Primary Urothelial Cells Research Articles

Abstract A026: PIN1 isomerase promotes the initiation and progression of bladder cancer through SREBP2-mediated cholesterol biosynthesis pathway

Abstract Bladder cancer, one of the most commonly diagnosed cancers worldwide, is associated with high morbidity, mortality, and a poor prognosis. The PIN1 phospho-dependent prolyl isomerase is frequently overexpressed in many human cancer types and affects tumor initiation and progression through disrupting the balance of oncoprotein and tumor suppressor protein signaling. However, the functional pSer/Thr.Pro protein substrates of PIN1 and its effects on downstream signaling in bladder carcinogenesis remain largely unknown. Phenotypically, we discovered that ablation of Pin1 in human/mouse bladder cancer cells and organoids formed from mouse primary urothelial cells resulted in decreased cell proliferation, stemness maintenance, cell invasion, migration, urothelium clearance capacity, and reduced free/total cholesterol levels in vitro. Re-expressing Pin1 in Pin1 knockout cells reversed the defects caused by lack of Pin1. Moreover, in vivo subcutaneous xenograft and allograft transplantation mouse models combined with an orthotopic implantation model were utilized to confirm a positive role for Pin1 in controlling tumor growth and potential metastasis. Therapeutically, a combination of the sulfopin PIN1 inhibitor and the simvastatin HMGCR inhibitor was shown to suppress cell proliferation in vitro and tumor growth in vivo synergistically. Mechanistically, we observed a negative enrichment of SREBP2-driven cholesterol metabolism pathways in PIN1 knockout cells via RNA-sequencing and used a suite of diverse molecular and biochemical approaches to show that PIN1 directly binds to JNK-dependent phosphorylated SREBP2 (Ser455) and vice versa. These results indicate that active cholesterol biosynthesis pathway in bladder cancer cells is regulated by PIN1-mediated isomerization and JNK-mediated phosphorylation of its key transcriptional factor SREBP2. These findings emphasize that PIN1 can act as a regulator and potential therapeutic target in bladder cancer. Citation Format: Xue Wang, Tony Hunter. PIN1 isomerase promotes the initiation and progression of bladder cancer through SREBP2-mediated cholesterol biosynthesis pathway [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 A026.

The absence of urinary diversion in radical cystectomy avoids early complications in hemodialysis patients

Background: Patients with end-stage renal disease who receive dialysis are considered a high-risk group for perioperative complications following anesthesia and surgery. However, for patients with anuria who will undergo radical cystectomy (RC), a urinary diversion is unnecessary. This study aimed to identify a safe surgical strategy by comparing oncologic outcomes and early complication rates in dialysis and nondialysis patients after RC. Materials and Methods: This study included 85 patients with primary urothelial cell carcinoma of the bladder who underwent RC at the Chang Gung Memorial Hospital, Keelung, Taiwan. Twenty-eight of these patients underwent regular hemodialysis. Overall survival and recurrence-free survival were compared to evaluate the oncologic outcomes. Complications at 3 months were graded using the Clavien–Dindo classification. Results: The overall survival and recurrence-free survival differences between dialysis and nondialysis patients were not significant (P = 0.686; P = 0.528). The degree of muscle-invasive disease was an independent factor affecting overall survival. The overall complication rates in the dialysis and nondialysis groups were 36% and 84%, respectively (P < 0.001). The major complication (Grades III–V) was 16% in the dialysis group and 28% in the nondialysis group (P = 0.241). The most common early complications were urinary tract infection and bowel kinetics change, and both were significantly lower in the dialysis group. A lower re-admission rate was also observed in the dialysis group. Conclusion: Lower rates of early complications and acceptable survival outcomes were observed in dialysis patients. Surgery can be conducted more aggressively, with confidence in suitable cases.

A biomimetic hyaluronic acid-silk fibroin nanofiber scaffold promoting regeneration of transected urothelium.

