Normal Urothelium Research Articles

The Role of MiR-124 in Bladder Cancer – A Review of the Literature

Abstract MicroRNAs (miRNAs) are a group of non-coding RNA molecules that have an important role in modulating the expression of genes involved in regulating cellular functions. A growing number of studies suggest the abnormal expression of microRNAs in different types of cancer cells. MiRNA-124 is a microRNA that is down-regulated in many types of cancer cells, including bladder cancer. Our objective is to provide a review of the key publications that studied the effect of miR-124 on bladder cancer. This review focus on the targets and different pathways of miR-124 that were identified in various studies and differences between their expressions in normal urothelium and tumor tissues. We also include data regarding urinary methylations levels of miR-124 and their role in bladder cancer diagnosis and prognosis. Subsequently, we establish future perspectives of miR-124 research and its promising role in bladder cancer.

Chloride Intracellular Channel Protein 1 (CLIC1) Ιs Over-expressed in Muscle Invasive Urinary Bladder Cancer.

Invasive bladder cancer mortality remains high despite progresses made in early diagnosis and surgical procedures. Thus, there is a need to define new markers for bladder cancer. CLIC1 has not been previously studied in bladder cancer and thus, we aimed to assess its immunohistochemical expression in relation to different stages of bladder cancer development. Immunohistochemistry for CLIC1 was applied in 50 cases of muscle invasive bladder cancer. CLIC1 was not expressed in the normal urothelium, but a strong reaction was observed in dysplastic urothelium, carcinoma in situ and in 94% of the cases with invasive urothelial carcinoma; however, it was not expressed in squamous cell carcinoma cases. No correlation was found between the immunohistochemical expression of CLIC1 and the stage and grade of the tumour. CLIC1 was overexpressed in urinary bladder dysplastic epithelium, carcinoma in situ and invasive carcinoma. CLIC1 constitutes a new potential marker of invasive bladder cancer.

Mutational signature modelling in vitro recapitulates bladder cancer initiation

Smoking is the best established risk factor for bladder cancer and direct mutagenesis by smoke carcinogens on the cellular genome can be described in clonal populations as mutational signatures. A mutational signature summarises a lifetime of DNA-damage (caused by interaction between mutagenic processes, tissue-specific gene transcription, and the DNA-repair machinery) as mutational classes in the context of their local genomic chemistry. Our aim was to establish a novel experimental model for understanding carcinogenic initiation in normal human urothelium. Finite normal human urothelial cell cultures were established in vitro and differentiated to form functional barrier epithelia capable of activating the smoke-derived procarcinogen benzo[a]pyrene (BaP), via CYP1A1, into mutagenic metabolites. New in vitro approaches were developed allowing chronic BaP-exposure, subsequent clonal expansion of NHU cells for whole genome DNAseq and mutational signature derivation. This approach retains tissue heterogeneity during the carcinogenic exposure, modelling the early selection pressures in urothelium that lead to clonal expansion and cancer. The single-base (SBS) and double-base signatures (DBS) derived from BaP-exposed urothelial tissues were dominated by C>A and CC>AA transversions, respectively. The InDel (ID) signature described cytosine deletions in homopolymer regions. Comparison to the Catalogue of Somatic Mutations in Cancer (COSMIC) using the Signal pipeline showed homology with SBS4, DBS2 and ID3, respectively; which have been detected by others in bladder cancer patients. Mutation enrichment in specific genes (eg KMT2D and CDKN1A) mirrored in situ observations by others; effectively modelling the competitive advantage mutations convey in the carcinogenic tissue environment. BaP is detected in urine, metabolically activated by urothelial CYP1A1, and therefore may be a bladder mutagen. However, C>A transversions contribute a minor fraction of all mutations recorded in bladder tumours. Widespread DNA damage caused by chronic BaP exposure did not trigger any of the APOBEC-like mutational processes (SBS2/13) that dominate bladder tumour genomes, indicating a missing factor that the new model system is well positioned to identify.

