Normal Bladder Urothelium Research Articles

Distinct Infiltration of T Cell Populations in Bladder Cancer Molecular Subtypes.

Bladder cancer is a heterogenous disease, and molecular subtyping is a promising method to capture this variability. Currently, the immune compartment in relation to subtypes is poorly characterized. Here, we analyzed the immune compartment in bladder tumors and normal bladder urothelium with a focus on T cell subpopulations using flow cytometry and RNA sequencing. The results were investigated in relation to tumor invasiveness (NMIBC/MIBC) and molecular subtypes according to the Lund Taxonomy system. Whereas the NMIBC/MIBC differed in the overall immune infiltration only, the molecular subtypes differed both in terms of immune infiltration and immune compartment compositions. The Basal/Squamous (Ba/Sq) and genomically unstable (GU) tumors displayed increased immune infiltration compared to urothelial-like (Uro) tumors. Additionally, the GU tumors had a higher proportion of regulatory T cells within the immune compartment compared to Uro tumors. Furthermore, sequencing showed higher levels of exhaustion in CD8+ T cells from GU tumors compared to both Uro tumors and the control. Although no such difference was detected at the transcriptomic level in Uro tumors compared to the controls, CD8+ T cells in Uro tumors showed higher expression of several exhaustion markers at the protein level. Taken together, our findings indicate that depending on the molecular subtype, different immunotherapeutic interventions might be warranted.

Mannose-targeting Concanavalin A-Epirubicin Conjugate for Targeted Intravesical Chemotherapy of Bladder Cancer.

Intravesical instillation of chemotherapeutic drugs such as epirubicin (EPI) is routinely used to prevent tumor recurrence and progression after transurethral resection of bladder tumor. However, the lack of tumor selectivity often causes severe damage to normal bladder urothelium leading to intolerable side effects. Here, we analyzed abnormal changes in glycosylation in bladder cancer and identified mannose as the most aberrantly expressed glycan on the surface of bladder cancer cell lines and human bladder tumor tissues. We then constructed a lectin-drug conjugate by linking concanavalin A (ConA) - a lectin that specifically binds to mannose, with EPI through a pH-sensitive linker. This ConA-EPI conjugate conferred EPI with mannose-targeting ability and selectively internalized cancer cells in vitro. This conjugate showed selective cytotoxicity to cancer cells in vitro and better antitumor activity in an orthotopic mouse model of bladder cancer. Our lectin-drug conjugation strategy makes targeted intravesical chemotherapy of bladder cancer possible.

The LINC01929/miR-6875-5p/ADAMTS12 Axis in the ceRNA Network Regulates the Development of Advanced Bladder Cancer.

Considering its speedy development and extremely low 5-year overall survival rate worldwide, bladder cancer (BCa) is one of the most common and highly malignant tumors. Increasing evidence suggests that protein-coding mRNAs and non-coding RNAs, including long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs), play an essential role in regulating the biological processes of cancer. To investigate the molecular regulation associated with poor prognosis during advanced BCa development, we constructed a competitive endogenous RNA (ceRNA) network. Using transcriptome profiles from The Cancer Genome Atlas and Gene Expression Omnibus databases, we performed differential expression (DE) analysis, weighted gene co-expression network analysis, functional enrichment analysis, survival analysis, prediction of miRNA targeting, and Pearson correlation analysis. Through layers of selection, 8 lncRNAs-28 mRNAs and 8 miRNAs-28 mRNAs pairs shared similar expression patterns, constituting a core ceRNA regulatory network related to the invasion, progression, and metastasis of advanced clinical stage (ACS) BCa. Subsequently, we conducted real time qPCR, western blotting, and immunohistochemistry to validate expression trend bioinformatics analysis on 3, 2, and 3 differentially expressed mRNAs, lncRNAs, and miRNAs, respectively. The most significantly differentially expressed LINC01929, miR-6875-5p and ADAMTS12 were selected for in vitro experiments to assess the functional role of the LINC01929/miR-6875-5p/ADAMTS12 axis. RNA pull-down, luciferase assays, and rescue assays were performed to examine the binding of LINC01929 and miR-6875-5p. Increasing trends in COL6A1, CDH11, ADAMTS12, LINC01705, and LINC01929 expression variation were verified as consistent with previous DE analysis results in ACS-BCa, compared with low clinical stage (LCS) BCa. Expression trends in parts of these RNAs, such as hsa-miR-6875-5p, hsa-miR-6784-5p, COL6A1, and CDH11, were measured in accordance with DE analysis in LCS-BCa, compared with normal bladder urothelium. Through experimental validation, the cancer-promoting molecule ADAMST12 was found to play a key role in the development of advanced BCa. Functionally, ADAMTS12 knockdown inhibited the progression of bladder cancer. Overexpression of LINC01929 promoted bladder cancer development, while overexpression of miR-6785-5p inhibited bladder cancer development. Mechanistically, LINC01929 acted as a sponge for miR-6785-5p and partially reversed the role of miR-6785-5p. Our findings provide an elucidation of the molecular mechanism by which advanced bladder cancer highly expressed LINC01929 upregulates ADAMTS12 expression through competitive adsorption of miR-6875-5p. It provides a new target for the prognosis and diagnosis of advanced bladder cancer.