This study was designed to investigate the regulatory effect of hyaluronic acid (HA)—coating silk fibroin (SF) nanofibers during epithelialization of urinary tract for urethral regeneration. The obtained electrospun biomimetic tubular HA‐SF nanofiber scaffold is composed of a dense inner layer and a porous outer layer in order to mimic adhesion and cavernous layers of the native tissue, respectively. A thin layer of HA‐gel coating was fixed in the inner wall to provide SF nanofibers with a dense and smooth surface nano‐topography and higher hydrophilicity. Compared with pure SF nanofibers, HA‐SF nanofibers significantly promoted the adhesion, growth, and proliferation of primary urothelial cells, and up‐regulate the expression of uroplakin‐3 (terminal differentiation keratin protein in urothelium). Using the New Zealand male rabbit urethral injury model, the scaffold composed of tubular HA‐SF nanofibers could recruit lumen and myoepithelial cells from the adjacent area of the host, rapidly reconstructing the urothelial barrier in the wound area in order to keep the urinary tract unobstructed, thereby promoting luminal epithelialization, smooth muscle bundle structural remodeling, and capillary formation. Overall, the synergistic effects of nano‐topography and biophysical cues in a biomimetic scaffold design for effective endogenous regeneration.

A database on differentially expressed microRNAs during rodent bladder healing

Urinary bladder wound healing relies on multiple biological events that are finely tuned in a spatial–temporal manner. MicroRNAs are small non-coding RNA molecules with regulatory functions. We hypothesized that microRNAs are important molecules in the coordination of normal urinary bladder wound healing. We aimed at identifying microRNAs expressed during bladder wound healing using Affymetrix global array for microRNA profiling of the rodent urinary bladder during healing of a surgically created wound. Results were validated in the rat bladders by real-time PCR (RT-PCR) using three of the differentially expressed (DE) microRNAs. The model was thereafter validated in human cells, by measuring the expression of eight of the DE microRNAs upon in vitro wound-healing assays in primary urothelial cells. Our results indicated that 508 (40%) of all rodent microRNAs were expressed in the urinary bladder during wound healing. Thirteen of these microRNAs (1%) were DE (false discovery rate (FDR) < 0.05, P < 0.05, |logfold|> 0.25) in wounded compared to non-wounded bladders. Bioinformatic analyses helped us to identify target molecules for the DE microRNAs, and biological pathways involved in tissue repair. All data are made available in an open-access database for other researchers to explore.

Intravesical CD74 and CXCR4, macrophage migration inhibitory factor (MIF) receptors, mediate bladder pain

Activation of intravesical protease activated receptor 4 (PAR4) leads to release of urothelial macrophage migration inhibitory factor (MIF). MIF then binds to urothelial MIF receptors to release urothelial high mobility group box-1 (HMGB1) and elicit bladder hyperalgesia. Since MIF binds to multiple receptors, we investigated the contribution of individual urothelial MIF receptors to PAR4-induced HMGB1 release in vivo and in vitro and bladder pain in vivo. We tested the effect of intravesical pre-treatment with individual MIF or MIF receptor (CD74, CXCR4, CXCR2) antagonists on PAR4-induced HMGB1 release in vivo (female C57/BL6 mice) and in vitro (primary human urothelial cells) and on PAR4-induced bladder hyperalgesia in vivo (mice). In mice, PAR4 induced HMGB1 release and bladder hyperalgesia through activation of intravesical MIF receptors, CD74 and CXCR4. CXCR2 was not involved in these effects. In primary urothelial cells, PAR4-induced HMGB1 release through activation of CD74 receptors. Micturition parameters in mice were not changed by any of the treatments. Urothelial MIF receptors CD74 and CXCR4 mediate bladder pain through release of urothelial HMGB1. This mechanism may set up persistent pain loops in the bladder and warrants further investigation. Urothelial CD74 and CXCR4 may provide novel targets for interrupting bladder pain.

