Bladder Urothelial Cell Research Articles

Abstract 1552: Tumorigenesis of basal muscle invasive bladder cancer was mediated by PTEN protein degradation resulting from SNHG1 upregulation

Abstract hosphatase and tensin homolog deleted on chromosome ten (PTEN) serves as a powerful tumor suppressor, and has been found to be downregulated in human bladder cancer (BC) tissues. Despite this observation, the mechanisms contributing to PTEN’s downregulation have remained elusive. In this study, we discovered that mice exposed to N-butyl-N-(4-hydroxybu-tyl)nitrosamine (BBN), a specific bladder chemical carcinogen, exhibited primary basal muscle invasive BC (BMIBC) accompanied by a pronounced reduction in PTEN protein expression in vivo. Utilizing a lncRNA deep sequencing high-throughput platform, along with gain- and loss-of-function analyses, we identified small nucleolar RNA host gene 1 (SNHG1) as a critical lncRNA that might drive the formation of primary BMIBCs in BBN-treated mice. Cell culture results further demonstrated that BBN exposure significantly induced SNHG1 in normal human bladder urothelial cell UROtsa. Notably, the ectopic expression of SNHG1 alone was sufficient to induce malignant transformation in human urothelial cells, while SNHG1 knockdown effectively inhibited anchorage-independent growth of human BMIBCs. Our detailed investigation revealed that SNHG1 overexpression led to PTEN protein degradation through its direct interaction with HUR. This interaction reduced HUR binding to ubiquitin-specific peptidase 8 (USP8) mRNA, causing degradation of USP8 mRNA and a subsequent decrease in USP8 protein expression. The downregulation of USP8, in turn, increased PTEN poly-ubiquitination and degradation, culminating in cell malignant transformation and BMIBC anchorage-independent growth. In vivo studies confirmed the downregulation of PTEN and USP8, as well as their positive correlations in both BBN-treated mouse bladder urothelium and tumor tissues of bladder cancer in nude mice.Our findings, for the first time, demonstrate that overexpressed SNHG1 competes with USP8 for binding to HUR. This competition attenuates USP8 mRNA stability and protein expression, leading to PTEN protein degradation, consequently, this process drives urothelial cell malignant transformation and fosters BMIBC growth and primary BMIBC formation. Citation Format: Chuanshu Huang, Tengda Li, Honglei Jin. Tumorigenesis of basal muscle invasive bladder cancer was mediated by PTEN protein degradation resulting from SNHG1 upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1552.

Tumorigenesis of basal muscle invasive bladder cancer was mediated by PTEN protein degradation resulting from SNHG1 upregulation

BackgroundPhosphatase and tensin homolog deleted on chromosome ten (PTEN) serves as a powerful tumor suppressor, and has been found to be downregulated in human bladder cancer (BC) tissues. Despite this observation, the mechanisms contributing to PTEN’s downregulation have remained elusive.MethodsWe established targeted genes’ knockdown or overexpressed cell lines to explore the mechanism how it drove the malignant transformation of urothelial cells or promoted anchorageindependent growth of human basal muscle invasive BC (BMIBC) cells. The mice model was used to validate the conclusion in vivo. The important findings were also extended to human studies.ResultsIn this study, we discovered that mice exposed to N-butyl-N-(4-hydroxybu-tyl)nitrosamine (BBN), a specific bladder chemical carcinogen, exhibited primary BMIBC accompanied by a pronounced reduction in PTEN protein expression in vivo. Utilizing a lncRNA deep sequencing high-throughput platform, along with gain- and loss-of-function analyses, we identified small nucleolar RNA host gene 1 (SNHG1) as a critical lncRNA that might drive the formation of primary BMIBCs in BBN-treated mice. Cell culture results further demonstrated that BBN exposure significantly induced SNHG1 in normal human bladder urothelial cell UROtsa. Notably, the ectopic expression of SNHG1 alone was sufficient to induce malignant transformation in human urothelial cells, while SNHG1 knockdown effectively inhibited anchorage-independent growth of human BMIBCs. Our detailed investigation revealed that SNHG1 overexpression led to PTEN protein degradation through its direct interaction with HUR. This interaction reduced HUR binding to ubiquitin-specific peptidase 8 (USP8) mRNA, causing degradation of USP8 mRNA and a subsequent decrease in USP8 protein expression. The downregulation of USP8, in turn, increased PTEN polyubiquitination and degradation, culminating in cell malignant transformation and BMIBC anchorageindependent growth. In vivo studies confirmed the downregulation of PTEN and USP8, as well as their positive correlations in both BBN-treated mouse bladder urothelium and tumor tissues of bladder cancer in nude mice.ConclusionsOur findings, for the first time, demonstrate that overexpressed SNHG1 competes with USP8 for binding to HUR. This competition attenuates USP8 mRNA stability and protein expression, leading to PTEN protein degradation, consequently, this process drives urothelial cell malignant transformation and fosters BMIBC growth and primary BMIBC formation.