Barrier-Forming Potential of Epithelial Cells from the Exstrophic Bladder

Bladder exstrophy (BEX) is a rare developmental abnormality resulting in an open, exposed bladder plate. Although normal bladder urothelium is a mitotically quiescent barrier epithelium, histologic studies of BEX epithelia report squamous and proliferative changes that can persist beyond surgical closure. The current study examined whether patient-derived BEX epithelial cells in vitro were capable of generating a barrier-forming epithelium under permissive conditions. Epithelial cells isolated from 11 BEX samples, classified histologically as transitional (n = 6) or squamous (n = 5), were propagated in vitro. In conditions conducive to differentiated tight barrier formation by normal human urothelial cell cultures, 8 of 11 BEX lines developed transepithelial electrical resistances of more than 1000 Ω.cm2, with 3 squamous lines failing to generate tight barriers. An inverse relationship was found between expression of squamous KRT14 transcript and barrier development. Transcriptional drivers of urothelial differentiation PPARG, GATA3, and FOXA1 showed reduced expression in squamous BEX cultures. These findings implicate developmental interruption of urothelial transcriptional programming in the spectrum of transitional to squamous epithelial phenotypes found in BEX. Assessment of BEX epithelial phenotype may inform management and treatment strategies, for which distinction between reversible versus intractably squamous epithelium could identify patients at risk of medical complications or those who are most appropriate for reconstructive tissue engineering strategies.

Abstract 6366: Novel imidazolium compounds as intravesical therapeutic agents for non-muscle invasive bladder cancer

Abstract Introduction: Intravesical therapy has critical role in the treatment of high-risk non-muscle invasive bladder cancer. Due to ongoing shortage of BCG and lack of effective alternatives, there is an urgent need for novel intravesical chemotherapeutic agents. In this study, we investigated novel imidazolium compounds on bladder cancer cell lines to characterize activity and mechanism of action and in a mouse model to assess their efficacy and toxicity as intravesical agents. Methods: Three different imidazolium compounds (IS23, TPP1 and TCK1) were screened for their activity against six bladder cancer cell lines at various concentrations and different exposure time. The GI50s for each compound were then used to assess induction of apoptosis in vitro and to study mechanism of action. In vivo efficacy and toxicity of these compounds was studied in normal mice. The BBN mouse model was used to assess in vivo efficacy of these compounds, as it recapitulates the histological and genetic characteristics of human bladder tumors. The drinking water of 6 to 8 weeks old mice was supplemented with 0.05% BBN for 12 weeks, then switched to regular drinking water. Mice were randomly assigned into 5 intravesical treatment groups with 50:50 male:female make up in each group: 1) IS23; 2) TPP1; 3) TCK1; 4) Gemcitabine; 5) vehicle. Drugs and vehicle were instilled for four weeks. Each mouse was subjected to excretory computed tomography urogram to assess the filling defects in the bladder before treatment and every two weeks following the treatment. Mice were euthanized if size of filling defect in urograms exceeded 1/3 of the bladder, developed hydronephrosis, or 4 weeks after completion of intravesical treatment, whichever came first. Results: All imidazolium compounds showed toxicity to bladder cancer cells, TCK1 and TPP1 being the most and least potent respectively. They were also highly effective in killing bladder cancer cells with a 15-min GI90-dose. They briskly induced apoptosis by directly causing Cytochrome C expulsion from isolated mitochondria. Intravesical imidazolium compounds did not cause significant histopathological changes in normal bladder compared to vehicle group, but TPP1 was found to be tumor specific in vivo. TPP1 induced apoptosis in tumor without affecting normal bladder urothelium. In BBN-induced mice, 4/4 IS23-treated mice had filling defects while 8/15, 5/6, 10/11, and 7/8 TPP1-, TCK1-, vehicle-, and gemcitabine-treated mice had filling defects on urograms consistent with tumor. TPP1- and TCK1-treated mice had smaller filling defects compared to vehicle group mice, which was also confirmed histopathologically. Conclusions: TPP1 has a novel and potent mechanism and is worthy of further study as an intravesical agent for the treatment of high-grade superficial bladder cancer. It appears to work through a mitochondrial-centric mechanism and have cancer-specificity. Citation Format: Uttam Satyal, Abhishek Srivastava, David J. Weader, Michael L. Stromyer, Marie R. Southerland, Wiley J. Youngs, Philip H. Abbosh. Novel imidazolium compounds as intravesical therapeutic agents for non-muscle invasive bladder cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6366.

