients when compared to urothelial papilloma patients(P<0.05).However,no significant differences regarding platelet count were observed among healthy controls,urothelial papilloma patients and bladder cancer patients enrolled in our study.PDW is a potential diagnostic indicator of tumor malignancy in bladder neoplasmPDW,as assessed using ROC analysis,assists in distinguishing bladder cancer from urothelial papilloma,MIBC from NMIBC,and high-grade bladder cancer from low-grade bladder cancer.The AUC regarding the prediction of bladder cancer,MIBC,and high-grade bladder cancer were 0.583(95%CI:0.507,0.659;P=0.033),0.612(95%CI:0.534,0.690;P=0.006),and 0.662(95%CI:0.588,0.736;P<0.001).The cutoff values of PDW for evaluating bladder cancer,MIBC,and high-grade bladder cancer were 11.25(sensitivity 71.1%,specificity 42.4%),11.95(sensitivity 51.6%,specificity 65.5%),and 11.95(sensitivity 59.6%,specificity 66.1%)respectively,according to the Youden index.The relationship between PDW and clinicopathological characteristics in bladder cancer patientsThe levels of PDW were significantly lower in patients with more advanced bladder cancer.Specifically,decreased PDW levels were observed in bladder cancer patients with higher platelet counts(>223×109/L,P<0.001),greater tumor size(>3cm,P=0.029),high tumor grade(P<0.001),more advanced TNM stages(P=0.018),MIBC(P=0.003),and higher G classification(P=0.022).Further grouping analysis according to TNM stages revealed that the PDW levels were associated with primary tumor stages(pT stages,P=0.011)and lymph node stages(pN stages,P=0.074).Furthermore,PDW level was lower in MIBC patients with lymph node metastasis compared with patients in NO stage,though the difference was not significant(pN stages in MI,P=0.125).No significant correlations were observed between PDW levels and age,gender,smoking history,WBC count,and multiplicity of tumor growth.According to the cutoff values of PDW in evaluating muscle invasiveness and tumor grade,bladder cancer patients were divided into two groups.Of the total 210 bladder cancer patients,116 patients(55.2%)had PDW<11.95,while 94 patients(44.8%)had PDW>11.95.Tumor size(P=0.042),tumor grade(P<0.001),TNM stages(P=0.005),pT stages(P<0.004),pN stages(P=0.044),muscle invasiveness(P=0.015),and G classification(P=0.004)showed significant differences between the two groups,whereas no significant differences in age,gender and smoking history were observed between these two groups of patients.Meanwhile,patients with PDW<11.95 showed considerably lower MPV levels(P<0.001),as well as higher platelet count(P=0.002),compared to patients with PDW>11.95.ConclusionsReduced preoperative PDW level is an indicator of malignancy and advanced bladder cancer stages,suggesting it as a potential biomarker in bladder cancer diagnosis.