Urothelial Bladder Research Articles

Changes of the Urothelial Barrier System in the Cyclophosphamide-Induced Cystitis in Rats by Using a Newly Established "Inside-Out" Urinary Bladder Preparation.

The study was aimed to establish the "inside-out" preparation with the urothelium and investigate the changes in urothelial permeability of the cyclophosphamide (CYP)-induced cystitis model in rats. In female rats with or without CYP injection, the isolated whole bladder was utilized as an "inside-out" preparation with the urothelium, which was created by reversing the bladder from a top portion. The preparation was fixed in the organ bath, and instilled with a Krebs solution (0.5 mL) through the bladder neck. After it was kept under an isovolumetric condition, high K+ (KCl: 50 mM) or acetylcholine (ACh: 10 μM) was added into the organ bath. In the normal bladder, the intravesical pressure of the inside-out preparation with the urothelium did not change with the addition of KCl or ACh. Contrarily, in the CYP-injected bladder 24 or 48 h after injection of CYP, the intravesical pressure of the inside-out preparation increased with the addition of KCl or ACh. Histological examinations showed a denuded and/or cracked surface of the urothelial layer, and the intensity of uroplakin III staining of the urothelial layer decreased in the CYP-injected rats. The study demonstrated the bladder urothelium has robust barrier mechanisms for preventing the absorption of water (urine) under the normal condition. However, these barrier mechanisms were disrupted in the CYP-induced cystitis, suggesting that water and urine insults can be permeabilized into the urinary bladder, specifically to the smooth muscle layer.

The Orexin OX2 Receptor-Dependent Pathway Is Implicated in the Development of Overactive Bladder and Depression in Rats Exposed to Corticosterone.

In the present study, we wanted to check whether TCS OX2 29 (TCS), a potent selective antagonist of OX2 receptors, would have positive effects in an animal model of detrusor overactivity co-existed with the depression-like state in Wistar male rats. The forced swim test with the measurement of spontaneous locomotor activity, conscious cystometry, determination of c-Fos expression in central micturition areas, and a set of biochemical analyses (with the use of urine, hippocampus, bladder urothelium, and detrusor muscle of tested animals) were carried out. The outcomes showed that a 7-day administration of TCS (3 mg/kg/day, subcutaneously) normalizes the cystometric parameters corresponding to overactivity of the detrusor and reverses the pro-depressive response. Furthermore, the antagonism of OX2 receptors restored the abnormal levels of overactive bladder markers (i.e., ATP, CGRP, OCT3, TRPV1, ROCK1, and VAChT), diminished neuronal overactivity in central micturition areas (i.e., pontine micturition center, ventrolateral periaqueductal gray, and medial preoptic area) as well as restored the altered hippocampal levels of CRF, cytokines (IL-1β, IL-6, IL-10, and TNF-α), and growth factors (BDNF and NGF) that reflected biochemical disturbances detected in depressed people. It seems that our findings open new perspectives regarding the implication of the orexin system in the functioning of the urinary bladder and in the pathophysiology of depression.

Is It Possible to Regenerate the Underactive Detrusor? Part 1 Molecular and Stem Cell Therapies Targeting the Urinary Bladder and Neural Axis ICI-RS 2024.