Correlation of miR-100 Expression with Clinopathological Parameters in Bladder Cancer

Objective: Bladder carcinoma is one of the most common tumors in the world and, despite the therapy currently available, most of the patients relapse. MicroRNA- 100 is down regulated in bladder cancer tissue in comparison to normal tissue; however, clinical significance of miR-100 in human bladder cancer has not been elucidated. The aim of study was to correlate miR 100 expression levels with tumor grade, stage and recurrence. Methods: Expression of miR-100 in 26 pairs of bladder cancer and adjacent normal tissues were analysed by Sybergreen RT-PCR. The expression levels of miR 100 in tumor and normal were normalised with 18s gene. The expression levels of miR 100 expression were analysed in fold change (DDCT method). The difference between groups were analysed by student t test and Anova. Results: Downregulation of miR-100 expression in tumor was an important finding in our study. MiR-100 expression were decreased (n=24) in tumour tissue in comparison with normal bladder urothelium. Mean expression of miR-100(fold change) was 0.33 ± 0.35 and significantly decreased on comparison to normal (p=0.001). There is no correlation of miR-100 expression levels with gender and age (p=0.583). Mean expression of miR-100 in low grade was 0.41 ± 0.38 whereas in high grade 0.22 ± 0.26 (p=0.182). Ta tumors had mean (fold change) 0.42 ± 0.39, T1 had mean value of 0.25 ± 0.30 and T 2 had mean value of 0.24 ± 0.30.(p=0.463) miR-100 expression levels were decreased in MIBC(T2) more than NMIBC (Ta,T1) (p=0.473).Mean levels 2^-DDCT (fold change) in recurrent tumors were 0.16 ± 0.19 whereas fold change in nonrecurring tumors was 0.40 ± 0.38.(p=0.045) 1 patient with T2 tumor underwent progression with all patients surviving till end of study. Conclusions: miR-100 expression is down regulated in Bladder cancer tissue than normal bladder urothelium. The miR-100 expression levels were decreased in all bladder tumors however reaching significance in recurrent bladder tumor, suggesting use of miR 100 as potential marker for further risk stratification and prognosis in treatment of urinary bladder cancer.

MicroRNA-100 expression correlation with clincopathological parameters in bladder cancer

Objective: - Bladder carcinoma is one of the most common tumors in the world and, despite the therapy currently available, most of the patients relapse. MicroRNA- 100 is down regulated in bladder cancer tissue in comparison to normal tissue; however, clinical significance of miR-100 in human bladder cancer has not been elucidated. The aim of study was to correlate miR 100 expression levels with tumor grade, stage and recurrence. Methods: - Expression of miR-100 in 26 pairs of bladder cancer and adjacent normal tissues were analysed by Sybergreen RT-PCR. The expression levels of miR 100 in tumor and normal were normalised with 18s gene. The expression levels of miR 100 expression were analysed in fold change (DDCT method). The difference between groups were analysed by student t test and Anova. Results: - Downregulation of miR-100 expression in tumor was an important finding in our study. MiR-100 expression were decreased (n=24) in tumour tissue in comparison with normal bladder urothelium. Mean expression of miR-100(fold change) was 0.33+/-0.35 and significantly decreased on comparison to normal (1) (p=0.001) There is no correlation of miR-100 expression levels with gender and age (p=0.583). Mean expression of miR-100 in low grade was 0.41+/-0.38 whereas in high grade 0.22+/-0.26 (p=0.182). Ta tumors had mean (fold change) 0.42+/- 0.39, T1 had mean value of 0.25 +/- 0.30 and T 2 had mean value of 0.24 +/- 0.30.(p=0.463) miR-100 expression levels were decreased in MIBC(T2) more than NMIBC (Ta,T1) (p=0.473).Mean levels 2^-DDCT (fold change) in recurrent tumors were 0.16+/-0.19 whereas fold change in nonrecurring tumors was 0.40 +/-0.38.(p=0.045) 1 patient with T2 tumor underwent progression with all patients surviving till end of study. Conclusions miR-100 expression is down regulated in Bladder cancer tissue than normal bladder urothelium. The miR-100 expression levels were decreased in all bladder tumors however reaching significance in recurrent bladder tumor, suggesting use of miR 100 as potential marker for further risk stratification and prognosis in treatment of urinary bladder cancer.