Gene expression changes of isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 affect carcinogenesis and survival probability

Isocitrate dehydrogenase (IDH) is an essential metabolic enzyme in the regulation of cellular metabolism. IDH gene encodes three protein isoforms, IDH1, IDH2, and IDH3, and the expression level of isoforms is altered in human cancer types. Examining the gene expression level of IDH is a therapeutic advantage that could help find a new target to use in cancer metabolism. The present study aimed to explore the gene expression level of IDH1 and IDH2 isoforms in the ten common human cancers using bioinformatic tools. In addition, the effect of gene expression changes on IDH1 and IDH2 on carcinogenesis and survival probability was examined in publicly available data deposited in the TCGA database. The results showed that the expression of IDH isoforms showed tissue-specific differences. IDH1 expression increased in esophageal and lung squamous cell carcinoma and lung and stomach adenocarcinoma tumors. Bladder urothelial, breast urothelial, and lung squamous cell carcinoma, colon, and lung adenocarcinoma displayed a significant upregulation of IDH2 expression. There was a direct relationship between the expression of IDH isoforms and the progression of various cancer types. High IDH1 expression led to decreased survival probability in esophageal carcinoma, lung, and stomach adenocarcinoma. Elevated IDH2 expression level led to decreased survival probability in bladder urothelial, breast urothelial, and lung squamous cell carcinoma and colon adenocarcinoma. In conclusion, all data showed that IDH1 could be a biomarker for esophageal carcinoma, lung and stomach adenocarcinoma, and IDH2 for bladder urothelial, breast urothelial, and lung squamous cell carcinoma, and colon adenocarcinoma.

Functional roles of tissue factor and EMMPRIN/CD147 present on bladder urothelial carcinoma-derived extracellular vesicles.

e16557 Background: Extracellular vesicles are involved in the homeostatic functions and extracellular matrix remodeling in the body. We previously found that the antithrombotic drug Tinzaparin, at clinical concentrations, induces the sufficient release of the tissue factor pathway inhibitor TFPI to functionally inhibit the prothrombotic effector tissue factor expressed on the vesicles released by malignant tumor cells. Treatment of in vitro cancer cells with Tinzaparin results in the inhibition of the tissue factor/ERK pathway. Our hypothesis is that vesicles derived from metastatic urinary tumors are enriched in tissue factor; therefore, their elusive functioning is affected by the suppressed tissue factor caused by TFPI. Here, we show that this observation may have major clinical relevance. Methods: To test the contribution of extracellular vesicles to the mechanism and extent of cell invasion, we assessed two human adherent bladder urothelial carcinoma cell lines, RT4 and J82, which represent non-muscle and muscle-invasive diseases, respectively. Urine samples were obtained from a cohort of 75 outpatients who underwent transurethral resection subdivided by the presence of cancer or muscle invasion. Vesicles were isolated using sequential centrifugation and further validated for their size, nanoparticle tracking analysis, and surface markers using flow cytometry. The tissue factor activity index levels of the vesicles and the potential for in vitro induction of matrix metalloproteinases (MMPs) were quantified using colorimetric enzymatic methods. The levels of phosphorylated and total Erk1/2 were measured using an in vitro kinase assay and western blot. Results: Vesicles derived from the invasive cells J82 displayed higher levels of the MMP inducer EMMPRIN/CD147. The distinct pattern of surface markers in muscle-invasive-derived vesicles, featuring enhanced potential for inducing Erk1/2 phosphorylation and in vitro MMP activation as key processes of extracellular matrix degradation, was drastically suppressed by a potent, specific small molecule CD147 inhibitor. There was higher tissue factor activity, a key initiator of coagulation, among the patients with bladder urothelial carcinoma than those without. In the subgroup analysis, the tissue factor activity index and CD147 were higher in patients with muscle-invasive disease than in patients with non-muscle-invasive disease, showing high accuracy in distinguishing patients regarding invasion potential. Conclusions: The tissue factor activity index of urine extracellular vesicles may be considered a marker of bladder urothelial carcinoma. In addition to tissue factor expression, CD147 is crucial in promoting the potential of extracellular vesicles to induce MMP-mediated extracellular matrix degradation and, consequently, cell invasion, thereby allowing metastatic disease progression.