Detrusor muscle weakness is commonly noted on urodynamics in patients with refractory voiding difficulty. No approved therapies have been proven to augment the strength of a detrusor voiding contraction. This subject was discussed by a think-tank at the International Consultation on Incontinence- Research Society (ICI-RS) meeting held in Bristol, June 2024. The discussions of the think-tank are being published in two parts. This first part discusses molecular and stem cell therapies targeting the urinary bladder and the neural axis. Senescence of the urothelium and extracellular ATP acting through P2X3 receptors might be important in detrusor underactivity. Several molecules such as parasympathomimetics, acotiamide, ASP8302, neurokinin-2 agonists have been explored but none has shown unequivocal clinical benefit. Different stem cell therapy approaches have been used, chiefly in neurogenic dysfunction, with some studies showing benefit. Molecular targets for the neural axis have included TRPV-4, Bombesin, and serotoninergic receptors and TAC-302 which induces neurite growth. Several options are currently being pursued in the search for an elusive molecular or stem cell option for enhancing the power of the detrusor muscle. These encompass a wide range of approaches that target each aspect of the contraction mechanism including the urothelium of bladder and urethra, myocyte, and neural pathways. While none of these have shown unequivocal clinical utility, some appear promising. Lessons from other fields of medicine might prove instructive. Not necessary. Not a clinical trial.

Impact of MicroRNA Depletion on Maintenance of Adult Bladder Urothelium

Impact of MicroRNA Depletion on Maintenance of Adult Bladder Urothelium

Nur77 protects the bladder urothelium from intracellular bacterial infection

Intracellular infections by Gram-negative bacteria are a significant global health threat. The nuclear receptor Nur77 (also called TR3, NGFI-B, or NR4A1) was recently shown to sense cytosolic bacterial lipopolysaccharide (LPS). However, the potential role for Nur77 in controlling intracellular bacterial infection has not been examined. Here we show that Nur77 protects against intracellular infection in the bladder by uropathogenic Escherichia coli (UPEC), the leading cause of urinary tract infections (UTI). Nur77 deficiency in mice promotes the formation of UPEC intracellular bacterial communities (IBCs) in the cells lining the bladder lumen, leading to persistent infection in bladder tissue. Conversely, treatment with a small-molecule Nur77 agonist, cytosporone B, inhibits invasion and enhances the expulsion of UPEC from human urothelial cells in vitro, and significantly reduces UPEC IBC formation and bladder infection in mice. Our findings reveal a new role for Nur77 in control of bacterial infection and suggest that pharmacologic agonism of Nur77 function may represent a promising antibiotic-sparing therapeutic approach for UTI.

PDE5 inhibitor potentially improves polyuria and bladder storage and voiding dysfunctions in type 2 diabetic rats.

Bladder dysfunction associated with type 2 diabetes mellitus (T2DM) includes urine storage and voiding disorders. We examined pathological conditions of the bladder wall in a rat T2DM model and evaluated the effects of the phosphodiesterase-5 (PDE-5) inhibitor tadalafil. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Long-Evans Tokushima Otsuka (LETO) rats were used as the T2DM and control groups, respectively. Tadalafil was orally administered for 12 weeks. Micturition behavior was monitored using metabolic cages, and bladder function was evaluated by cystometry. Bladder blood flow was evaluated by laser speckle imaging, and an organ bath bladder distention test was used to measure adenosine triphosphate (ATP) release from the bladder urothelium. The expression levels of vesicular nucleotide transporter (VNUT), hypoxia markers, pro-inflammatory cytokines and growth factors in the bladder wall were measured using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Bladder wall contractions in response to KCl and carbachol were monitored using bladder-strip tests. With aging, OLETF rats had higher micturition frequency and greater urine volume than LETO rats. Although bladder capacity was not significantly different, non-voiding bladder contraction occurred more frequently in OLETF rats than in LETO rats. Bladder blood flow was decreased and ATP release was increased with higher VNUT expression in OLETF rats than in LETO rats. These effects were suppressed by tadalafil administration, with accompanying decreased HIF-1α, 8-OHdG, IL-6, TNF-α, IGF-1, and bFGF expression. The impaired contractile responses of bladder strips to KCl and carbachol in OLETF rats with aging were restored by tadalafil administration. The T2DM rats had polyuria, increased ATP release induced by decreased bladder blood flow and impaired contractile function. PDE5 inhibition improved these changes and may prevent T2DM-associated urinary frequency and bladder storage and voiding dysfunctions.