Correlation of miR-100 Expression with Clinopathological Parameters in Bladder Cancer

Objective: Bladder carcinoma is one of the most common tumors in the world and, despite the therapy currently available, most of the patients relapse. MicroRNA- 100 is down regulated in bladder cancer tissue in comparison to normal tissue; however, clinical significance of miR-100 in human bladder cancer has not been elucidated. The aim of study was to correlate miR 100 expression levels with tumor grade, stage and recurrence. Methods: Expression of miR-100 in 26 pairs of bladder cancer and adjacent normal tissues were analysed by Sybergreen RT-PCR. The expression levels of miR 100 in tumor and normal were normalised with 18s gene. The expression levels of miR 100 expression were analysed in fold change (DDCT method). The difference between groups were analysed by student t test and Anova. Results: Downregulation of miR-100 expression in tumor was an important finding in our study. MiR-100 expression were decreased (n=24) in tumour tissue in comparison with normal bladder urothelium. Mean expression of miR-100(fold change) was 0.33 ± 0.35 and significantly decreased on comparison to normal (p=0.001). There is no correlation of miR-100 expression levels with gender and age (p=0.583). Mean expression of miR-100 in low grade was 0.41 ± 0.38 whereas in high grade 0.22 ± 0.26 (p=0.182). Ta tumors had mean (fold change) 0.42 ± 0.39, T1 had mean value of 0.25 ± 0.30 and T 2 had mean value of 0.24 ± 0.30.(p=0.463) miR-100 expression levels were decreased in MIBC(T2) more than NMIBC (Ta,T1) (p=0.473).Mean levels 2^-DDCT (fold change) in recurrent tumors were 0.16 ± 0.19 whereas fold change in nonrecurring tumors was 0.40 ± 0.38.(p=0.045) 1 patient with T2 tumor underwent progression with all patients surviving till end of study. Conclusions: miR-100 expression is down regulated in Bladder cancer tissue than normal bladder urothelium. The miR-100 expression levels were decreased in all bladder tumors however reaching significance in recurrent bladder tumor, suggesting use of miR 100 as potential marker for further risk stratification and prognosis in treatment of urinary bladder cancer.

Detection of carcinogen-induced bladder cancer by fluorocoxib A

BackgroundConventional cystoscopy can detect advanced stages of bladder cancer; however, it has limitations to detect bladder cancer at the early stages. Fluorocoxib A, a rhodamine-conjugated analog of indomethacin, is a novel fluorescent imaging agent that selectively targets cyclooxygenase-2 (COX-2)-expressing cancers.MethodsIn this study, we have used a carcinogen N-butyl-N-4-hydroxybutyl nitrosamine (BBN)-induced bladder cancer immunocompetent mouse B6D2F1 model that resembles human high-grade invasive urothelial carcinoma. We evaluated the ability of fluorocoxib A to detect the progression of carcinogen-induced bladder cancer in mice. Fluorocoxib A uptake by bladder tumors was detected ex vivo using IVIS optical imaging system and Cox-2 expression was confirmed by immunohistochemistry and western blotting analysis. After ex vivo imaging, the progression of bladder carcinogenesis from normal urothelium to hyperplasia, carcinoma-in-situ and carcinoma with increased Ki67 and decreased uroplakin-1A expression was confirmed by histology and immunohistochemistry analysis.ResultsThe specific uptake of fluorocoxib A correlated with increased Cox-2 expression in progressing bladder cancer. In conclusion, fluorocoxib A detected the progression of bladder carcinogenesis in a mouse model with selective uptake in Cox-2-expressing bladder hyperplasia, CIS and carcinoma by 4- and 8-fold, respectively, as compared to normal bladder urothelium, where no fluorocoxib A was detected.ConclusionsFluorocoxib A is a targeted optical imaging agent that could be applied for the detection of Cox-2 expressing human bladder cancer.