Expression patterns of the immune checkpoint ligand CD276 in urothelial carcinoma

BackgroundCD276 is an immune checkpoint molecule. Elevated CD276 expression by urothelial carcinoma is associated with poor prognosis, but little is known about its expression across different tumor stages. We therefore investigated CD276 expression in bladder cancer (BC) cells and in tissue samples of BC stages from pT2 to pT4.MethodsCD276 expression was explored in 4 urothelial cancer cell lines and 4 primary normal urothelial cell populations by quantitative RT-PCR, Western blot and flow cytometry. CD276 was investigated in bladder tumors from 98 patients by immunohistochemistry using a score (0–300) incorporating both, staining intensity and area of CD276 staining. Normal appearing urothelium in the bladder of the same patients served as controls.ResultsThe urothelial carcinoma cell lines expressed significantly higher levels of CD276 on transcript (p < 0.006), total protein levels (p < 0.005), and on the cell surface (p < 0.02) when compared to normal urothelial cells. In pT2–T4 tumor tissue samples, CD276 was overexpressed (median score 185) when compared to corresponding healthy tissues from the same patients (median score 50; p < 0.001). No significant differences in CD276 expression were recorded in late, locally advanced ≥ pT3a tumors (median score 185) versus organ-confined < pT3a tumors (median score 190), but it was significantly lower in the normal urothelial tissue associated with ≥ pT3a tumors (median score 40) versus < pT3a tumors (median score 80; p < 0.05).ConclusionCD276 expression is significantly elevated in urothelial carcinoma cells in all stages but varies between individuals considerably. Reduced CD276 expression in normal urothelial cells may imply that these cells would be protected from CD276-mediated immuno therapies.

Modulation of BAG3 expression in human normal urothelial cells by Diuron.

Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] is a substituted urea herbicide, carcinogenic for the rat urinary bladder. It has been hypothesized that Diuron cytotoxicity, resulting in regenerative proliferation, leads to urothelial hyperplasia and, finally, to bladder tumors, but molecular mechanisms of carcinogenesis have not still fully investigated. Here, we report the results of a study aimed at verifying the involvement of BAG3, an intracellular protein expressed in several tumors, in the Diuron-induced carcinogenesis. For this purpose, we analyzed the effect of Diuron on human primary urothelial cells and on human dermal fibroblasts. We found that while high concentrations of Diuron have a cytotoxic effect in human primary urothelial cells, in the same cells, noncytotoxic concentrations of the herbicide induce BAG3 expression. These findings show that BAG3 is a molecular target of Diuron and unravel the possible involvement of BAG3 protein in bladder carcinogenesis induced by the herbicide. In addition, these results suggest that BAG3 might be a potential early biomarker of damage induced by chronic exposure to Diuron.

Targeting IL-11 in the treatment of BK virus-associated haemorrhagic cystitis-A promising new approach.

The BK polyomavirus (BKPyV) has pathogenic relevance especially in immunocompromised patients. No causal therapy has been established yet. Therefore, new therapeutic targets need to be identified in experimental studies. A 3D organotypic cell culture model with primary urothelial cells and fibroblasts was used as infection model. The detection of virus replication was performed with quantitative polymerase chain reaction (qPCR), and immunohistochemistry (IHC) was also used for analysis. Interleukin levels were measured by enzyme‐linked immunosorbent assay (ELISA). Interestingly, the signal transducer and activator of transcription 3 (STAT3) pathway seems to be activated during infection with BKPyV, for example phosphorylated STAT3 is significantly (P < 0.0001) elevated on day 6 following infection. Therefore, we performed ELISAs for involved interleukins in STAT3 pathway. Interleukin 11 (IL‐11) was significantly (P = 0.026) elevated at day 9. Subsequently, 3D cultures were treated with IL‐11 neutralizing antibody. At day 9 following infection, the median virus replication rate is 4.4 × 106 copies/ml. The difference to replication rate without treatment was significantly lower at day 6 (P < 0.0001) and at day 9 (P < 0.0001), respectively. STAT3 pathways seem to be involved during BKPyV infection and need further investigation in experimental studies. A very promising target for treatment might be IL‐11.