Heterogeneity-analysis of molecular subtypes of muscle-invasive bladder cancer and their precursor lesions in multiregion mapped whole-organ bladders

In the recent years, molecular subtypes of urothelial bladder cancer have been described which seem to have a predictive effect for the efficacy of chemotherapy regimens and therefore are now under clinical investigation. Due to this clinical implication and possible decision guidance, the aim of our study is to characterize in which extent precursor lesions and tumors exhibit a heterogeneity of molecular subtypes, which possibly could influence response to chemotherapy regimens. 23 positions of three whole-organ mapped bladder specimens were histomorphologically reviewed for classifying location of normal urothelium, precursor lesions and different areas of the tumor. Immunohistochemical analysis of luminal (CK20, GATA3, FOXA1) and basal markers (CD44, CK14 and CK5/6) was carried out in selected precursor lesions and tumors to map the distribution of luminal and basal subtypes across the different positions/lesions. Out of three analyzed multiregion mapped bladder tumors, one showed a heterogenous luminal and double-negative molecular subtype in multiple tumor positions as well as a mixed morphology including conventional urothelial and neuroendocrine areas. Moreover, among the second analyzed bladder tumor, all tumor positions showed a high expression of basal markers whereas the included Carcinoma in situ demonstrated a luminal subtype with almost total absence of basal markers. The third analyzed mapped bladder tumor demonstrated a homogenous luminal molecular subtype in all precursor and tumorous lesions. This first analysis of three multiregion mapped bladder tumors shows divergent results of subtype distribution: Heterogeneity of subtypes can be observed, but other tumors and associated precursor lesions show a homogenous distribution of subtypes. To further elucidate how often subtype heterogeneity occurs in whole multiregion mapped bladder tumors, data of further bladder specimens will be presented at the meeting.

Transition of ANXA10 expression is a useful diagnostic and prognostic marker in upper tract urothelial carcinoma

Little is known about the diagnostic and prognostic markers of upper urinary tract urothelial cancer (UTUC) because of its rarity. ANXA10, a calcium dependent phospholipid binding protein, is differentially expressed in several cancers. It has been reported that ANXA10 expression is a urinary mRNA biomarker for detection of bladder urothelial cancer (BUC), and loss of ANXA10 expression is associated with BUC progression. To clarify the significance of ANXA10 in UTUC, we studied ANXA10 expression with immunohistochemistry (IHC). We analyzed ANXA10 by IHC in 117 respective cases of UTUC treated by radical nephroureterectomy without neoadjuvant chemotherapy. ANXA10 positivity (ANXA10+) was defined as >10 % of nuclear staining. ANXA10 expression was weak in normal urothelium of upper tract and was positive in 39/117 (33%) of UTUC. ANXA10+ was more frequently in tumors with pure UC (36%, p<0.05), papillary morphology (50%, p<0.01), histological grade 1/2 (G1/2: 57%, p<0.01) and pTa/is/1 stage (55%, p<0.01) than in those with histological variants (0%), nodular morphology (9%), G3 (16%) and pT2/3/4 (13%), respectively. Patients with ANXA10+ showed better cancer specific survival (CSS) than those with ANXA10- (P<0.05). With IHC, ANXA10+ was detected more in TP53 negative cases (44%, P<0.01) and Uroplakin 3 (UPK3) positive cases (44%, p= 0.089) than in TP53 positive cases (6%) and UPK3 negative cases (28%), respectively. In TCGA dataset of MIBC, higher ANXA10 expression was correlated with papillary morphology, lower grade/stage, luminal papillary subtype and wild type of TP53. ANXA10 expression increases during carcinogenesis, and is observed more frequently in papillary UC with lower grade and stage. However, its expression decreases during cancer progression. Transition of ANXA10 expression in UTUC mimicking BC is clinically useful for decision-making.

Expression of the epigenetic H3K27me3 modifier genes KDM6A and EZH2 in patients with upper tract urothelial carcinoma.

The development of upper tract urothelial carcinoma (UTUC) has been associated with the ingestion of aristolochic acid (AA) in Chinese herbs. The tumors are more malignant and patients have a worse prognosis in China compared with that in Western countries. Recently, whole-genome and exome sequencing of AA-associated UTUCs found that the most frequently mutated gene was lysine demethylase 6A (KDM6A). However, its biological role and clinical significance have not yet been defined in patients with UTUC in China. A total of 108 surgically resected UTUC samples were obtained. Using immunohistochemistry, the protein expression level of KDM6A in the tumors was investigated together with the clinical and pathological characteristics of the patients, including survival times. In the present study, the expression level of KDM6A was significantly lower in UTUC specimens compared with that in samples from the normal urothelium. Lower KDM6A expression was also found to be significantly associated with a higher tumor grade and shorter cancer-specific and disease-free survival times (P=0.023 and P=0.033, respectively). In addition, using immunohistochemical analysis, no positive association was found between KDM6A expression and the expression of H3K27me3 or histone-lysine N-methyltransferase EZH2, a histone methyltransferase that generates H3K27me3. The results of the present study indicated that decreased KDM6A expression level was significantly associated with tumor grade and decreased survival time in UTUC, suggesting that KDM6A expression could be used as a prognostic marker in patients with UTUC in China.