Surface-Enhanced Raman Scattering Nanoparticles for Multiplexed Imaging of Bladder Cancer Tissue Permeability and Molecular Phenotype

Bladder cancer has the highest recurrence rate of all cancers due in part to inadequate transurethral resection. Inadequate resection is caused by the inability of cystoscopes to detect invisible lesions during the resection procedure. To improve detection and resection of nonmuscle invasive bladder cancer, we quantified the ability of a surface-enhanced Raman nanoparticle and endoscope system to classify bladder tissue as normal or cancerous. Both antibody-based (active) and tissue permeability-based (passive) targeting mechanisms were evaluated by topically applying nanoparticles to ex vivo human bladder tissue samples. Multiplexed molecular imaging of CD47 and Carbonic Anhydrase 9 tumor proteins gave a receiver operating characteristic area under the curve (ROC AUC of 0.93 (0.75, 1.00). Furthermore, passively targeted nanoparticles enabled tissue classification with an ROC AUC of 0.93 (0.73, 1.00). Passively targeted nanoparticles penetrated 5-fold deeper and bound to tumor tissue at 3.3-fold higher concentrations in cancer compared to normal bladder urothelium, suggesting the existence of an enhanced surface permeability and retention effect in human bladder cancer.

MP65-09 NEAR INFRARED PHOTOIMMUNOTHERAPY USING ANTI-CD47 ANTIBODY FOR HUMAN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2018MP65-09 NEAR INFRARED PHOTOIMMUNOTHERAPY USING ANTI-CD47 ANTIBODY FOR HUMAN BLADDER CANCER Bernhard Kiss, Kelly McKenna, Robert Ertsey, Nynke Sjoerdtje Van den Berg, Kathleen Mach, Eben Rosenthal, and Joseph Liao Bernhard KissBernhard Kiss More articles by this author , Kelly McKennaKelly McKenna More articles by this author , Robert ErtseyRobert Ertsey More articles by this author , Nynke Sjoerdtje Van den BergNynke Sjoerdtje Van den Berg More articles by this author , Kathleen MachKathleen Mach More articles by this author , Eben RosenthalEben Rosenthal More articles by this author , and Joseph LiaoJoseph Liao More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2075AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Near infrared photoimmunotherapy (PIT) is a new molecular-targeted cancer therapy that uses a specific photosensitizer based on a near-infrared (NIR) phthalocyanine dye, IRDye700DX, conjugated to a monoclonal antibody. CD47 is a cell surface protein that elicits a ″don′t eat me signal″ to prevent macrophage engulfment and is expressed on over 80% of bladder cancer tumors but absent on the luminal cell layer of normal bladder urothelium. Thus, targeting CD47 for PIT has the potential to selectively induce cell death in CD47 expressing tumor cells as well as increase phagocytosis of tumor cells. METHODS The anti-CD47 antibody (B6H12) was conjugated to IRDye700DX NHS ester and purified using a dialysis spin column. Anti-CD47-IRDye700 concentration was determined with a Coomassie Plus protein assay. The molecular weight of the conjugated antibody was verified by SDS-page and IRDye700 conjugation verified using the 700nm fluorescence channel of a PEARL imager. Binding of anti-CD47-IRDye700DX to CD47 was verified by ELISA. Three different human urothelial cell lines (UMUC3, HT-1376 and 639V) were used for in vitro NIR-PIT experiments. 639V cells were stably transfected with GFP using a lentiviral vector. After plating in a 96well plate and incubation with 10 µg/ml of anti-CD47-IRDye700 or unconjugated anti-CD47 antibody (1h on ice), cells were irradiated with a red light-emitting diode (LED) at 670-710nm wavelength at different energy levels. The cytotoxic effects of NIR-PIT with anti-CD47-IRDye700DX were determined by propidium iodide staining and flow cytometry. To evaluate macrophage activity, a phagocytosis assay using human macrophages and analyzed by flow cytometry was performed. RESULTS Cancer-cell death significantly increased with anti-CD47-IRDye700DX in a light-dose dependent manner in all 3 human bladder cancer cell lines. Significant cancer-cell death was first observed with 2J exposure and when exposed to 40J of NIR light over 90% of 639V and UMUC3 cells and over 50% of HT1376 died. Phagocytosis of cancer-cells incubated with anti-CD47-IRDye700DX and irradiated with 8J was significantly increased compared to anti-CD47-antibody alone (p=0.0002). There was no cytotoxicity associated with anti-CD47 antibody alone with NIR light, with NIR light alone or with anti-CD47-IRDye700DX without NIR light. CONCLUSIONS NIR-PIT with anti-CD47-IRDye700 shows significantly increased cancer-cell cytotoxicity and significantly increased cancer-cell phagocytosis making this combination a highly promising novel approach for molecular targeted bladder cancer therapy. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e862 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Bernhard Kiss More articles by this author Kelly McKenna More articles by this author Robert Ertsey More articles by this author Nynke Sjoerdtje Van den Berg More articles by this author Kathleen Mach More articles by this author Eben Rosenthal More articles by this author Joseph Liao More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The relation between Ring Box-1 protein overexpression and tumor grade and stage in bladder urothelial cell carcinoma.