Ex vivo near-infrared targeted imaging of human bladder carcinoma by ICG-anti-CD47.

The low detection rate of early-stage and small tumors remains a clinical challenge. A solution to this unmet need is urgently warranted for the accurate diagnosis and treatment of bladder cancer (BC). This study aimed to evaluate the feasibility of CD47 as a target for optical molecular imaging of human BC and conduct preliminary ex vivo imaging experiments. Using indocyanine green (ICG) and a CD47 antibody (anti-CD47), we synthesized a new targeted fluorescent probe ICG-anti-CD47. A total of 25 patients undergoing radical cystectomy were prospectively included in ex vivo imaging experiments. Following surgery, the freshly isolated bladder specimens were incubated with ICG-anti-CD47, and images were captured under white light and near-infrared (NIR) light. Standard histopathologic evaluation was performed, and findings were correlated with those of CD47-targeted NIR molecular imaging. Based on the ex vivo imaging experiments, 23 and 2 patients were pathologically diagnosed with bladder urothelial carcinoma and bladder squamous cell carcinoma, respectively. There were no adverse effects of ICG-anti-CD47 on the histological structure of the tumor and normal uroepithelium. In the NIR grayscale images, the mean fluorescence intensity of the tumor tissue was significantly higher than that of the adjacent normal background tissue, which markedly improved tumor visualization. Anti-CD47-targeted NIR molecular imaging may be a feasible and powerful strategy for the accurate diagnosis of BC. Nevertheless, larger-scale randomized trials are warranted to verify the present findings.

O036 Does Cxbladder accurately exclude bladder urothelial cell malignancy on diagnostic and surveillance cystoscopy? a prospective multi-centre study

Abstract Introduction and Objectives Health systems incur significant costs from bladder cancer. Diagnostic and surveillance protocols are intensive. Urine cytology and Computed tomography (CT) have low sensitivity as screening and surveillance tools. Cxbladder is a non-invasive urinary biomarker approved for use in Australia, with high reported sensitivity in bladder cancer detection. The purpose of this study was to determine the accuracy of Cxbladder as a screening and surveillance tool in patients with suspected and established bladder cancer, respectively. Methods A prospective trial (88 participants) was performed at Gosford and North Gosford Hospitals (New South Wales, Australia). Inclusion criteria were: patients undergoing investigation for macroscopic haematuria or bladder cancer surveillance. Patients were asked to perform a Cxbladder test prior to either flexible or rigid cystoscopy. Exclusion criteria were: patients aged < 18 years, or patients that had undergone transurethral resection of the bladder within five months. Testing kits were supplied at no cost by Pacific Edge Ltd. Cxbladder results were then compared with cystoscopic findings, the prevailing gold-standard for bladder cancer diagnosis. Results Sensitivity and specificity of Cxbladder for detection of bladder cancer were 76.5% and 45.1% respectively. Positive and negative predictive values (PPV/NPV) were 31.7% and 85% respectively. False negative cases comprised low-grade papillary tumours only. Cxbladder had 100% NPV in detection of high-risk bladder cancer. Conclusion There is evidence to support Cxbladder as a screening tool in the detection of high-risk bladder cancer. Further studies involving larger populations are required to validate its accuracy. Take-home message The purpose of this study was to determine the accuracy of Cxbladder as a screening and surveillance tool in patients with suspected and established bladder cancer, respectively. Sensitivity and specificity of Cxbladder for detection of bladder cancer were 76.5% and 45.1% respectively, whilst a 100% negative predictive value was obtained in detection of high-risk bladder cancer.

Abstract 1337: Pan-cancer analysis of immune-associated genes and pathways dysregulated by tobacco reveals osteopontin as a key mediator of smoking-associated carcinogenesis

