The Role Of SNF5 Downregulation In Progression Of Bladder Cancer

BackgroundThe global cancer statistics shows that bladder cancer(BC)causes an estimated 430,000 new cases and 170,000 deaths each year.Now BC becomes the primary disease in urology department and the most common urinary tumor among men in China.Epidemiological studies have shown that the number of young BC patients is increasing,resulting in heavy burden for the society.BC can be divided into non-muscle invasive bladder cancer(NMIBC)and muscle invasive bladder cancer(MIBC),according to depth of myometrial invasion.The former is characterized and the high risk for recurrence which requires long-term follow-up,but NMIBC has a favorable prognosis.MIBC is prone to metastasis and confers a poor outcome.The 2-year mortality of untreated or mistreated BC patients is 85%.Surgical resection remains the primary treatment for BC.Recently,immune checkpoint blockade has been successfully used in a variety of cancers and transformed conventional cancer treatment.Therefore,immunotherapy has become a milestone in the therapy of various malignancies.Despite advances in treatment of cancer,the 5-year survival rate of BC has not yet improved over the past three decades.On the one hand,high heterogeneity of BC leads to different prognoses even among the BC patients with the same stage.Secondly,patients with NMIBC are required long-term follow-up due to a high incidence of recurrence.Long-term tracking disease activity and progression accurately is necessary for a better understanding of disease progression in NMIBC.However,the follow-up causes heavy financial burden and influences the life quality of the patients.Hence,the long duration also contributes to poor adherence.It is reported that less than 0.02% of high-grade NMIBC patients receive standard treatment.Moreover,the staging of BC depends on the sample quality of surgical resection.However,it is difficult for pathologists to distinguish T1 from T2 stages,which has great implications for therapy for patients.Therefore,it is urgent to investigate the mechanism of BC initiation and development,and search robust biomarkers and potential therapeutic targets for early detection and proper treatment.Uncontrolled cell proliferation and metastasis are the main features in cancer.DNA replication,transcription,repair and recombination are crucial for cell proliferation.Chromatin remodeling complexes alter chromatin structure by changing position and local density of nucleosomes in all living creatures.The yeast switch in mating type/sucrose non fermentation(SWI/SNF)complex was first discovered and isolated from yeast,and is highly conserved across the eukaryotic kingdom.SWI/SNF complex consists of 29 subunits.SNF5,the core subunit of the SWI/SNF complex,caused widespread concern for frequently inactivation by biallelic mutations in 95% of Atypical Teratoid and Rhabdoid Tumors(AT/RTs).Previous studies demonstrated that the interaction between SNF5 and c-MYC has been involved in cell proliferation and cell cycle.As for cancer metastasis,upregulated Rho A induced by SNF5 deficiency has been reported to alter cell motility and modify actin stress fibers in AT/RTs.In addition,in myeloid leukemia,SNF5 downregulation induces Rac GTPase activation,thereby promoting cell migration.Up to date,the role of SNF5 in BC is still unclear.ObjectiveThe aim of this study was to explore SNF5 expression pattern and the mechanism of SNF5 in the progression of BC.This work gave a deep insight into the role of SNF5 in BC biological characteristics and searched the new potential biomarkers and therapeutic targets,which may provide a basis for individualized treatment.Methods1.TCGA database and GEO database(array data from GSE13507 and GSE121711)were used to analyze the m RNA level of SNF5 in BC samples and normal tissues.TCGA-BLCA dataset was utilized to determine the survival differences.The BC patients were divided into high expression group and low expression group by using survival and survminer R packages.Combined with the clinical characteristics of BC patients,the differences of SNF5 in different AJCC stages,grades,T stages,N stages and M stages of BC patients,and that in BC cell lines and normal urothelial cell line were analyzed.In addition,the IHC of 157 clinical BC samples from our hospital was also performed for analyzing the association of SNF5 with age,gender and myometrial invasion.2.SNF5 silencing 5637 and T24 cells,together with SNF5 overexpressing T24 cells were established.CCK-8 assays and colony-formation assays were conducted to test cell proliferation.The effect of SNF5 depletion was determined in tumor xenograft models by subcutaneously injected SNF5 knockdown or the negative control BC cells.The size and weight of tumor were recorded,and Ki-67 and Cleaved caspase-3 expression were detected by IHC.GSEA was used to analyzing the potential signaling pathway influenced by low SNF5 expression.Wound-healing and Transwell chamber assays were performed.The EMT related molecules were examined by Western blotting.Last,CCK-8,colony-formation,wound-healing and Transwell assays were performed with T24 SNF5 overexpressing cells.3.GSEA was used to analyze the potential signaling pathway low SNF5 expression involved in.Western blotting and IHC were used to analyze the expression of STAT3 and pSTAT3(Y705).STAT3 inhibitor S3I-201 was utilized in colony-formation,wound-healing and Transwell assays