Macroscopic somatic clonal expansion in morphologically normal human urothelium.

Knowledge of somatic mutation accumulation in normal cells, which is essential for understanding cancer development and evolution, remains largely lacking. In this study, we investigated somatic clonal events in morphologically normal human urothelium (MNU; epithelium lining the bladder and ureter) and identified macroscopic clonal expansions. Aristolochic acid (AA), a natural herb-derived compound, was a major mutagenic driving factor in MNU. AA drastically accelerates mutation accumulation and enhances clonal expansion. Mutations in MNU were widely observed in chromatin remodeling genes such as KMT2D and KDM6A but rarely in TP53, PIK3CA, and FGFR3 KMT2D mutations were found to be common in urothelial cells, regardless of whether the cells experience exogenous mutagen exposure. Copy number alterations were rare and largely confined to small-scale regions, along with copy-neutral loss of heterozygosity. Single AA-associated clones in MNU expanded to a scale of several square centimeters in size.

Mutational selection in normal urothelium.

Mutations in normal tissue point to causes of DNA damage and set the stage for cancer

Abstract 3147: Non-invasive detection of aristolochic acid exposure using ultra-sensitive duplex sequencing

Abstract Aristolochic acids (AA) are a potent class of mutagen that occur in plants of the genera Aristolochia and Asarum. Exposures to AA are caused both by intentional ingestion of traditional herbal medicines containing Aristolochia and through inadvertent consumption from contaminated crops. The carcinogenic potential and epidemiologic connection of AA exposure with upper-tract urothelial cancers (UTUC), liver cancer and kidney failure have been known for years; however, the extent of AA exposure and its influence on cancer risk and mortality have yet to be fully explored in many world populations. Using whole exome sequencing of tumor samples, a recent study determined that 78% of HCCs from Taiwan exhibit distinct mutation signatures of AA exposure (Ng, Poon, Huang, et al., 2017). Exposure to AA is geographically widespread and there is a major opportunity for primary and secondary prevention of AA-associated diseases if it were possible to detect the exposure through non-invasively sampled body fluids such as blood or urine sediment. Although AA adducts can be measured in body fluids, this measurement only reflects recent ingestion, not the life-integrated exposure that better approximates disease risk. A mutation burden-based assay could circumvent this problem; however, a major challenge is that AA-mediated mutations are extremely rare in bulk tissue or fluid samples from non-tumor sources. Duplex Sequencing (DS) is a tag-based NGS error-correction method that enables the detection of ultra-rare mutations that exist at the mutation frequency of normal tissues (~10e-8). DS can be applied to DNA from any tissue of any organism without the need for tumor formation or ex vivo cloning. We developed a DS-based assay that measures mutations across a 50 kb genome-representative panel and applied it to the tissues and liquid biopsies of cancer patients of known AA-exposure status. We sampled the blood, urine, kidney, normal-adjacent urothelium and ureteral tumors from 12 UTUC patients where 6 of the patients were exposed to AA, and 6 were not. Under blinded conditions, we positively identified AA mutation signatures in the normal adjacent urothelium tissues of 5 AA-exposed patients. Remarkably, we also detected AA signatures in all available blood and urine samples from the same patients. The proportion of mutations attributed to AA-exposure ranged from 2.5% in blood to 67.8% in kidney. These mutations, on the order of one-in-a-million, were exposure-related, not tumor derived. We did not detect AA mutation signatures in any samples from the non-AA-exposed patients. To our knowledge, this is the first time an AA mutation signature has been detected in non-invasively sampled body fluids. DS is a promising approach for the detection of mutagenic signatures caused by environmental carcinogens, and we foresee it being a powerful metric that can be used to measure life-integrated carcinogenic processes and cancer risk. Citation Format: Fang Yin Lo, Charles Valentine, Elizabeth Schmidt, Lindsey Williams, Arnoud Boot, Steve Rozen, Jesse Salk. Non-invasive detection of aristolochic acid exposure using ultra-sensitive duplex sequencing [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 3147.