Abstract Background: Smoking remains a primary etiological agent of 12 different cancers. However, current understanding of the pathogenesis and progression of these smoking-induced cancers remains unclear. Though vaping has been proposed as a safer alternative to cigarette smoking, more research is needed to fully understand its potential to cause cancer. In particular, the chemicals in e-cigarette (e-cig) vapor have previously been demonstrated to cause immune dysregulation, similar to conventional cigarette smoke. In this study, we aimed to investigate the immune-associated (IA) genomic and transcriptomic landscapes of a panel of smoking-induced cancers in order to identify common and unique elements among these cancers and to determine whether certain immune dysregulations may be shared between smoking and vaping. Methods: Whole genome sequencing data from tumors and adjacent normal samples was obtained from The Cancer Genome Atlas (TCGA) to compare the IA landscapes of 5 different smoking-induced cancers: head and neck squamous cell carcinoma (HNSCC), esophageal carcinoma (ESCA), bladder urothelial carcinoma (BLCA), lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC). Differentially expressed genes were identified and correlated to tumor stage and patient survival. Gene Set Enrichment Analysis and ReactomeFi were used to correlate IA gene expression to cancer pathway enrichment. The REVEALER algorithm was used to correlate gene expression to copy number variations and mutations. Results: We found that the genomic landscapes of smoking-induced cancers were largely unique. However, Osteopontin (OPN), an IA gene capable of regulating the EGFR and CD44 signaling cascades, was found to be upregulated along with its downstream targets in HNSCC, LUAD, LUSC, and ESCA smoking patients. Such upregulation was correlated to poorer prognosis for all 4 cancers. In vitro study indicated an upregulation of OPN and its downstream targets in cell lines exposed to both e-cig vapor and cigarette smoke. Conclusions: Despite few similarities among the IA landscapes of these smoking-induced cancers, we suggest the importance of OPN upregulation in HNSCC, LUSC, LUAD, and ESCA pathogenesis caused by smoking and, potentially, vaping. Citation Format: Jaideep Chakladar, Neil Shende, Wei Tse Li, Weg M. Ongkeko. Pan-cancer analysis of immune-associated genes and pathways dysregulated by tobacco reveals osteopontin as a key mediator of smoking-associated carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1337.

Genomic classification of clinically advanced major genito-urinary cancers (GUca) based on methylthioadenosine phosphorylase (MTAP) genomic loss.

164 Background: Novel treatments for clinically advanced GUca including castrate resistant prostate ca (PC), bladder urothelial ca (BUC) and clear cell renal ca (ccRCC) are widely needed. Recently, the targeting of cancer cells with arginine accumulation caused by MTAP loss has emerged as a new synthetic lethality-based anti-cancer program. Methods: 8,436 mCRPC, 2,683 BUC and 841 ccRCC underwent hybrid-capture based comprehensive genomic profiling (CGP) to evaluate all classes of genomic alterations (GA). Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on up to 114 loci. PD-L1 tumor cell expression was determined by IHC (Dako 22C3). Results: 1.3% of PC, 24.2% of BUC and 5.5% of ccRCC featured MTAP loss. There were no significant age or gender differences associated with MTAP loss. CDKN2A/B loss ranged from 94% in ccRCC to > 99% in PC and BUC. The GA/tumor frequencies were similar when CDKN2A/B GA are excluded. In PC, GA in TP53, PTEN and BRCA1 were more frequent, while GA in AR, CDK12, RB1 and BRCA2 were less frequent in cases with MTAP loss. In BUC, GA in TSC1 and FGFR3 were more frequent and GA in RB1and TP53 were less frequent in cases with MTAP loss. In ccRCC, GA in NF2 were more frequent in cases with MTAP loss, while GA in VHL and PBRM1 were less frequent in cases with MTAP loss. “Targetable” kinase GA were rare in all groups, except for FGFR3 GA in MTAP loss BUC. Immunotherapy (IO) putative biomarkers varied among tumors, with MSI-high status less frequent and TMB ≥ 10 mut/Mb more frequent in BUC MTAP-intact than BUC with MTAP loss. PD-L1 expression was similar except for high PD-L1 expression more frequent in MTAP-intact BUC. Conclusions: When compared with PC and ccRCC, the clinical development of novel drugs, such as PRMT5 and MTA2 inhibitors in GUca will likely be focused on BUC given the 24% frequency of MTAP loss in that tumor type. CGP of PC, BUC and ccRCC reveal significant differences in GA in MTAP-intact and tumors with MTAP loss, which may impact future combinatorial clinical trial designs.[Table: see text]

The expression of GGCT in the bladder urothelial cell carcinoma and its clinical significance