Novel 3D organotypic urothelial cell culture model for identification of new therapeutic approaches in urological infections

Purpose3D organotypic cell cultures offer the possibility to study cell growth in a more in vivo like situation. To our knowledge no 3D culture of primary urothelial cells has been established yet. BK Polyomavirus (BKPyV), replicating in urothelial cells, may cause haemorrhagic cystitis in immunocompromised patients. Primary endpoints of this studyEstablishment of a 3D organotypic cell culture of primary urothelial cells and fibroblasts; use of this model as infection model for archetype BKPyV; description of first parts of viral life cycle with identification of therapeutic targets. MethodsThis is an experimental study. Primary urothelial cells were purchased from CellnTec, Bern, Switzerland; fibroblasts were isolated from the ureter of patients with no urothelial malignancy in their medical history. As main methods we used quantitative real-time PCR and immunohistochemistry. Outcomes were analysed using SPSS 23.0. ResultsWe were able to develop a 3D organotypic culture for primary urothelium. An infection with archetype BKPyV was established in this model with virus replication rates up to 6.41 × 108 copies/ml on day 9 following Infection. Interestingly, proliferation rate of the urothelial cells is significantly (p = 0.049 at day 6 following infection) elevated while cells are losing differentiation under infection. Phosphorylated STAT3 is also significantly elevated (p < 0.0001) during infection. ConclusionsThe established of urothelial 3D cultures is a new method to study several urothelial diseases. The archetype BKPyV infection model is novel and the first method to study archetype viral life cycle. The STAT3 pathway might be an interesting target for the development of a causal therapy.

Histamine induces peripheral and central hypersensitivity to bladder distension via the histamine H1 receptor and TRPV1.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a common chronic pelvic disorder with sensory symptoms of urinary urgency, frequency, and pain, indicating a key role for hypersensitivity of bladder-innervating sensory neurons. The inflammatory mast cell mediator histamine has long been implicated in IC/BPS, yet the direct interactions between histamine and bladder afferents remain unclear. In the present study, we show, using a mouse ex vivo bladder afferent preparation, that intravesical histamine enhanced the mechanosensitivity of subpopulations of afferents to bladder distension. Histamine also recruited "silent afferents" that were previously unresponsive to bladder distension. Furthermore, in vivo intravesical histamine enhanced activation of dorsal horn neurons within the lumbosacral spinal cord, indicating increased afferent signaling in the central nervous system. Quantitative RT-PCR revealed significant expression of histamine receptor subtypes (Hrh1-Hrh3) in mouse lumbosacral dorsal root ganglia (DRG), bladder detrusor smooth muscle, mucosa, and isolated urothelial cells. In DRG, Hrh1 was the most abundantly expressed. Acute histamine exposure evoked Ca2+ influx in select populations of DRG neurons but did not elicit calcium transients in isolated primary urothelial cells. Histamine-induced mechanical hypersensitivity ex vivo was abolished in the presence of the histamine H1 receptor antagonist pyrilamine and was not present in preparations from mice lacking transient receptor potential vanilloid 1 (TRPV1). Together, these results indicate that histamine enhances the sensitivity of bladder afferents to distension via interactions with histamine H1 receptor and TRPV1. This hypersensitivity translates to increased sensory input and activation in the spinal cord, which may underlie the symptoms of bladder hypersensitivity and pain experienced in IC/BPS.

Expression of components of the urothelial cholinergic system in bladder and cultivated primary urothelial cells of the pig