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.

Abstract PR02: Profiling intratumoral heterogeneity of bladder cancer subtypes at the single-cell level using machine-learning assisted histopathology

Abstract Purpose: Machine-learning assisted histopathology using markers of basal and luminal differentiation was employed to profile the intratumoral heterogeneity of bladder cancer from cystectomy patients and predict disease-free survival in this high-risk patient population. Methods: Urothelial carcinomas are biologically heterogeneous and vary greatly in clinical progression as well as treatment response. Delineation of molecular subtypes by gene expression analysis of luminal and basal markers has indicated differential outcomes associated with basal and luminal subtypes. However, histologic validation of this classification using protein markers (basal = KRT5/6, P63; luminal = KRT20/GATA3) has been challenging. While using multiplex-immunofluorescence to subtype a retrospective cystectomy cohort (a TMA of 380 patients), we determined that nearly 50% of tumors did not exhibit cytokeratin markers. Subtyping was further confounded by frequent loss of basal-to-luminal stratification and the emergence of intratumoral spatial heterogeneity with the basal and luminal subtypes being completely intermixed throughout the tumor. These observations caused us to hypothesize that previously undefined but clinically relevant subtypes might exist. To address this challenge we developed a single-cell image analysis pipeline that leveraged machine learning to classify molecular subtype and spatial heterogeneity within each tumor. Using the informatics software KNIME we achieved single-cell segmentation and extracted 285 features for 5 protein markers (P63, GATA3, collagen, nuclear stain, and pan-cytokeratin) from each ~20,000 cells contained in 2 cores of tumor and adjacent benign for each patient. Under guidance from a pathologist, definitive urothelial cells (luminal, intermediate, and basal cells) as well as stromal cells were selected from 25 cores normal urothelium to form the ground truth for XGboost-based machine-learning. Summary Findings: Single-cell profiling with machine learning on transcription factors could classify basal and luminal subtypes with greater than 97% accuracy according to validation in normal urothelium using keratin markers. While we were able to recapitulate differential survival associated with a pure basal subtype, it was the intratumoral heterogeneity of basal and luminal cells that was the predominant driver of disease-free survival. Conclusion: A newly identified bladder cancer subtype defined by intratumoral heterogeneity is a clinically relevant driver of disease-free survival. This abstract is also being presented as Poster B22. Citation Format: Adel Eskaros, Tatiana Novitskaya, Andries Zijlstra. Profiling intratumoral heterogeneity of bladder cancer subtypes at the single-cell level using machine-learning assisted histopathology [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr PR02.

Microtubule-organizing center-mediated structural atypia in low- and high-grade urothelial carcinoma

Urothelial carcinoma (UC) comprises two subtypes, low grade (LG-UC) and high grade (HG-UC), with different pathological and clinical behavior. LG-UC and HG-UC are classified based on cellular and structural atypia of pathological findings. The mechanisms responsible for maintaining structural atypia, such as the disturbance of nuclear polarity, remain unclear. In this study, we studied microtubule-organizing center (MTOC)-mediated nuclear polarity in UC subtypes. We evaluated six cases with normal urothelium (NU), 10 LG-UC cases, and 10 HG-UC cases by double immunofluorescence staining of γ-tubulin as a marker of MTOC and E-cadherin as a marker of each cell border. The number and position of γ-tubulin dots of expression in more than 100 cells per case were assessed using the spatial relationship with the nucleus and surface-basal axis. We found one γ-tubulin dot in most normal and tumor cells, and more than two γ-tubulin dots in 4.6% of NU cells, 6.1% of LG-UC cells, and 9.8% of HG-UC cells. More than three γ-tubulin dots were found only in 1.2% of HG-UC cells. Surface side positioning of γ-tubulin was found in 77.4% of normal urothelial cells, 63.8% of LG-UC cells, and 39.2% of HG-UC cells, whereas aberrant lateral and basal side positioning of γ-tubulin was found in 22.6% of normal urothelial cells, 36.1% of LG-UC cells, and 60.8% of HG-UC cells. We concluded that numerical and positional aberrations of MTOC in UC cases were strongly correlated with both cellular and structural atypia as well as abnormal cell proliferation.