Objective: To evaluate the expression and clinical significance of γ-glutamylcyclotransferase (GGCT) in patients with bladder urothelial cell carcinoma. Methods: Immunohistochemical staining for GGCT were performed on tissue sections of 86 patients with bladder urothelial cell carcinoma and 10 normal controls, and the correlations between GGCT and clinicopathological characteristics and the prognosis were analyzed. Results: The positive rate of the expression of GGCT in 86 cases of bladder urothelial cell carcinoma was 61.6% (53/86). GGCT protein was located mainly in cancer cell cytoplasm, and it can be seen in the nucleus of the tumor cells in some cases. The level of GGCT expression was positively related to pathological classification (P<0.001), stage (P=0.020), and tumor size (P=0.025). Immunohistochemical semiquantitative analysis showed that the expression of GGCT in patients with T1 stage of non-muscle invasion bladder urothelial cell carcinoma was significantly higher than that with Ta stage (P=0.034). Kaplan-Meier analysis showed that the expression of GGCT was correlated with the recurrence-free survival in patients with non-muscle invasive bladder cancer, the recurrence-free survival rate was lower in the GGCT positive group (P=0.029). Multivariate COX regression analysis showed that the pathological stage (OR=5.029, P=0.009) and the number of tumors (OR=3.320, P=0.024)were the independent risk factors for recurrence-free survival in patients with early urothelial cell carcinoma of the bladder. Conclusions: The expression of GGCT is significantly increased in bladder urothelial cell carcinoma and is related to the malignant biological behavior and progression of tumor. Patients with GGCT positive early bladder tumor are inclined to recur.

WNT7B represses epithelial-mesenchymal transition and stem-like properties in bladder urothelial carcinoma

BackgroundRecurrence and metastasis are the major problems of bladder urothelial carcinoma, which mainly attribute to tumor cell stemness, epithelial-mesenchymal transition (EMT) and chemoresistance. MethodsTCGA database was interrogated for gene mRNA expression in bladder urothelial carcinoma samples. CCLE database was interrogated for gene mRNA expression in bladder cancer cell lines. The correlation between two genes was analyzed by Pearson statistics. 37 human bladder urothelial carcinoma specimens were adopted for immunohistochemistry. Bladder cancer cells RT4, J82, and UM-UC-3 were used to carry out loss and gain of function studies. Kaplan-Meier method was performed to analyze the overall survival. FindingsWNT7B is downregulated in high-grade bladder urothelial carcinomas. Low WNT7B expression is associated with unfavorable prognosis. Loss and gain of function studies showed that WNT7B inhibits bladder urothelial carcinoma cell EMT, stem-like properties and chemoresistance. FZD5, a specific receptor for WNT7B, mediates WNT7B signaling. ELF3 is a downstream component of WNT7B signaling, which transcriptionally modulates NOTCH1, a tumor suppressor in bladder urothelial carcinoma. InterpretationThese data demonstrate that WNT7B/FZD5-ELF3-NOTCH1 signaling functions as a tumor-suppressing pathway in bladder urothelial carcinoma.

Androgen receptor signalling confers clonogenic and migratory advantages in urothelial cell carcinoma of the bladder.

Bladder urothelial cell carcinoma (UCC) incidence is about three times higher in men compared with women. There are several indications for the involvement of hormonal factors in the aetiology of UCC. Here, we provide evidence of androgen signalling in UCC progression. Microarray and qPCR analysis revealed that the androgen receptor (AR) mRNA level is upregulated in a subset of UCC cases. In an AR‐positive UCC‐derived cell line model, UM‐UC‐3‐AR, androgen treatment increased clonogenic capacity inducing the formation of big stem cell‐like holoclones, while AR knockdown or treatment with the AR antagonist enzalutamide abrogated this clonogenic advantage. Additionally, blockage of AR signalling reduced the cell migration potential of androgen‐stimulated UM‐UC‐3‐AR cells. These phenotypic changes were accompanied by a rewiring of the transcriptome with almost 300 genes being differentially regulated by androgens, some of which correlated with AR expression in UCC patients in two independent data sets. Our results demonstrate that AR signals in UCC favouring the development of an aggressive phenotype and highlights its potential as a therapeutic target for bladder cancer.

The sustaining of fluorescence in photodynamic diagnosis after the administration of 5-aminolevulinic acid in carcinogen-induced bladder cancer orthotopic rat model and urothelial cancer cell lines