to validation the involvement of p-STAT3(Y705)in promotive effected by silencing SNF5.4.The potential drugs,AKT inhibitor VIII、AZD0530,gefitinib,were screened out after establishing the IC50 estimated model on chem drugs.Then the differences of IC50 of chem drugs were compared between low SNF5 and high SNF5 group.Moreover,EZH2 inhibitor GSK126 was exposed to SNF5 silencing and negative groups to test the resistance to cisplatin.Results1.The results obtained from TCGA and GEO database showed that the SNF5 expression level in BC tissues significantly higher than that in normal samples,even in the comparison between tumor samples and paracancerous tissues.The analysis was also stratified by race,gender,age and AJCC stages.Consistently,compared to normal tissues,upregulated SNF5 expression was observed in BC tissues.As for survival analyses in TCGA-BLCA and GSE13507,patients with low SNF5 expression level showed a much more shorter survival time than those with high SNF5 expression.At the m RNA level,by integrating RNA sequencing data into clinical characteristics,we found that patients with lymphatic metastasis had significantly lower SNF5 expression than those without lymphatic metastasis.Similarly,two metastatic BC cell lines in CCLE database,253 J and 253J-BV,had the lower SNF5 expression than other BC cell lines.Moreover,q-PCR and western blotting demonstrated that T24 cells,which derived from a more advanced tumor grade(Grade III)than 5637 cells(Grade II),which showed the highest SNF5 expression among the BC cell lines.The association of SNF5 with clinicopathological characteristics in our clinical cohort showed that SNF5 expression significantly correlated with lymph node metastasis.2.SNF5 downregulation facilitates BC cell proliferation and migration.Compared with control group,T24 and 5637 cells with decreased SNF5 expression showed accelerated cell proliferation and increased clonogenicity.The tumor growth rate was significantly faster and tumor weights were remarkably increased in the T24 SNF5-depleted group than in the control group.Likewise,the growth of xenografts from 5637 SNF5-depleted cells grew much faster and weighed significantly more than those formed by negative group cells.Further IHC staining revealed that tumor samples from T24 and 5637 SNF5-depleted cells had higher levels of Ki67 expression compared with control group,but no difference in the expression of cleaved caspase-3.GSEA suggested that epithelial-mesenchymal transition(EMT)related gene sets were enriched in low SNF5 expression group.Then wound-healing assay to evaluate the effect of SNF5 on cell migration.The results revealed that the migratory speed of T24 and 5637 cells was enhanced after SNF5 depletion,compared with the counterparts.Consistently,Transwell migration assay showed that the number of migrating cells was significantly increased after SNF5 knock-down.Moreover,western blotting showed that vimentin and snail,the EMT signature molecules,were elevated in SNF5-depletion cells,which was accompanied with by obvious downregulation of E-cadherin.However,no difference was observed in cell proliferation and cell migration in SNF5 overexpression T24 cells,compared to negative control cells.3.It was STAT3 activation induced by SNF5 knock-down that facilitated cell proliferation and migration.First,JAK/STAT signaling pathway was significantly enriched in low SNF5 expression group.Thus,the expression of STAT3 and p-STAT3(Y705)were detected by western blotting and the results presented the upregulation of p-STAT3(Y705)rather than STAT3.Similarly,IHC staining confirmed that the expression of p-STAT3(Y705)in SNF5-depleted inoculated tumor tissues was significantly elevated than that in control group.To verify whether the SNF5-induced enhancements were mediated by STAT3 activation,BC cells were treated with the STAT3 selective inhibitor S3I-201.The finding demonstrated that S3I-201 dampened the promotive effect on cell proliferation and cell migration.4.By establishing a predictive model based on the GDSC cell line datasets,we then calculated the estimated IC50 for the patients in the TCGA database to nominate candidate drugs based on the SNF5 expression.The results indicated that IC50 for cisplatin,gemcitabine,doxorubicin and mitomycin C in patients with low SNF5 expression was higher than high SNF5 expression group.Meanwhile,a significantly lower estimated IC50 in the SNF5 lowexpression group than that of high-expression group for some of inhibitors,including gefitinib,AZD0530,and AKT inhibitor VIII,where low SNF5 expression could indicate more sensitivity.Subsequently,we validated the effects of these three drugs on the established cells.The results showed that attenuated SNF5 expression conferred greater sensitivity for gefitinib in both T24 and 5637 SNF5 knockdown cells.However,the difference in sensitivity to AKT inhibitor VIII was only observed in T24 SNF5 depleted cells and no difference to AZD-0530 in 5637 and T24.ConclusionIn this study,we found that the m RNA and protein levels of SNF5 were up-regulated in BC.The survival time of patients with BC harboring low SNF5 expression was significantly shortened,when compared with high expression group.Enhanced proliferation and migration were mediated by STAT3 activation,which could accelerate progression of BC.Importantly,gefitinib or cisplatin plus EZH2 inhibitor regimens showed the promise to be novel strategies in personalized BC treatment.