Combined lectin- and immuno-histochemistry (CLIH) for applications in cell biology and cancer diagnosis: Analysis of human urothelial carcinomas.

Lectin histochemistry (LHC) and immunohistochemistry (IHC), which demonstrate the composition and localisation of sugar residues and proteins in cell membranes, respectively, are generally used separately. Using these two methods, we previously demonstrated that malignant transformation of urothelial cells results in the alterations of protein glycosylation and reduced expression of urothelium-specific integral membrane proteins uroplakins (UPs). However, the correlation between these changes was not studied yet. To evaluate this correlation, we developed innovative method, which we named Combined Lectin- and Immuno-Histochemistry (CLIH). We used human biopsies of 6 normal urothelia and 9 papillary urothelial carcinomas, i.e. 3 papillary urothelial neoplasms of low malignant potential (PUNLMP), 3 non-invasive papillary urothelial carcinomas of low grade (pTa, l.g.), and 3 invasive papillary urothelial carcinomas of high grade (pT1, h.g.). We tested five different protocols (numbered 1-5) of CLIH on paraffin and cryosemithin sections and compared them with LHC and IHC performed separately. Additionally, we carried out western and lectin blotting with antibodies against UPs and lectins Amaranthus caudatus agglutinin (ACA), Datura stramonium agglutinin (DSA), and jacalin, respectively. We showed that incubation with primary antibodies first, followed by the mixture of secondary antibodies and lectins is the most efficient CLIH method (protocol number 5). Additionally, 300 nm thick cryo-semithin sections enabled better resolution of co-localisation between sugar residues and proteins than 5 mm thick paraffin sections. In the normal urothelium, CLIH showed co-localisation of lectins ACA and jacalin with UPs in the apical plasma membrane (PM) of superficial umbrella cells. In papillary urothelial carcinomas, all three lectins (ACA, DSA and jacalin) labelled regions of apical PM, where they occasionally co-localised with UPs. Western and lectin blotting confirmed the differences between normal urothelium and papillary urothelial carcinomas. Our results show that CLIH, when used with various sets of lectins and antigens, is a useful, quick, and reliable method that could be applied for basic cell biology research as well as detailed subtyping of human urothelial carcinomas.

TERT C228T mutation in non-malignant bladder urothelium is associated with intravesical recurrence for patients with non-muscle invasive bladder cancer.

Telomerase reverse transcriptase (TERT) promoter mutations are frequently found in tumors or urine from patients with urothelial carcinoma (UC). TERT promoter mutations are also detected in urine from patients with no evidence of cancer but are associated with subsequent UC development. Mutations in the TERT promoter are thought to be present in nonmalignant urothelium (NMU) during early stages of tumor formation prior to pathological change, but this has not been proven directly. In this proof‐of‐concept study, we investigated the clinical utility of TERT promoter mutation analysis in NMU of patients with non‐muscle‐invasive bladder cancer (NMIBC). This single‐institute study included 53 primary tumors and 428 systematic bladder biopsy specimens from 54 patients with NMIBC. All patients underwent systematic random biopsy and transurethral resection of the bladder tumor. Genomic DNA was analyzed for TERT C228T and C250T mutations using droplet digital PCR (ddPCR). The association between TERT promoter mutation of NMU and bladder recurrence was examined by the Kaplan–Meier method and Cox proportional hazards model. Of the 54 patients, 16 (29.6%) had a TERT C228T mutation and three (5.6%) had a TERT C250T mutation in NMU. Of 428 biopsy specimens, the TERT C228T mutation was detected in 9% (31/364) of normal urothelium, 27% (4/15) of urothelial dysplasia (UD), 50% (9/18) of UD suspicious for carcinoma in situ (CIS), and 58% (18/31) of CIS. During follow‐up (median: 3.7 years), 22 (40.7%) patients experienced bladder recurrence and five (9.3%) experienced disease progression. Cox proportional hazard analysis showed that TERT C228T mutation in NMU was significantly associated with bladder recurrence after adjustment for cofounding factors (P = 0.0128). Thus, TERT C228T mutation was detected in NMU, which was a reliable independent prognostic factor of bladder tumor recurrence.