Uncoupling as initiating event in mitochondrial dysfunction after diuron exposure

Diuron, a herbicide derived from urea, has been shown to induce urinary bladder urothelial tumors in rodents, leading the U.S. Environmental Protection Agency (USEPA) to designate it as a ‘known/likely’ human carcinogen. In our laboratory, a series of studies investigating the carcinogenic mode of action (MoA) of Diuron have consistently revealed its cytotoxic effects on the urinary bladder urothelium. Prolonged exposure to relatively high doses of Diuron results in urothelial necrosis, regenerative hyperplasia, and eventually, the development of tumors. The hypothesis posited is that Diuron and its metabolites exert toxicity by causing damage to mitochondria, a phenomenon referred to as mitotoxicity. Our research focuses on evaluating how Diuron and its metabolites affect mitochondria isolated from both the urothelium and the liver, the primary organ for Diuron biotransformation. In this context, we present and discuss data pertaining to mitochondria isolated from the liver of Wistar rats exposed to Diuron or its metabolites 3-(3,4-diclorofenil)-1-metilureia (DCPMU) or 3,4-dichloroaniline (DCA) at concentrations ranging from 0.5 to 500 µM in vitro. The findings indicate that, at concentrations of 100 and 500 µM, the tested chemicals induce uncoupling of oxidative phosphorylation, as evidenced by the dissipation of mitochondrial membrane potential and basal oxygen consumption. Notably, at 500 µM, DCA causes mitochondrial swelling, a morphofunctional indicator of severe organelle damage. These outcomes underscore the classification of Diuron and its metabolites, DCA and DCPMU, as mitotoxic to liver cells, given the pronounced mitochondrial dysfunction they induce.

Comprehensive Genomic Analysis of Puerarin in Inhibiting Bladder Urothelial Carcinoma Cell Proliferation and Migration.

Bladder urothelial carcinoma (BUC) ranks second in the incidence of urogenital system tumors, and the treatment of BUC needs to be improved. Puerarin, a traditional Chinese medicine (TCM), has been shown to have various effects such as anti-cancer effects, the promotion of angiogenesis, and anti-inflammation. This study investigates the effects of puerarin on BUC and its molecular mechanisms. Through GeneChip experiments, we obtained differentially expressed genes (DEGs) and analyzed these DEGs using the Ingenuity® Pathway Analysis (IPA®), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway enrichment analyses. The Cell Counting Kit 8 (CCK8) assay was used to verify the inhibitory effect of puerarin on the proliferation of BUC T24 cells. String combined with Cytoscape® was used to create the Protein-Protein Interaction (PPI) network, and the MCC algorithm in cytoHubba plugin was used to screen key genes. Gene Set Enrichment Analysis (GSEA®) was used to verify the correlation between key genes and cell proliferation. A total of 1617 DEGs were obtained by GeneChip. Based on the DEGs, the IPA® and pathway enrichment analysis showed they were mainly enriched in cancer cell proliferation and migration. CCK8 experiments proved that puerarin inhibited the proliferation of BUC T24 cells, and its IC50 at 48 hours was 218μmol/L. Through PPI and related algorithms, 7 key genes were obtained: ITGA1, LAMA3, LAMB3, LAMA4, PAK2, DMD, and UTRN. GSEA showed that these key genes were highly correlated with BUC cell proliferation. Survival curves showed that ITGA1 upregulation was associated with poor prognosis of BUC patients. Our findings support the potential antitumor activity of puerarin in BUC. To the best of our knowledge, bioinformatics investigation suggests that puerarin demonstrates anticancer mechanisms via the upregulation of ITGA1, LAMA3 and 4, LAMB3, PAK2, DMD, and UTRN, all of which are involved in the proliferation and migration of bladder urothelial cancer cells.