BackgroundPorcine urinary bladders are widely used for uro-pharmacological examinations due to their resemblance to the human organ. However, characterisations of the porcine urothelium at the molecular level are scarce up to now. As it has become clear over the last years that this tissue plays an important role in the signaling-pathways of the bladder, we examined whether the transporter and receptor pattern (with focus on the transmitter acetylcholine) is comparable to the human urothelium. With regard to in vitro studies, we also investigated if there is a difference between the native tissue and cultivated primary urothelial cells in culture.MethodsUrothelium from German Landrace and Göttingen Minipig bladders was collected. One part of the German Landrace tissue was used for cultivation, and different passages of the urothelial cells were collected. The actual mRNA expression of different transporters and receptors was examined via quantitative real-time PCR. These included the vesicular acetylcholine transporter (VAChT), the choline acetyl transferase (ChAT), organic cation transporters 1–3 (OCT1–3), organic anion transporting polypeptide 1A2 (OATP1A2), P-glycoprotein (ABCB1), the carnitine acetyl-transferase (CarAT), as well as the muscarinic receptors 1–5 (M1–5).ResultsThere is a strong qualitative resemblance between the human and the porcine urothelium with regard to the investigated cholinergic receptors, enzymes and transporters. CarAT, OCT1–3, OATP1A2 and ABCB1 could be detected in the urothelium of both pig races. Moreover, all 5 M-receptors were prominent with an emphasis on M2 and M3. VAChT and ChAT could not be detected at all. Cultures of the derived urothelial cells showed decreased expression of all targets apart from ABCB1 and CarAT.ConclusionsBased on the expression pattern of receptors, transporters and enzymes of the cholinergic system, the porcine urinary bladder can be regarded as a good model for pharmacological studies. However, cultivation of primary urothelial cells resulted in a significant drop in mRNA expression of the targets. Therefore, it can be concluded that the intact porcine urothelium, or the whole pig bladder, may be appropriate models for studies with anticholinergic drugs, whereas cultivated urothelial cells have some limitation due to significant changes in the expression levels of relevant targets.

The Outcome of Post-Chemotherapy Retroperitoneal Lymph Node Dissection in Patients with Metastatic Bladder Cancer in the Retroperitoneum.

Purpose: While a definitive cure can be achieved by radical cystectomy and pelvic lymph node dissection in select patients with regional lymphadenopathy, the benefit remains uncertain in patients who present with non-regional metastases. We analyzed the survival outcomes of post-chemotherapy retroperitoneal lymph node dissection.Materials and Methods: We reviewed our institutional database and identified 13 patients with radiographically evident or biopsy proven retroperitoneal nodal metastases with a significant response to chemotherapy. These patients underwent consolidative surgery with concomitant or delayed retroperitoneal lymph node dissection. The primary endpoints were progression-free survival and disease-specific survival from the time of retroperitoneal lymph node dissection.Results: All patients had primary urothelial cell carcinoma. Twelve patients underwent concomitant radical cystectomy, pelvic and retroperitoneal lymph node dissection. Seven patients (54%) had residual disease in the retroperitoneum and the median number of retroperitoneal nodes containing metastases was 4 (IQR 2–6). Six (86%) developed disease recurrences within 2 years of surgery and 5 (71%) died of cancer. Of the 6 patients without residual disease in the retroperitoneum, 2 (33%) developed recurrences and died of disease progression. The 2-year disease-specific survival was worse for patients with residual disease in the retroperitoneum than those without residual retroperitoneal disease (34%, 95% CI 5–68 vs 50%, 95% CI 6–85).Conclusions: The presence of retroperitoneal nodal metastases at post-chemotherapy retroperitoneal lymph node dissection is a poor prognosticator. Consolidative surgery with retroperitoneal lymph node dissection provides important prognostic information and may be therapeutic in a very small subset of these patients.

NLRP3 Promotes Diabetic Bladder Dysfunction and Changes in Symptom-Specific Bladder Innervation.

The NLRP3 inflammasome senses diabetic metabolites and initiates inflammation implicated in diabetic complications and neurodegeneration. No studies have investigated NLRP3 in diabetic bladder dysfunction (DBD), despite a high clinical prevalence. In vitro, we found that numerous diabetic metabolites activate NLRP3 in primary urothelial cells. In vivo, we demonstrate NLRP3 is activated in urothelia from a genetic type 1 diabetic mouse (Akita) by week 15. We then bred an NLRP3-/- genotype into these mice and found this blocked bladder inflammation and cystometric markers of DBD. Analysis of bladder innervation established an NLRP3-dependent decrease in overall nerve density and Aδ-fibers in the bladder wall along with an increase in C-fiber populations in the urothelia, which potentially explains the decreased sense of bladder fullness reported by patients and overactivity detected early in DBD. Together, the results demonstrate the role of NLRP3 in the genesis of DBD and suggest specific NLRP3-mediated neuronal changes can produce specific DBD symptoms.