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.

Abstract B12: Carcinogen exposure alters keratin 5 expression and K5-Cre recombination in transgenic mouse urothelium

Abstract In the urinary bladder, the normal urothelium is divided into three subpopulations of cells: superficial umbrella cells, intermediate cells, and a basal layer, each with distinct expression patterns of uroplakins (UP) and keratin 5 (K5). Many current transgenic mouse models (Tg) for urinary bladder cancer rely on uroplakin or keratin promoters to drive inducible Cre-lox recombination in basal or luminal bladder urothelial cells. Further, many models utilize the chemical carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), to drive or accelerate tumorigenesis. A major limitation in Tg model systems where genes are knocked out is the requirement for highly penetrant and reproducible Cre activity and subsequent gene deletion in order to see a phenotype. This problem is compounded in long-term experiments such as BBN carcinogenesis that can run for up to six months after Cre induction. We have previously shown that Trim29 (tripartite motif containing 29) is sufficient to drive invasive bladder cancer formation in Tg models and in human bladder tumors. We have now developed a Tg system utilizing K5-Cre-ERT2 to target Trim29 for deletion in K5-expressing basal urothelial cells. In this model, tamoxifen exposure results in Cre expression and Trim29 deletion in K5 basal cells. Whether this would translate into complete loss of Trim29 in the urothelium over time and what effect BBN might have on tamoxifen induction of K5-Cre and loss of Trim29 were unclear. We found that exposure of K5-Cre-ERT2, Trim29 flox/flox mice to tamoxifen for six weeks resulted in loss of Trim29 expression only in the K5+ basal urothelium and that these K5+ Trim29 -/- basal cells did not give rise to intermediate cells or umbrella cells during the six months of follow-up. In contrast, mice treated with 0.05% BBN in drinking water for six months after induction of Cre produced full thickness of Trim29 knockout (KO) in 50% of mice (8/16 mice). Our results indicate that in a nonstressed state, K5 basal cells give rise to other basal cells but do not repopulate intermediate or umbrella cell layers. Furthermore, exposure to BBN resulted in the proliferation of basal cells and the replacement of intermediate cells by basal precursors in the urothelium. These results suggest that K5-Cre promoters may have different effects on transgene recombination in the bladder in the presence or absence of BBN, which induces more universal expression of Cre and its subsequent recombination events. These findings should be considered when interpreting and developing BBN-induced bladder cancer Tg models. Citation Format: Alan Kelleher, Yin Wang, Luke Broses, Sylvia Zelenka-Wang, Phillip L. Palmbos. Carcinogen exposure alters keratin 5 expression and K5-Cre recombination in transgenic mouse urothelium [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B12.

Two diagnostic criteria of optical spectroscopy for bladder tumor detection: Clinical study using 5-ALA induced fluorescence and mathematical modeling.

The study proposes to improve bladder cancer diagnosis by photodynamic diagnosis (PDD) using red-light excitation (632.8 nm) of 5-ALA induced-protoporphyrin IX. Employing 9 patients' bladders, two types of signals were used to improve diagnostic accuracy for malignancy and we also present numerical modeling of the scattering coefficient to provide biological explanation of the results obtained. Two modalities of bladder cancer spectral diagnosis are presented: conventional PDD and intensity assessment of the diffusely reflected laser light by fiber-optic spectroscopy. Experiments are done in clinical conditions and as a series of numerical simulations. High-grade cancerous bladder tissues display twice a higher relative fluorescence intensity (mean value 1, n = 9) than healthy (0.39, n = 9), dysplastic (0.44, n = 5) tissues and CIS (0.39, n = 2). The laser back-scattering signal allows to discriminate most effectively high-grade cancerous and dysplastic tissues from normal. Numerical modeling of diffuse reflectance spectra reveals that spectral behavior of the back-scattered light depends on both, nuclear size and nuclear density of tumoral cells. Unlike the fluorescence signal, where its value is higher in the case of pathological tissues, the tendency of the laser signal to, both, decrease or increase in comparison with the signal from normal urothelium, should be perceived as a sign towards neoplasm. Numerical simulation reveals that such a double-analysis at a multiwavelength mode potentially may be used to provide diagnostic accuracy.