Vitexin Induces Apoptosis and Ferroptosis and Suppresses Malignant Proliferation and Invasion of Bladder Urothelial Carcinoma through PI3K/AKT-Nrf2 Axis

Background: Bladder urothelial carcinoma (BUC) is a type of malignant urinary system. Although several strategies have been applied in the treatment of BUC, its survival remains unsatisfactory, especially in the patients with advanced BUC. Vitexin, a natural flavonoid has exhibited the inhibitory effect on various tumors, however, its effect on BUC is still unclear. Objective: This study aimed to explore the effect of vitexin on the progression of BUC. Methods: The toxicity of vitexin on T24 and 5637 cells was detected by cell counting kit-8 (CCK-8). The effects of vitexin on proliferation, apoptosis, invasion, epithelial-mesenchymal transition (EMT) and ferroptosis in BUC cells were determined by CCK-8, flow cytometry, western blot, transwell and immunofluorescence assays. Additionally, the related mechanism was explored by examining the expression of the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT)-nuclear factor-erythroid 2 related factor 2 (Nrf2) pathway. Besides, in vivo validation was performed in the xenografted mice. Results: Vitexin reduced the BUC cell viability and enhanced the apoptosis rate and the relative protein expression of p53 and cleaved-caspase3. Also, vitexin decreased the invasion number, and increased the relative protein expression of E-cadherin with the decreased N-cadherin protein level in T24 and 5637 cells. Besides, vitexin promoted the levels of ROS and MDA, while reduced the GSH level. Vitexin also increased the level of iron, but decreased the relative protein expression of xCT and GPX4. Erastin further increased the vitexin-induced iron levels, whereas inverse outcomes were observed in the application of ferrostatin-1. Additionally, vitexin decreased the relative protein levels of PI3K, p-AKT/AKT, and nuclear Nrf2, while increased the relative protein level of cytoplasmic Nrf2. Overexpression of PI3K notably inverted the effect of vitexin on cell viability, apoptosis, invasion, level of ROS and iron. Furthermore, vitexin reduced the tumor volume and weight of xenografted mice. Vitexin decreased the protein level of N-cadherin, while increased apoptosis rate of xenografted mice. In addition, vitexin reduced the relative protein levels of PI3K, p-AKT/AKT, and nuclear Nrf2 with the enhanced relative protein expression of cytoplasmic Nrf2 in xenografted mice. Moreover, vitexin decreased the relative protein expression of xCT and GPX4 and the GSH level, whereas increased the MDA level in xenografted mice. Conclusion: Vitexin suppressed malignant proliferation and invasion and induced apoptosis and ferroptosis of BUC involving in PI3K/AKT-Nrf2 pathway.

Elucidating the pan-oncologic landscape of S100A9: prognostic and therapeutic corollaries from an integrative bioinformatics and Mendelian randomization analysis