NKA enhances bladder-afferent mechanosensitivity via urothelial and detrusor activation.

Tachykinins are expressed within bladder-innervating sensory afferents and have been shown to generate detrusor contraction and trigger micturition. The release of tachykinins from these sensory afferents may also activate tachykinin receptors on the urothelium or sensory afferents directly. Here, we investigated the direct and indirect influence of tachykinins on mechanosensation by recording sensory signaling from the bladder during distension, urothelial transmitter release ex vivo, and direct responses to neurokinin A (NKA) on isolated mouse urothelial cells and bladder-innervating DRG neurons. Bath application of NKA induced concentration-dependent increases in bladder-afferent firing and intravesical pressure that were attenuated by nifedipine and by the NK2 receptor antagonist GR159897 (100 nM). Intravesical NKA significantly decreased bladder compliance but had no direct effect on mechanosensitivity to bladder distension (30 µl/min). GR159897 alone enhanced bladder compliance but had no effect on mechanosensation. Intravesical NKA enhanced both the amplitude and frequency of bladder micromotions during distension, which induced significant transient increases in afferent firing, and were abolished by GR159897. NKA increased intracellular calcium levels in primary urothelial cells but not bladder-innervating DRG neurons. Urothelial ATP release during bladder distention was unchanged in the presence of NKA, whereas acetylcholine levels were reduced. NKA-mediated activation of urothelial cells and enhancement of bladder micromotions are novel mechanisms for NK2 receptor-mediated modulation of bladder mechanosensation. These results suggest that NKA influences bladder afferent activity indirectly via changes in detrusor contraction and urothelial mediator release. Direct actions on sensory nerves are unlikely to contribute to the effects of NKA.

Interleukin-6/Stat3 signaling has an essential role in the host antimicrobial response to urinary tract infection

The signaling networks regulating antimicrobial activity during urinary tract infection (UTI) are incompletely understood. Interleukin-6 (IL-6) levels increase with UTI severity, but the specific contributions of IL-6 to host immunity against bacterial uropathogens are unknown. To clarify this we tested whether IL-6 activates the Stat3 transcription factor, to drive a program of antimicrobial peptide gene expression in infected urothelium during UTI. Transurethral inoculation of uropathogenic Escherichia coli led to IL-6 secretion, urothelial Stat3 phosphorylation, and activation of antimicrobial peptide transcription, in a Toll-like receptor 4-dependent manner in a murine model of cystitis. Recombinant IL-6 elicited Stat3 phosphorylation in primary urothelial cells invitro, and systemic IL-6 administration promoted urothelial Stat3 phosphorylation and antimicrobial peptide expression invivo. IL-6 deficiency led to decreased urothelial Stat3 phosphorylation and antimicrobial peptide mRNA expression following UTI, a finding mirrored by conditional Stat3 deletion. Deficiency in IL-6 or Stat3 was associated with increased formation of intracellular bacterial communities, and exogenous IL-6 reversed this phenotype in IL-6 knockout mice. Moreover, chronic IL-6 depletion led to increased renal bacterial burden and severe pyelonephritis in C3H/HeOuJ mice. Thus, IL-6/Stat3 signaling drives a transcriptional program of antimicrobial gene expression in infected urothelium, with key roles in limiting epithelial invasion and ascending infection.

Spheroid Cultures of Primary Urothelial Cancer Cells: Cancer Tissue-Originated Spheroid (CTOS) Method.