Ring Box Protein-1 (RBX-1), a component of SCF E3 ubiquitin ligases, has a crucial role in bladder urothelial cell carcinoma (UCC) carcinogenesis and progression. In the present study, it is aimed to determine the expression of RBX-1 protein in bladder UCC and the association between tumor grade, stage and RBX-1 expression. Ninety UCC samples and 20 samples containing foci of normal bladder urothelium were recruited and analyzed immunohistochemically in terms of RBX-1 expression. Immuno-reactivity scoring system (IRS) was used to determine RBX-1 expression levels. RBX-1 overexpression was associated with high tumor grade (p= 0.001) and advanced stage (p= 0.001). pT1 tumors showed higher RBX-1 expression than pTa tumors. pT2 tumors showed not only higher expression than pTa tumors but also higher expression than the total of pTa and pT1 groups combined. There was no statistically significant relation between RBX-1 expression and patient gender (p= 0.116) or age (p= 0.191). In bladder UCC, RBX-1 overexpression is associated with high tumor grade and advanced stage and represents biological potential of invasiveness and aggressive disease. Results of the present study have to be supported with further studies to reveal clinical and therapeutic implications of RBX-1 overexpression in bladder UCC.

The Relation between Survivin Gene Expression and Urinary Bladder Cancer Disease

Purpose: The aim of this study is to investigate the expression of survivin gene in patients suffering from urinary bladder cancer, thus contributing to further understanding of the molecular etiology of the disease by assessment of Survivin gene expression on both RNA and protein levels in urinary bladder tissue samples and Correlate between its expression and pathological prognostic parameters of patients with urinary bladder cancer risk. Methods: In the present study 26 cases of urinary bladder cancer and 26 cases of normal tissues far from tumor sites in the urinary bladder of the same patients (as control samples) were collected, also 10 paraffin-embedded tissue samples from consecutive of archival bladder specimens patients and 10 tissue samples that of healthy tissue from the same individual as control group were collected for immunohistochemical detection of survivin protein and real time PCR for survivin mRNA detection. Results: In normal tissues of control samples no expression for survivin gene has been noticed. However, in patients affected with bladder cancer the survivin expression was highly increased by increasing degree of tumor malignancy as indicated by real time PCR for survivin mRNA and immunohistochemistry for survivin protein. Conclusion: From this study, it is concluded that survivin protein was not expressed in normal bladder urothelium but was present in a high percentage in tissue samples of patients suffering from bladder cancer and its expression is associated with disease recurrence, tumor grade, progression and mortality.

GON4L Drives Cancer Growth through a YY1-Androgen Receptor-CD24 Axis.

In principle, the inhibition of candidate gain-of-function genes defined through genomic analyses of large patient cohorts offers an attractive therapeutic strategy. In this study, we focused on changes in expression of CD24, a well-validated clinical biomarker of poor prognosis and a driver of tumor growth and metastasis, as a benchmark to assess functional relevance. Through this approach, we identified GON4L as a regulator of CD24 from screening a pooled shRNA library of 176 candidate gain-of-function genes. GON4L depletion reduced CD24 expression in human bladder cancer cells and blocked cell proliferation in vitro and tumor xenograft growth in vivo Mechanistically, GON4L interacted with transcription factor YY1, promoting its association with the androgen receptor to drive CD24 expression and cell growth. In clinical bladder cancer specimens, expression of GON4L, YY1, and CD24 was elevated compared with normal bladder urothelium. This pathway is biologically relevant in other cancer types as well, where CD24 and the androgen receptor are clinically prognostic, given that silencing of GON4L and YY1 suppressed CD24 expression and growth of human lung, prostate, and breast cancer cells. Overall, our results define GON4L as a novel driver of cancer growth, offering new biomarker and therapeutic opportunities. Cancer Res; 76(17); 5175-85. ©2016 AACR.

Bladder cancer cells secrete while normal bladder cells express but do not secrete AGR2