The calcium-binding protein S100A9 has emerged as a pivotal biomolecular actor in oncology, implicated in numerous malignancies. This comprehensive bioinformatics study transcends traditional boundaries, investigating the prognostic and therapeutic potential of S100A9 across diverse neoplastic entities. Leveraging a wide array of bioinformatics tools and publicly available cancer genomics databases, such as TCGA, we systematically examined the S100A9 gene. Our approach included differential expression analysis, mutational burden assessment, protein interaction networks, and survival analysis. This robust computational framework provided a high-resolution view of S100A9’s role in cancer biology. The study meticulously explored S100A9’s oncogenic facets, incorporating comprehensive analyses of its relationship with prognosis, tumor mutational burden (TMB), microsatellite instability (MSI), DNA methylation, and immune cell infiltration across various tumor types. This study presents a panoramic view of S100A9 expression across a spectrum of human cancers, revealing a heterogeneous expression landscape. Elevated S100A9 expression was detected in malignancies such as BLCA (Bladder Urothelial Carcinoma), CESC (Cervical squamous cell carcinoma and endocervical adenocarcinoma), COAD (Colon adenocarcinoma), ESCA (Esophageal carcinoma), and GBM (Glioblastoma multiforme), while reduced expression was noted in BRCA (Breast invasive carcinoma), HNSC (Head and Neck squamous cell carcinoma), and KICH (Kidney Chromophobe). This disparate expression pattern suggests that S100A9’s role in cancer biology is multifaceted and context-dependent. Prognostically, S100A9 expression correlates variably with patient outcomes across different cancer types. Furthermore, its expression is intricately associated with TMB and MSI in nine cancer types. Detailed examination of six selected tumors—BRCA, CESC, KIRC (Kidney renal clear cell carcinoma), LUSC (Lung squamous cell carcinoma), SKCM (Skin Cutaneous Melanoma); STAD (Stomach adenocarcinoma)—revealed a negative correlation of S100A9 expression with the infiltration of most immune cells, but a positive correlation with neutrophils, M1 macrophages, and activated NK cells, highlighting the complex interplay between S100A9 and the tumor immune environment. This bioinformatics synthesis posits S100A9 as a significant player in cancer progression, offering valuable prognostic insights. The data underscore the utility of S100A9 as a prognostic biomarker and its potential as a therapeutic target. The therapeutic implications are profound, suggesting that modulation of S100A9 activity could significantly impact cancer management strategies.

Mitochondrial viability in neurogenic bladder urothelium after sigmoidocolocystoplasty. Implications for persistent vesicoureteral reflux.

We investigated whether inflammatory cell infiltration (ICI), fibrosis, and mitochondrial viability of the neurogenic bladder urothelium are involved in the mechanism of persistent vesicoureteral reflux (VUR) after sigmoidocolocystoplasty (SCP). Bladder biopsies obtained 1994-2023 from 62 neurogenic bladder patients were examined by hematoxylin and eosin for ICI, Masson's trichrome for fibrosis, and immunofluorescence for urothelial growth differentiation factor 15 (GDF15; a mitochondrial stress-responsive cytokine) (positive/negative) and heat shock protein 60 (HSP60; a mitochondrial matrix marker) (strong ≥ 50%/weak≤ 50%) expression. GDF15 + /weak HSP60 indicated compromised mitochondrial viability. Cystometry measured neobladder compliance/capacity. Mean ages (years) at SCP and bladder biopsies were 9.4 ± 4.6 and 14.2 ± 7.1, respectively. VUR was present in 38/62 patients (51 ureters) at SCP and resolved with SCP alone in 4/38 patients, with SCP and ureteroneocystostomy in 17/38, and persisted in 17/38. Fibrosis was significantly denser in GDF15 + (n = 24)/weak HSP60 (n = 31) compared with GDF15- (n = 38)/strong HSP60 (n = 31) (p < 0.001 and p < 0.01, respectively). Differences in ICI were significant for GDF15 + vs. GDF15- (p < 0.05) but not for HSP60. Patients with VUR after SCP had higher incidence of GDF15 + /weak HSP60 compared with cases without VUR (p < 0.05 and p < 0.001, respectively). Viability of mitochondria appears to be compromised with possible etiologic implications for VUR persisting after SCP.

The upregulation of POLR3G correlates with increased malignancy of bladder urothelium