Increasingly, it has been recognized that studying cancer samples from individual patients is important for the development of effective therapeutic strategies and in endeavors to overcome therapy resistance. Primary cultures of cancer cells acutely dissected from individual patients can provide a platform that enables the study and characterization of individual tumors. To that end, we have developed a method for preparing cancer cells in the form of multi-cellular spheroids. The cells can be derived from patient tumors (primary cells), from patient-derived xenografts, or from genetically- or chemically induced animal tumors. This method of culturing spheroids composed of cells derived from cancer tissues can be applied to various types of cancer, including urothelial cancer. The method is based on the principle of retaining cell-cell contact throughout cancer cell preparation and culturing. The first step is a partial digestion of the tumor specimen into small fragments; these fragments spontaneously form spheroidal shapes within several hours. The spheroid is referred to as a cancer tissue-originated spheroid (CTOS). The advantage of the CTOS method is that it allows one to prepare pure cancer cells at high yield. CTOSs can be stably cultured in serum-free conditions. The CTOS method can be applied to drug sensitivity assays, drug screening, and analyses of intracellular signaling. Moreover, the CTOS method provides a platform for studying the nature of cancer cell clusters.

SETD6 regulates NF-κB signaling in urothelial cell survival: Implications for bladder cancer.

Non-muscle invasive bladder cancer has a high recurrence rate of 45-70%, progressing to muscle invasive disease in about 15% of those patients over a 5-year period. Administration of the mycobacterium, Bacillus Calmette-Guerin (BCG) that induces local inflammation resulting in tumor remission in responsive patients is frequently used for treatment. BCG-treated patients with NF-κB del/del genotype have an increased risk of recurrence suggesting an important role of NF-κB in bladder cancer. Since protein methyltransferases play critical roles in modulating chromatin structure and gene expression, we screened a focused array of epigenetic modification genes to identify differential expression between normal urothelial and bladder cancer cells. We found and validated high expression of the SET-domain-containing protein methyltransferase, SETD6. SETD6 monomethylates NF-κB-p65 at lysine 310. Our results show that primary urothelial cells and normal bladder tissue have nearly undetectable message and protein level of SETD6 that increases in transformed urothelial cells and is further increased in bladder cancer cells and tissues. Overexpression of SETD6 in transformed urothelial cells increased cell survival and colony formation while knockdown in cancer cells decreased both parameters. Luciferase reporter assays showed that SETD6 induced the canonical NF-κB signaling pathway. Further, the use of catalytic SETD6 and IκBα mutant shows that SETD6 positively regulates survival by affecting p65 message, protein level and its function as determined by increased expression of NF-κB target genes. Our findings suggest that SETD6 plays an important role in NF-κB regulation and may have an important role in NF-κB-mediated local inflammatory response following BCG treatment.

AB276. SPR-03 In vitro evaluation of hydrostatic pressure on ATP release and Caspase-1 activation in rat urothelial cells

ObjectiveBladder outlet obstruction (BOO) is projected to affect approximately 1.1 billion men and women by 2018. While BOO results from a multitude of etiologies, they are all characterized by chronically elevated storage and voiding pressures, along with inflammation in the bladder tissue, which can lead to bladder fibrosis and overactive bladder symptoms. Previous research in our lab demonstrated that ATP release by rat primary urothelial cells increased in response to elevated pressure (10–15 cmH2O) in vitro. Thus, we hypothesize that the release of ATP by urothelial cells upon exposure to pathological pressures in BOO initiates a cascade of events that includes the formation of the NLRP3 inflammasome and caspase-1 activation. In the present study, we examined the effects of elevated pressure on ATP-purinergic signaling and caspase-1 activation in rat urothelial cells in vitro.MethodsUsing a custom built pressure system, rat urothelial cell line MYP3 cells (1.2×106 cells/well in F-12 K medium) were exposed to pressure conditions that represent both pathological storage and voiding pressures: 15 cmH2O 60 min, 15, 40 and 75 cmH2O 1 min. Cells that were prepared in a similar manner but maintained under atmospheric pressure served as a control. In addition, MYP3 cells that were exposed to a hypotonic condition (240 mOsm) and cells treated with 1.25 mM ATP served as two positive controls. After exposure to pressure, the supernatant media was collected and the extracellular ATP concentration was measured using a luciferin-luciferase assay kit (Life Technologies). Cells were then lysed and intracellular caspase-1 activity was measured using an established method. The pressure experiments (40 cmH2O 1 min) were repeated in the presence of a P2X7 antagonist [100 µM A-438079 (R&D Systems)] to determine the mechanism for pressure-induced caspase-1 activation.ResultsExposure of MYP3 cells to hydrostatic pressure for 1 min at 15, 40 and 75 cmH2O resulted in a 3-, 3.5- and 4.5-fold increase in extracellular ATP levels, respectively, compared to the 0 cmH2O control. When these cells were exposed to a hypotonic solution, ATP release increased by 6-fold compared to the isotonic control. Exposure of MYP3 cells to pressure also resulted in up to 1.3-fold increases in caspase-1 levels, which were similar to the positive controls, indicating caspase-1 levels are maximized after exposure to pathologic pressure conditions.ConclusionsThe significant increase in ATP release indicates that hydrostatic pressure is a good mechanical stimulus to model BOO in vitro. The acute caspase-1 responses after 1-minute exposure to pressure suggest that high-pressure voiding may be an important trigger of the NLRP3 inflammasome in BOO. The results that demonstrate exposure to pressure and treatment with ATP both result in caspase-1 activation implicate autocrine purinergic signaling as a mediator of NLRP inflammasome formation.Funding Source(s)NIH (R01DK103534, P20GM103444), NSF (1264579)