Anterior gradient 2 (AGR2) is a cancer-associated secreted protein found predominantly in adenocarcinomas. Given its ubiquity in solid tumors, cancer-secreted AGR2 could be a useful biomarker in urine or blood for early detection. However, normal organs express and might also secrete AGR2, which would impact its utility as a cancer biomarker. Uniform AGR2 expression is found in the normal bladder urothelium. Little AGR2 is secreted by the urothelial cells as no measurable amounts could be detected in urine. The urinary proteomes of healthy people contain no listing for AGR2. Likewise, the blood proteomes of healthy people also contain no significant peptide counts for AGR2 suggesting little urothelial secretion into capillaries of the lamina propria. Expression of AGR2 is lost in urothelial carcinoma, with only 25% of primary tumors observed to retain AGR2 expression in a cohort of lymph node-positive cases. AGR2 is secreted by the urothelial carcinoma cells as urinary AGR2 was measured in the voided urine of 25% of the cases analyzed in a cohort of cancer vs. non-cancer patients. The fraction of AGR2-positive urine samples was consistent with the fraction of urothelial carcinoma that stained positive for AGR2. Since cancer cells secrete AGR2 while normal cells do not, its measurement in body fluids could be used to indicate tumor presence. Furthermore, AGR2 has also been found on the cell surface of cancer cells. Taken together, secretion and cell surface localization of AGR2 are characteristic of cancer, while expression of AGR2 by itself is not.

Novel somatic mutations identified by whole-exome sequencing in muscle-invasive transitional cell carcinoma of the bladder.

Transitional cell carcinoma (TCC) is the one of the most commonly observed types of cancer globally. The identification of novel disease-associated genes in TCC has had a significant effect on the diagnosis and treatment of bladder cancer; however, there may be a large number of novel genes that have not been identified. In the present study, the exomes of two individuals who were diagnosed with muscle-invasive TCC (MI-TCC) were sequenced to investigate potential variants. Subsequently, following algorithm and filter analysis, Sanger sequencing was used to validate the results of deep sequencing. Immunohistochemistry (IHC) was employed to observe the differences in HECT, C2 and WW domain-containing E3 ubiquitin protein ligase 1 (HECW1) protein expression between tumor tissues and para-carcinoma tissues. A total of 6 nonsynonymous mutation genes were identified in MI-TCC, identified as copine VII, RNA binding motif protein, X-linked-like 3, acyl-CoA synthetase medium-chain family member 2A, HECW1, zinc finger protein 273 and trichohyalin. Furthermore, 5 cases were identified to possess a HECW1 gene mutation in 61 MI-TCC specimens, and all of these were point mutations located at exon 11 on chromosome 7. The mutation categories of HECW1 had 4 missense mutations and 1 nonsense mutation. IHC revealed that HECW1 protein was expressed at significantly increased levels in MI-TCC compared with normal bladder urothelium (P<0.001). The present study provided a novel approach for investigating genetic changes in the MI-TCC exome, and identified the novel mutant gene HECW1, which may possess a significant role in the pathogenesis of TCC.

The effect of thermochemotherapy with mitomycin C on normal bladder urothelium, an experimental study.

To investigate the effects of thermochemotherapy with mitomycin C (MMC) on normal rabbit bladder urothelium and to compare it with standard intravesical MMC and hyperthermia with normal saline. Twenty-four male New Zealand rabbits, with a mean weight of 2.7 kg (in weight of 2.1–4.3 kg), were divided into three groups, each containing eight rabbits. Thermotherapy with only normal saline was performed in the first group, standard intravesical MMC was performed in the second group, and thermotherapy with MMC was performed in the last group. A week after the primary procedure, total cystectomy was performed and tissue samples were evaluated. The presence of epithelial vacuolar degeneration (p = 0.001), epithelial hyperplasia (p = 0.000), subepithelial fibrosis (p = 0.001) and hemorrhagic areas in the connective tissue (p = 0.002) was observed statistically significantly higher in the standard MMC group than in thermotherapy with normal saline group. There was almost a significant difference among standard MMC and normal saline group in terms of vascular congestion in the connective tissue (p = 0.08). Presence of epithelial vacuolar degeneration (p = 0.002), epithelial hyperplasia (p = 0.002), subepithelial fibrosis (p = 0.030), hemorrhagic areas (p = 0.011) and vascular congestion (p = 0.36) in the connective tissue was observed statistically significantly higher in the thermochemotherapy with MMC group than in standard intravesical MMC group. Polymorphonuclear cell infiltration was not considerable in any of the groups, and there was no significant difference between each groups (p = 0.140). Administration of intravesical MMC causes a toxic effect on the normal urothelium of the bladder rather than an inflammatory reaction. Heating MMC significantly increased this effect.

Utility of SAM68 in the progression and prognosis for bladder cancer.

