The Role And Mechanism Of SQLE In Bladder Urothelial Cancer

BackgroundBladder cancer(BC)is the most common urinary system cancer,and smoking is the main risk factor for the formation of this tumor.Most BC are urothelial carcinomas,which are divided into non-muscle invasive bladder cancer(NMIBC)and muscle invasive bladder cancer(MIBC).Approximately 75% of patients are affected by non-muscle invasive bladder cancer(NMIBC),of which 50% are classified as low grade.In most cases,muscle invasive BC belongs to high grade.In the past three decades,there were few progress in treating high grade muscle invasive bladder cancer except for cisplatin based chemotherapy.Currently,a few new treatment agents were approved.Therefore,only a more in-depth understanding of the molecular mechanisms of the occurrence and the development of high grade BC can we develop better treatments for bladder cancer.Squalene epoxidase(SQLE)is one of the key rate-limiting enzymes in the first step of cholesterol biosynthesis,catalyzing the stereospecific conversion of non-sterol intermediate squalene to 2,3(S)-oxysqualene.In our previous urine High-throughput metabolic assay,cholesterol was found to be elevated in BC and linked closely to higher grade.By combining matched transcriptional data and immunohistochemistry assay,we linked the expression of SQLE in BC tissue and urine cholesterol level.SQLE functions as an oncogene in a variety of malignant tumors and is necessary for the occurrence,development and metastasis of cancer.Nevertheless,there is no research on the function and molecular mechanism of SQLE in bladder cancer.Purpose1.To systematically predict and detect the expression of SQLE in clinical samples of bladder cancer,and analyze the relationship between the expression level of SQLE and the proliferation,grade and prognosis of bladder cancer;2.To explore the effects of SQLE on the proliferation,migration,cell cycle and subcutaneous tumor formation in bladder cancer cells;3.To verify the effects of SQLE's specific inhibitor NB-598 on the proliferation,cycle and migration of BC cells.To test drug sensitivity of NB-598,Gemcitabine and cisplatin in bladder cancer organoids and explore the potential mechanism of drug response;4.To explore the related pathways the SQLE exert its cancer-promoting function and build a signature derived from SQLE associated transcriptomic signature for the diagnosis of high grade disease and for the prognosis prediction.Method1.Urine metabolomics was used to detect the cholesterol content in the urine of patients with bladder cancer and explore the relationship between its level and tumor grade;Evaluate the SQLE expression in BC tissue using IHC and analysis the association between urine cholesterol and tissue SQLE level;Using IHC and data mining of public BC transcriptome to explore the correlation between cell proliferation markers and SQLE.Using IHC to analysis the association between SQLE expression and tumor grade or prognosis of BC patients.2.The SQLE was silenced by small hairpin RNA(sh RNA);CCK8 experiment was used to detect the cell viability;Flow Cyto Metry was used to analysis the effect of SQLE knockdown on cell cycle;Transwell migration chamber was employed to detect the ability of cell migration;Nude mouse subcutaneous tumor formation assay was used to detect the ability of cell subcutaneous tumor formation;3.SQLE's specific inhibitor NB-598 was applied to block the function of SQLE;CCK8 experiment was used to detect the cell viability;Flow Cyto Metry was used to analysis the cell cycle;Transwell migration chamber was employed to detect the ability of cell migration;Three bladder cancer organoids were developed to evaluate the potential drug response of NB-598 in BC and compare NB-598 with gemcitabine and cisplatin.4.The mechanism of SQLE in bladder cancer was predicted by using GSEA analysis in five public BC transcriptome data set.The association between high SQLE expression and cell cycle related/MTORC1 pathways was specifically evaluated.A pathway phosphorylation protein array was used to screen altered phosphorylation protein in the cell knocking down SQLE.Western bolt was used to validate the altered phosphorylation protein.Using RNA sequencing to find the altered genes and pathways in NB-598 treated bladder cancer cells.The main findings were validated by Q-PCR and western bolt.Using logistic regression and multivariate COX regression separately to build and evaluate the NB-598 associated gene expression signature in diagnosing high grade BC and predicting patients' prognosis respectively.Result1.Compared with