BackgroundThe administration of 5-aminolevulic acid hydrochloride (5-ALA·HCl) 3 h (range: 2–4 h) before photodynamic diagnosis (PDD) is recommended for detecting bladder tumors. However, there is insufficient evidence on the time duration for the fluorescence of PDD after oral administration of 5-ALA. We investigated the sustainability of the photodynamic effect and protoporphyrinⅨ (PpⅨ) after 5-ALA administration in a carcinogen-induced bladder tumor rat model and bladder cancer cell lines. MethodsThe carcinogen-induced bladder tumor orthotopic rat model was established by the administration of N-butyl-N-(4-hydroxybutyl) nitrosamine. ResultsRed fluorescence was visible 2–8 h after the oral administration of 5-ALA in the carcinogen-induced bladder tumor rat model. Plasma and intratissue PpⅨ (nM) progressed to a higher level at 2 h and remained almost constant 2–8 h after oral administration of 5-ALA. The peak fluorescence intensity of PpⅨ was observed 3–4 h after the administration of 5-ALA in bladder cancer cell lines. The accumulated PpⅨ remained for 4 h after the removal of 5-ALA in UMUC3 cells. It was not clearly visible 3 h after the removal of 5-ALA in MGHU3 and T24 cells. The expression level of ferrochelatase was significantly lower in UMUC3 cells than in other cells. Our findings suggest that 5-ALA-assisted PDD (ALA-PDD) can aid in detecting non-muscle-invasive bladder cancer 2–8 h after 5-ALA administration. ConclusionUrologists might not be required to make excess effort to start ALA-PDD-assisted transurethral resection of bladder tumor after the administration of 5-ALA.

Clinicopathological Significance of Estrogen Receptor β and Estrogen Synthesizing/Metabolizing Enzymes in Urothelial Carcinoma of Urinary Bladder.

Sex-specific differences in the incidence of urinary bladder carcinomas are well known, and the possible involvement of sex steroids has been proposed. We previously reported the association of the loss of androgen receptors and androgen-producing enzymes with tumor progression of urinary bladder cancer patients. Clinically, the selective estrogen receptor modulators (SERMs) were reported to suppress the progression of these tumors but the status of estrogen receptors (ERs) has not been well studied in patients with bladder urinary cancer. Moreover, not only ERs but also estrogen-related enzymes, such as aromatase, steroid sulfatase (STS), and estrogen sulfotransferase (EST), have been reported in the biological/clinical behavior of various hormone-dependent carcinomas but not studied in urinary bladder carcinoma. Therefore, in this study, we immunolocalized ERs as well as estrogen metabolizing enzymes in urinary bladder carcinoma and performed immunoblotting and cell proliferation assays using the bladder urothelial carcinoma cell line, T24. The results revealed that the loss of STS and aromatase was significantly correlated with advanced stages of the carcinoma. In vitro studies also revealed that T24 cell proliferation rates were significantly ameliorated after treatment with estradiol or diarylpropionitrile (DPN). EST and aromatase were also significantly correlated with the nuclear grade of the carcinoma. The results of our present study, for the first time, demonstrated that biologically active estrogens that bind to ERs could suppress tumor progression and the inactive ones could promote its progression and the potential clinical utility of SERM treatment in selective patients with urinary bladder carcinoma.

Educational Case: Bladder Urothelial Cell Carcinoma TNM Stage, Prognosis and Management

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040. 1

Effects of miR-599 targeting YAP1 on proliferation, invasion and apoptosis of bladder urothelial carcinoma cells

The aim of this study was to investigate the effects of microRNA (miR)-599 targeting Yes-associated protein 1 (YAP1) on the proliferation, invasion and apoptosis of bladder urothelial carcinoma cell line J82. J82 cells cultured in vitro were divided into miR-nc group (transfected with analog negative control miR-NC), miR-599 group (transfected with miR-599 analog), miR-599 + pcDNA3.1 group (co transfected with miR-599 analog and pcDNA3.1 empty vector), miR-599 + pcDNA3.1-YAP1 group (co transfected with miR-599 analog and pcDNA3.1-YAP1 over expression vector), the targeting relationship between miR-599 and YAP1 was detected by double luciferase reporter gene assay, the expression level of miR-599 was detected by real-time fluorescence quantitative PCR, the expression of YAP1 was detected by Western blot, cell proliferation was detected by MTT method, cell invasion was detected by Transwell's cell experiment, and apoptosis was detected by flow cytometry. The miR-599 was able to bind to YAP1 targetingly; compared with those in miR-NC group, miR-599 expression level and apoptosis rate in miR-599 group were significantly higher, however, the expression level of YAP1 protein and the abilities of cell proliferation and invasion were significantly lower (P < 0.05); there was no significant difference in the above indexes between miR-599 group and miR-599 + pcDNA3.1 group (P > 0.05); compared with those in miR-599 + pcDNA3.1 group, the expression level of YAP1 protein and the abilities of cell proliferation and invasion in miR-599 + pcDNA3.1-YAP1 group were significantly higher, while the apoptotic rate was significantly lower (P < 0.05). MiR-599 may inhibit the proliferation, invasion and promote apoptosis of bladder urothelial carcinoma J82 cells by targetingly regulating YAP1.