Bladder cancer remains a significant health challenge due to its high recurrence and progression rates. This study aims to evaluate the role of POLR3G in the development and progression of bladder cancer and the potential of POLR3G to serve as a novel therapeutic target. We constructed a bladder cancer model in Wistar rats by administering N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), which successfully induced a transition from normal mucosa to hyperplasia and ultimately to urothelial carcinoma. We observed a progressive upregulation of POLR3G expression during the bladder cancer development and progression. To investigate the functional role of POLR3G, we performed functional experiments in bladder cancer cell lines. The results demonstrated that knocking down POLR3G significantly inhibited cell proliferation, migration, and invasion. We further conducted RNA sequencing on POLR3G-knockdown bladder cancer cells, and Metascape was employed to perform the functional enrichment analysis of the differentially expressed genes (DEGs). Enrichment analysis revealed the enrichment of DEGs in the RNA polymerase and apoptotic cleavage of cellular proteins pathways, as well as their involvement in the Wnt and MAPK signaling pathways. The downregulation of Wnt pathway-related proteins such as Wnt5a/b, DVL2, LRP-6, and phosphorylated LRP-6 upon POLR3G knockdown was further confirmed by Western blotting, indicating that POLR3G might influence bladder cancer behavior through the Wnt signaling pathway. Our findings suggest that POLR3G plays a crucial role in bladder cancer progression and could serve as a potential therapeutic target. Future studies should focus on the detailed mechanisms by which POLR3G regulates these signaling pathways and its potential as a biomarker for early detection and prognosis of bladder cancer.

Reduction in lower urinary tract mucosal microtrauma as an effect of reducing eyelet sizes of intermittent urinary catheters

Intermittent catheterization (IC) utilizing conventional eyelets catheters (CECs) for bladder drainage has long been the standard of care. However, when the tissue of the lower urinary tract comes in close proximity to the eyelets, mucosal suction often occurs, resulting in microtrauma. This study investigates the impact of replacing conventional eyelets with a drainage zone featuring multiple micro-holes, distributing pressure over a larger area. Lower pressures limit the suction of surrounding tissue into these micro-holes, significantly reducing tissue microtrauma. Using an ex vivo model replicating the intra-abdominal pressure conditions of the bladder, the intra-catheter pressure was measured during drainage. When mucosal suction occurred, intra-catheter images were recorded. Subsequently affected tissue samples were investigated histologically. The negative pressure peaks caused by mucosal suction were found to be very high for the CECs, leading to exfoliation of the bladder urothelium and breakage of the urothelial barrier. However, a micro-hole zone catheter (MHZC) with a multi-eyelet drainage zone showed significantly lower pressure peaks, with over 4 times lower peak intensity, thus inducing far less extensive microtraumas. Limiting or even eliminating mucosal suction and resulting tissue microtrauma may contribute to safer catheterizations in vivo and increased patient comfort and compliance.

Keratin 5 basal cells are temporally regulated developmental and tissue repair progenitors in bladder urothelium.

Urothelium forms a distensible yet impermeable barrier, senses and transduces stimuli, and defends the urinary tract from mechanical, chemical, and bacterial injuries. Biochemical and genetic labeling studies support the existence of one or more progenitor populations with the capacity to rapidly regenerate the urothelium following injury, but slow turnover, a low mitotic index, and inconsistent methodologies obscure progenitor identity. The progenitor properties of basal keratin 5 urothelial cells (K5-UCs) have been previously investigated, but those studies focused on embryonic or adult bladder urothelium. Urothelium undergoes desquamation and apoptosis after birth, which requires postnatal proliferation and restoration. Therefore, we mapped the fate of bladder K5-UCs across postnatal development/maturation and following administration of cyclophosphamide to measure homeostatic and reparative progenitor capacities, respectively. In vivo studies demonstrate that basal K5-UCs are age-restricted progenitors in neonates and juveniles, but not in adult mice. Neonatal K5-UCs retain a superior progenitor capacity in vitro, forming larger and more differentiated urothelial organoids than adult K5-UCs. Accordingly, K5-UC transcriptomes are temporally distinct, with enrichment of transcripts associated with cell proliferation and differentiation in neonates. Induction of urothelial proliferation is sufficient to restore adult K5-UC progenitor capacity. Our findings advance the understanding of urothelial progenitors and support a linear model of urothelial formation and regeneration, which may have significant impact on therapeutic development or tissue engineering strategies.NEW & NOTEWORTHY Fate mapping reveals an important linear relationship, whereby bladder basal urothelial cells give rise to intermediate and superficial cells in an age-restricted manner and contribute to tissue repair. Neonatal basal cells reprise their role as superior progenitors in vitro and display distinct transcriptional signatures, which suggest progenitor function is at least partially cell intrinsic. However, the urothelium progenitor niche cannot be overlooked, since FGF7 rescues adult basal cell progenitor function.