Downregulation of TET1 Promotes Bladder Cancer Cell Proliferation and Invasion by Reducing DNA Hydroxymethylation of AJAP1.

Ten-eleven translocation 1 (TET1) is a member of methylcytosine dioxygenase, which catalyzes 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5 hmC) to promote the demethylation process. The dysregulated TET1 protein and 5 hmC level were reported to either suppress or promote carcinogenesis in a cancer type-dependent manner. Currently, the role of TET1 in the development of urinary bladder cancer (UBC) and its underlying molecular mechanisms remain unclear. Herein, we found that TET1 expression was downregulated in UBC specimens compared with normal urothelium and was inversely related to tumor stage and grade and overall survival, suggesting its negative association with UBC progression. TET1 silencing in UBC cells increased cell proliferation and invasiveness while the ectopic expression of wild-type TET1-CD, but not its enzymatic inactive mutant, reversed these effects and suppressed tumorigenicity in vivo. In addition, as a direct regulator of TET1 activity, vitamin C treatment increased 5 hmC level and inhibited the anchorage-independent growth and tumorigenicity of UBC cells. Furthermore, we found that TET1 maintained the hypomethylation in the promoter of the AJAP1 gene, which codes for adherens junction-associated protein 1. The downregulation of AJAP1 reversed TET1-CD-induced nuclear translocation of β-catenin, thus inhibiting the expression of its downstream genes. In human UBC specimens, AJAP1 is frequently downregulated and positively associated with TET1. Notably, low expression levels of both TET1 and AJAP1 predict poor prognosis in UBC patients. In conclusion, we found that the frequently downregulated TET1 level reduces the hydroxymethylation of AJAP1 promoter and subsequently activates β-catenin signaling to promote UBC development. The downregulation of both TET1 and AJAP1 might be a promising prognostic biomarker for UBC patients.

Biogenic silver nanoparticles: In vitro and in vivo antitumor activity in bladder cancer

Bladder cancer is the fifth most common disease in the United States, and the treatment and alternatives for patients have not changed in the last decades. Silver nanoparticles (AgNP) have been used in the treatment of various cancer, mainly because of the antineoplastic activity; however, their use and the molecular mechanisms towards bladder cancer still unexplored. Therefore, this work aims to evaluate the in vitro and in vivo antitumoral mechanisms of biogenic silver nanoparticles synthesized from Fusarium sp. First, AgNP showed cytotoxicity in a dose- and time-response relationship and detailed analysis demonstrated the induction of cell death via apoptosis, also inhibiting cell migration and proliferation in bladder carcinoma cell line 5637. Next, it was evaluated the antitumoral activity of AgNP against non-muscle invasive bladder cancer (NMIBC). Bladder cancer was chemically induced with N-methyl-N-nitrosourea (MNU) on C57BL/6JUnib female mice and treated by intravesical route with AgNP concentrations of 0.5, 0.2, and 0.05 mg/mL. Finally, treatment with AgNP (0.05 mg/mL) led to 57.13% of tumor regression, with 14.28% of the animals showing normal urothelium, and 42.85% showing flat hyperplasia, considered to be a benign lesion. Overall, these findings demonstrated that AgNP might be a cost-effective alternative and promising candidate for the treatment of bladder cancer.

Evaluation of a Ruthenium coordination complex as photosensitizer for PDT of bladder cancer: Cellular response, tissue selectivity and in vivo response

AbstractTLD1433, a Ru(II) polypyridyl complex, was recently introduced. It can be activated in the 520 to 540 nm range. Here we report on the use of TLD1433 for the treatment of bladder carcinomas in vitro and in an in vivo orthotopic model. The electrostatic properties of the normal urothelium vs a tumor compromised urothelium provide for a high selective uptake ratio. In vitro, a 1000 difference in the LD50 between dark and 22.5 J cm−2 irradiation at 530 nm was achieved for two bladder cell lines (T24 and AY‐27). In dogs and rats the plasma concentration for TLD1433 was below 100 ng kg−1 at 72 hours following intravenous injection at a dose of up to 3.5 mg kg−1 and below 10 ng kg−1 following intrabladder instillation for 1 hour at a concentration of 6 mg mL−1. Successful tumor necrosis close to 1.5 mm in depth was achieved.