Validity of whole genomes sequencing results in neoplasms in precision medicine

ObjectiveTo compare the whole genomes sequencing (WGS) results in the 100K Genomes project with the results of routine molecular diagnostics in precision medicine.Materials and methodsWe analysed 374 cancers including a...

MP54-19 THE DISRUPTED CIRCADIAN RHYTHM OF THE FATTY ACID METABOLITE, PALMITOYLETHANOLAMIDE, INDUCES NOCTURIA THROUGH G PROTEIN-COUPLED RECEPTOR 55 ACTIVATION IN THE BLADDER

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology (MP54)1 Apr 2020MP54-19 THE DISRUPTED CIRCADIAN RHYTHM OF THE FATTY ACID METABOLITE, PALMITOYLETHANOLAMIDE, INDUCES NOCTURIA THROUGH G PROTEIN-COUPLED RECEPTOR 55 ACTIVATION IN THE BLADDER Tatsuya Ihara*, Takahiko Mitsui, Sachiko Tsuchiya, Mie Kanda, Satoru Kira, Hiroshi Nakagomi, Manabu Kamiyama, Yoichi Shinozaki, Norifumi Sawada, Shuichi Koizumi, and Masayuki Takeda Tatsuya Ihara*Tatsuya Ihara* More articles by this author , Takahiko MitsuiTakahiko Mitsui More articles by this author , Sachiko TsuchiyaSachiko Tsuchiya More articles by this author , Mie KandaMie Kanda More articles by this author , Satoru KiraSatoru Kira More articles by this author , Hiroshi NakagomiHiroshi Nakagomi More articles by this author , Manabu KamiyamaManabu Kamiyama More articles by this author , Yoichi ShinozakiYoichi Shinozaki More articles by this author , Norifumi SawadaNorifumi Sawada More articles by this author , Shuichi KoizumiShuichi Koizumi More articles by this author , and Masayuki TakedaMasayuki Takeda More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000916.019AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: It is reported that the bladder functions are under the regulation of clock genes, which regulate circadian rhythm in organisms, and dysregulation of circadian bladder function can cause nocturia. In humans, some of the fatty acid metabolite levels are higher in nocturia patient plasma compared to non-nocturia patient. Especially, Palmitoylethanolamide (PEA) showed highest level, which are known as an agonist of G protein-coupled receptor 55 (GPR55). In the present study, we investigated the dynamics and effect of PEA on GPR55 in the mice bladder to evaluate the relationship between nocturia and lipid metabolism. METHODS: Male C57BL/6 (WT) mouse and Clock mutant mouse (ClockΔ19/Δ19) were used. ClockΔ19/Δ19 mouse deficit a function of Clock, which is one of clock genes, and showed nocturia phenotype. They were bred 12 h dark/light condition. PEA concentration in the mouse serum was measured using LS/MS every 8 hour for 2 days and compared between WT and ClockΔ19/Δ19 mice. PEA (10 mg/kg) was administered intraperitoneally (ip) in WT mice under the constant dark condition, and urination pattern was recorded for 2 days using metabolic cages. GPR55 distribution in mice was evaluated using Immunofluorescence staining, RT-PCR, and Western blotting. The PEA induced intracellular Ca2+ variations was measured in the mouse primary cultured urothelial cells (UCs) using Fura2-AM Ca2+ imaging. RESULTS: Serum PEA concentration fluctuated with circadian rhythm in WT mice, which was lower during light phase. However, those in ClockΔ19/Δ19 mice were in constant and higher-level during sleep phase. The voiding frequency was increased, and bladder capacity was decreased after PEA ip in WT mice. GPR55 was predominant at bladder urothelial cell layer. The intracellular Ca2+ was increased after PEA stimulation, also under extracellular Ca2+ free condition in UCs. These changes were inhibited by an endoplasmic reticulum (ER) inhibitor (Fig. 1). CONCLUSIONS: These results indicated that clock gene abnormalities cause higher serum PEA level in mice. PEA can activate GPR55 in the bladder urothelium, then mobilizes Ca2+ in the ER to cytoplasm and triggers the micturition reflex. The development of new drugs for GPR55 may leads to nocturia treatment. Source of Funding: no competing financial interests. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e799-e799 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tatsuya Ihara* More articles by this author Takahiko Mitsui More articles by this author Sachiko Tsuchiya More articles by this author Mie Kanda More articles by this author Satoru Kira More articles by this author Hiroshi Nakagomi More articles by this author Manabu Kamiyama More articles by this author Yoichi Shinozaki More articles by this author Norifumi Sawada More articles by this author Shuichi Koizumi More articles by this author Masayuki Takeda More articles by this author Expand All Advertisement PDF downloadLoading ...