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.

Abstract PR001: TRIM29 is required for basal bladder cancer invasive progression

Abstract TRIM29 (Tripartite Motif 29) also known ATDC, is an E3 ubiquitin ligase frequently upregulated in muscle-invasive bladder cancer (MIBC). We have previously shown TRIM29 global overexpression in a genetically engineered mouse model (GEMM) is sufficient to ` bladder cancer development and progression to MIBC. Other studies strongly suggest cytokeratin-5/14 (Krt5+/Krt14+) expressing basal-stem progenitor cells in the urothelium are the cells of origin for MIBC in mice. We therefore hypothesized that Trim29 expression in Krt5+ basal urothelial cells is required for development of MIBC in mice. To test this, we generated a GEMM harboring a Trim29 conditional KO allele and tamoxifen responsive Cre under control of the bovine Krt5promoter. Exposure of Krt5-CreERT2 Trim29flox/flox mice to tamoxifen resulted in activation of Cre and loss of Trim29 expression only in the Krt5+ basal urothelium. To induce bladder tumorigenesis, we utilized the urothelial specific carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in drinking water at 0.05%. After 6 months, tumor invasion was staged by a genitourinary pathologist based on depth of invasion: T1 (stromal invasive), T2 (muscle invasive) and T3 (deep muscle/peri-vesicular invasion). In agreement with previous studies, BBN exposure resulted in loss of Krt5-negative intermediate and superficial cells, and expansion of Krt5+/Krt14+ basal cells across the urothelium. In combination with a tamoxifen treatment this led to total Trim29 KO in the urothelium. All male Trim29 WT mice exposed to BBN developed Trim29+ bladder tumors, &amp;gt;50% of which demonstrated extensive progression to muscle-invasion (57% n=23). In contrast, only 16% of mice with Trim29 loss in Krt5+ basal cells developed MIBC (n=18). As an E3 ubiquitin ligase TRIM29 is known to target immune adaptor molecules, notably the stimulator of interferon genes (STING), for proteasomal degradation. We hypothesized TRIM29 targets STING during invasive progression and supports the establishment of an immune-privileged niche at the bladder cancer invasive front. We find TRIM29 expression in human bladder tissues is upregulated in the urothelium under stress and in MIBC tumors. In mice, BBN exposure upregulated Trim29 expression in the bladder urothelium which correlated with low urothelial Sting. In contrast, Krt5+ Trim29 loss resulted in significantly elevated Sting in the bladder urothelium. Multiplex immunofluorescence imaging and immunotyping of Trim29 KO mouse tissues revealed peritumoral enrichment of CD4+ and CD8+ T cells, CD206+ immunosuppressive macrophages, and CD4+FoxP3+ regulatory T cells. Taken together, these results suggest that TRIM29-mediated downregulation of STING in the bladder urothelium results in decreased immune surveillance at the tumor-stromal interface which expedites invasion and tumor burden. Citation Format: Alan Kelleher, Luke Broses, Yin Wang, Marian Henderson, Mark Day, Aaron Udager, Phillip Palmbos. TRIM29 is required for basal bladder cancer invasive progression [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR001.