non-bladder cancer patients,bladder cancer patients have higher urine cholesterol content(72 normal vs 109 tumors,P<0.001,published data),and the urine cholesterol content of high-grade patients is higher than that of low-grade patients.SQLE expression in matched tissue sample was correlated with urine cholesterol level.SQLE expression was higher in high grade BC tissue compared to low grade BC tissue(P<0.001).In our previous 10 pairs of bladder cancer RNAseq data,SQLE was elevated in tumor tissue compared with normal urothelial.High SQLE in BC tissue was further validated in three public data sets.IHC analysis indicated that SQLE was up-regulated in the tumor budding and the invasive front.Tumor associated vessels may have higher SQLE compare to the tumor free areas.IHC indicated that in the 109 BC patients,SQLE expression was higher in high grade patients.This finding was validated in the additional six public data sets,including one single cell transcription data set.High SQLE was an independent predictor for worse overall survival and disease specific survival in the 113 patients with survival information.SQLE protein expression was positively associated with KI67 indicated by IHC analysis in the 113 patients.SQLE m RNA expression was positively correlated with MKI67,TOP2 A and PCNA m RNA expression in two public data sets..2.Down regulation of SQLE by sh RNA reduced the proliferation,migration and the subcutaneous tumors growth of bladder cancer cells.SQLE knockdown blocked cell cycle progression of 5637 and J82 cells.3.NB-598 inhibited the proliferation and migration and blocked cell cycle progression of 5637 and J82 cells.One BC organoid responded well to NB-598,even better than the gemcitabine or cisplatin used as single agent or in combination.The other BC organoid was also sensitive to NB-598.However,one organoid was not sensitive to NB-598.The association of tumor SQLE expression level and drug response of bladder cancer organoid needs to be further investigated.4.GSEA analysis in the five public data indicated that high SQLE was associated various oncologic and metabolic pathways,in which the HALLMARK?G2M?CHECKPOINT and HALLMARK?MTORC1?SIGNALING were common pathways among different data sets.The phosphorylation protein array also found the down regulation of p-m TOR and p-p70S6 K in SQLE knockdown cells,which was validated by western blot.RNA sequencing showed that the down regulated genes by NB-598 were associated with proteasome,spliceosome,RNA transport,cell cycle,TCA cycle and pathways associated with transcription activity.The up-regulated genes by NB-598 were particularly associated with various types of metabolic pathways,indicating the metabolic reprogramming of NB-598 treated cells.The up-regulation of ERBB pathway indicated that the combination of NB-598 and ERBB targeted agents may improve the drug response of NB-598.5.Two gene expression signature was build using the top down regulated genes by NB-598.The Changhai NB-598 full signature consists of 104 genes(log2fc <-1.2,FDR<0.05)and the Changhai NB-598 core signature has 27 gens(log2fc <-2.0,FDR<0.05).Both of the two signatures were able to diagnosis high grade tumor in TCGA,GSE13507 and E-MTAB-4321 data sets(all AUCs were above 0.79).These signatures were also effective in predicting prognosis of BC patients as indicated by a comprehensive survival analysis in seven public data sets,including one with the purpose of predicting BCG response in high grade T1 tumors.ConclusionSQLE was up regulated in BC patients and correlated with high tumor grade.The SQLE expression level was positively correlated with urine cholesterol level and tumor proliferation markers.High SQLE expression was an independent predictor of worse OS and DSS of BC patients.Down regulation of SQLE activity by sh RNA or NB-598 inhibited cell proliferation and migration,and blocked cell cycle progression.NB-598 may serve as target therapy agent as indicated by bladder cancer organoid drug assay.NB-598 may be more effective than gemcitabine and cisplatin.SQLE down regulation in tumor cells was associated with reduced activity of m TOR pathway.NB-598 down regulated oncologic pathways in bladder cancer cell and forced metabolic reprogramming.The gene expression signature named Changhai NB-598 full signature and Changhai NB-598 core signature were both promising biomarkers for diagnosing high grade tumor and predicting patients' prognosis,including predicting BCG response.Further investigation of SQLE and the expression signature associated with it will provide new insights into the diagnosis and treatment of BC.