Cigarette smoke induces the pyroptosis of urothelial cells through ROS/NLRP3/caspase-1 signaling pathway.

Cell death and inflammation are involved in the development of bladder dysfunction. Pyroptosis is programmed cell death, causing cytotoxic effects and local inflammation. As one of the biggest health threats in the world, smoking is also closely related to urinary system diseases. The aims of this study were to investigate the role of NLRP3 inflammasome-mediated pyroptosis in the bladder after cigarette smoke exposure. The expression of NLRP3 inflammasome and the activity of caspase-1 in bladder tissue was investigated after cigarette smoke exposure. In vitro, bladder urothelial cells were stimulated by cigarette smoke extract and then the activity of caspase-1 and the expression of NLRP3 inflammasome were measured. The role of oxidative stress was also assessed. The activity of caspase-1 in bladder tissue increased by 50% after cigarette smoke exposure. Cigarette smoke caused oxidative stress injury and the activation of NLRP3 inflammasome. In addition, reactive oxygen species (ROS) inhibitor N-acetyl-cysteine alleviated the pyroptosis of urothelial cells. Cigarette smoke-induced pyroptosis of bladder tissue by activating ROS/NLRP3/caspase-1 signaling pathway. Inhibition of bladder urothelial cell pyroptosis may be a new approach to alleviate bladder damage caused by smoking.

Osmotic regulation of UT-B urea transporters in the RT4 human urothelial cell line.

Facilitative UT‐B urea transporters play important physiological roles in numerous tissues, including the urino‐genital tract. Previous studies have shown that urothelial UT‐B transporters are crucial to bladder function in a variety of mammalian species. Using the RT4 bladder urothelial cell line, this study investigated the potential osmotic regulation of human UT‐B transporters. Initial end‐point PCR experiments confirmed expression of both UT‐B1 and UT‐B2 transcripts in RT4 cells. Western blotting analysis revealed glycosylated UT‐B protein to be highly abundant and immunolocalization experiments showed it was predominantly located on the plasma membrane. Further PCR experiments suggested that a 48 hr, NaCl‐induced raise in external osmolality increased expression of UT‐B transcripts. Importantly, these NaCl‐induced changes also significantly increased UT‐B protein abundance (p < .01, n = 7, ANOVA), whereas mannitol‐induced changes in external osmolality had no effect (NS, n = 4, ANOVA). Finally, similar increases in both UT‐B RNA expression and protein abundance were observed with urea‐induced changes to external osmolality (p < .05, n = 4, ANOVA). In conclusion, these findings strongly suggest that increases in external osmolality, via either NaCl or urea, can regulate human urothelial UT‐B transporters.

Cotinine, a major nicotine metabolite, induces cell proliferation on urothelium in vitro and in vivo

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. In a previous study, nicotine enhanced rat urinary bladder carcinogenesis using a rat urinary bladder two-stage carcinogenesis model. In the present study, nicotine metabolites (cotinine, trans-3’-hydroxy cotinine and N’-nitrosonornicotine) were evaluated in a cell proliferation assay using urinary bladder urothelial cell lines. Cotinine (0.1 to 1 mM) induced the highest cell proliferation compared to the others, including nicotine, in three bladder cancer cell lines (RT4, T24 and UMUC3 cells). By Western blot, cotinine induced phosphorylation of Stat3 and expression of cyclin D1 in UMUC3 cells. The cell proliferation induced by cotinine was blocked by inhibitors of nicotinic receptors (10 nM SR16584 or 10 μM methyllycaconitine citrate) and Stat3 (100 nM stattic). In an in vivo study, cotinine (13, 40 and 120 ppm) in drinking water also induced cell proliferation and simple hyperplasia in urinary bladder and renal pelvis urothelium of rats, but to a lesser degree compared to nicotine (40 ppm). Cytotoxicity detected by scanning electron microscopy and apoptosis in the bladder urothelium were induced by nicotine but not cotinine. These data suggest that cotinine is able to induce urothelial cell proliferation both in vitro and in vivo, and high urinary concentrations may enhance urothelial carcinogenesis.