BackgroundMuscle invasive bladder cancer (MIBC) is often lethal and non-MIBC (NMIBC) can recur and progress, yet prognostic markers are currently inadequate. SAM68, a member of RNA-binding proteins, has been reported to contribute to progression of other cancers. The aim of this study is to investigate the potential utility of SAM68 in the progression and prognosis of bladder cancer.MethodsQuantitative PCR and immunohistochemistry were utilized to examine the expression of SAM68 in ten pairs of MIBC and adjacent normal bladder urothelium, and eight pairs of MIBC and non-MIBC (NMIBC) tissues from the same patient. Moreover, SAM68 protein expression level and localization were examined by immunohistochemistry in 129 clinicopathologically characterized MIBC samples. Prognostic associations were determined by multivariable analysis incorporating standard prognostic factors.ResultsSAM68 expression was elevated in MIBC tissues compared with adjacent normal bladder urothelium, and was increased at both transcriptional and translational levels in MIBC tissues compared with NMIBC tissues of the same patient. For MIBC, high expression and nucleus-cytoplasm co-expression of SAM68 were associated with higher T-stage, higher N-stage and worse recurrence-free survival. Five-year recurrence-free survival was 80% and 52.9% for MIBC patients with low and high SAM68 expression, respectively (p = 0.001). SAM68 nucleus-cytoplasm co-expression associated with worse 5-year recurrence-free survival rate (49.2%) than SAM68 expression confined to the nucleus (82.5%) or cytoplasm (75.5%) alone. On multivariable analysis SAM68 expression level, SAM68 nucleus-cytoplasm co-expression, T-stage, and N-stage were all independent prognostic factors for recurrence-free survival of MIBC patients.ConclusionsSAM68 expression is increased in MIBC when compared to normal urothelium and NMIBC, and appears to be a potentially useful prognostic marker for MIBC.

Association between PSCA mRNA expression levels and rs2294008 polymorphism in transitional cell cancer of the bladder.

Prostate stem cell antigen (PSCA) was originally identified as a gene that is overexpressed in prostate cancer, and correlates with prostate cancer progression and prognosis. Recently, a significant association has been identified between the PSCA rs2294008 (C>T) polymorphism and the risk of developing bladder cancer. Therefore, the present study investigated the different expression levels of PSCA mRNA in transitional cell carcinoma (TCC) of the bladder and normal bladder tissue. Furthermore, the association between PSCA mRNA expression levels in TCC and different rs2294008 (C>T) genotypes and various clinicopathological features, including tumor stage and grade, were evaluated. Reverse transcription-quantitative polymerase chain reaction was performed on 80 TCC samples and 38 samples of normal bladder urothelium from TCC patients who underwent transurethral resection of bladder tumor or radical cystectomy at the Beijing Friendship Hospital (Beijing, China) between September 2010 and May 2011. Genomic DNA was extracted from tumor tissue and sequenced to determine the rs2294008 (C>T) genotype. PSCA mRNA expression was detected in all samples (100%); however, tumor samples exhibited significantly higher PSCA expression levels compared with the normal urothelium samples (P=0.038). PSCA mRNA expression was positively correlated with the histological grade of the tumor (G1-2 vs. G3; P=0.001); however, no significant difference was detected between patients with superficial (Ta or T1) and muscle-invasive (≥pT2) tumors (P=0.250). Thus, PSCA mRNA expression levels were associated with TCC and tumor histological grade, but not the tumor stage. Additionally, PSCA mRNA expression levels were significantly higher in T allele carriers compared with CC homozygous patients (P=0.001), indicating that the presence of the T allele may increase PSCA mRNA expression. Therefore, rs2294008 (C>T) may be associated with the biological properties of TCC and, thus, future research should focus on the physiological function of PSCA and the mechanism of rs2294008.

NOV is upregulated and promotes migration and invasion in bladder cancer

NOV is pro-tumourigenic via epithelial-mesenchymal transition (EMT) in several malignancies but is not studied in bladder cancer (BCa). Whether NOV is responsible for bladder carcinogenesis and the underlying mechanism is unclear. Using immunohistochemical staining, we quantified expressions of NOV, pS6, Vimentin and E-cadherin in 66 bladder cancer and 10 normal bladder urothelium samples. EMT was profiled by EMT index (EMTi) calculated as the ratio of Vimentin to E-cadherin. In vitro and in vivo studies were carried out to profile the role of NOV in the tumourigenesis of BCa. NOV was upregulated in bladder cancer compared to normal tissue, and its expression was correlated to pS6 and EMTi. Expression of NOV was higher in recurrent and multiple tumours and was increased with progression of tumour grade. NOV expression was also higher in BCa cell lines. Silence of NOV attenuated EMT, decreased invasion and migration of BCa cells. Silence of NOV also inhibited xenograft tumour growth and decreased tumour EMT. NOV is pro-tumourigenic in bladder cancer especially in nonmuscle-invasive entities (NMIBC). NOV may promote carcinogenesis via promotion of EMT and association with increased mTOR activity.