Abstract 5726: The potential anti-cancer activity of dual TTK/PLK1 inhibitor, BAL0891, in bladder cancer

Abstract Background: BAL0891 is a novel dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1) which affects mitotic spindle assembly checkpoint integrity resulting in aberrant mitotic progression of cancer cells. Since unlike other organs, bladder urothelium is highly regenerative in certain circumstances, there are high chances of cell cycle inhibitors to have anti-cancer activity over bladder cancer. In this study, we investigated the therapeutic potential of BAL0891 in bladder cancer compared to single TTK or PLK1 inhibitor and discovered the best therapeutic combination partner for BAL0891. Methods: We performed meta-analysis of the mRNA expression levels of bladder cancer using Gene Expression Profiling Interactive Analysis 2 (GEPIA2). Representative bladder cancer cell lines (253J, 253J-BV, J82, RT4, T24, J82, HT1197, and HT1376) were treated with the dual protein kinase inhibitors, BAL0891, in comparison with TTK inhibitor (BAY1217389) or PLK1 inhibitor (onvansertib). BAL0891 was combined with CDK4/6 inhibitor, another cell cycle inhibitor working at different point from BAL0891. Cell viability was assessed using CCK-8 assay during 72 hours after treatment. Expression levels of TTK and PLK1 were measured by immunoblotting. Cell cycle analysis and cell apoptosis detection were performed on T24 cell. Results: The mRNA expression levels of both TTK (16.7 times) and PLK1 (10.0 times) were significantly upregulated only in bladder cancers compared to normal bladder tissues (P&amp;lt;0.05). Compared to TTK or PLK1 inhibitor, BAL0891 was sensitive in 75% of bladder cancer cell lines (IC50 &amp;lt;0.1 uM) and significantly reduced proliferation of bladder cancer cell lines depending on the dosage. TTK and PLK1 expression levels of cancer cells were associated with the sensitivity to BAL0891. BAL0891 significantly induced necrosis and increased in polyploidy and G2/M arrest in T24 cell. The anti-cancer activity of BAL0891 was further enhanced in certain cell lines (RT4, and J82) by combination of CDK4/6 inhibitor. Conclusion: This is the first study to suggest the potent anti-cancer activity of BAL0891 in bladder cancer. BAL0891 effectively worked on bladder cancer by two tracks which not only induced G2/M arrest but also increased in polyploidy. Furthermore, BAL0891 in combination with CDK4/6 inhibitor promoted anti-cancer effects. Lastly, this study suggests the use of TTK and PLK1 as the predictive biomarker for BAL0891. These data support further investigation of BAL0891 for the treatment of bladder cancer patients. Citation Format: Jee Soo Park, Ji-eun Lee, Myung Eun Lee, Jongchan Kim, Won Sik Jang, Jae Jung Lee, Jun Seung Lee, Minkyung Kang, Won Sik Ham. The potential anti-cancer activity of dual TTK/PLK1 inhibitor, BAL0891, in bladder cancer [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 5726.

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.

Insulin receptor signaling engages bladder urothelial defenses that limit urinary tract infection

Urinary tract infections (UTIs) commonly afflict people with diabetes. To better understand the mechanisms that predispose diabetics to UTIs, we employ diabetic mouse models and altered insulin signaling to show that insulin receptor (IR) shapes UTI defenses. Our findings are validated in human biosamples. We reportthat diabetic mice have suppressed IR expression and are more susceptible to UTIs caused by uropathogenic Escherichia coli (UPEC). Systemic IR inhibition increases UPEC susceptibility, while IR activation reduces UTIs. Localized IR deletion in bladder urothelium promotes UTI by increasing barrier permeability and suppressing antimicrobial peptides. Mechanistically, IR deletion reduces nuclear factor κB (NF-κB)-dependent programming that co-regulates urothelial tight junction integrity and antimicrobial peptides. Exfoliated urothelial cells or urine samples from diabetic youths show suppressed expression of IR, barrier genes, and antimicrobial peptides. These observations demonstrate that urothelial insulin signaling has a role in UTI prevention and link IR to urothelial barrier maintenance and antimicrobial peptide expression.

Retracted: Pancancer Analysis of Neurovascular-Related NRP Family Genes as Potential Prognostic Biomarkers of Bladder Urothelial Carcinoma.

[This retracts the article DOI: 10.1155/2021/5546612.].