MP83-15 EFFECT OF GEMCITABINE AND MITOMYCIN ON CANCER STEM CELLS IN UROTHELIAL CARCINOMA CELLS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV1 Apr 2016MP83-15 EFFECT OF GEMCITABINE AND MITOMYCIN ON CANCER STEM CELLS IN UROTHELIAL CARCINOMA CELLS Rani Ojha, Shrawan Singh, Arup K Mandal, and Vivekanand Jha Rani OjhaRani Ojha More articles by this author , Shrawan SinghShrawan Singh More articles by this author , Arup K MandalArup K Mandal More articles by this author , and Vivekanand JhaVivekanand Jha More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2197AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To investigate the effect of chemotherapeutic agents on the behavior of cancer stem cell (CSC) subsets-side population in primary urothelial carcinoma cells (UCC). METHODS Low-grade urothelial carcinoma (LGUC), non muscle invasive high-grade (HGUC), muscle invasive high grade (MIUC) and bladder cancer cell lines (T24 and UM-UC-3) were used. Normal urothelial cells (NUC) were used as a control. Cytotoxicity of gemcitabine and mitomycin was measured by MTT assay in T24 and UM-UC-3 cells. Stemness properties were measured by isolation and characterization of side population (SP) using hoechst staining, by studying the expression of stemness genes using reverse transcriptase PCR analysis and tumor sphere forming ability. Effect of autophagy inhibition or induction on cell viability was measured by annexin-V/PI staining and caspase activity. RESULTS The number of SP was significantly higher in MIUCC (11.7%, p<0.05) and HGUCC (8.9%, p<0.05) as compared to LGUCC (4.2%) (Fig1A). SP cells showed more CD44 expression and tumor spheroid forming ability (Fig1B). Following treatment with gemcitabine or mitomycin, significantly increased number of SP cells was found in LGUCC (9.78%), HGUCC (13.56%), MIUCC (20.45%) and bladder cancer cell lines at 24 hours. In NUC only 1.7% SP cells were found and there was no increase in SP cells (Fig1C). Treatment with gemcitabine or mitomycin caused increased expression of stemness marker in SP (p<0.001) of HGUCC, LGUCC and MIUCC as compared to SP of NUC cells (Fig1D, E). SP of UCC cells treated with gemcitabine or mitomycin for 24 hours showed lower cell death as compared to NSP cells. Higher autophagic response was observed in SP of UCC in the presence of chemo-therapeutic agents as compared to NUC (Fig1F,G). Pharmacological inhibition or siRNA-mediated inhibition of autophagy led to decreased number of SP cells (Fig1H) and tumor sphere forming ability (Fig1I) with concomitant increase in caspase-mediated cell death (Fig1 J,K) in SP of UCC. CONCLUSIONS The present study is the first to show that urothelial cancer cells exposed to chemotherapeutic agents increases the stemness properties in UCC and could be the underlying cause of resistance and relapse of the tumor. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1086-e1087 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Rani Ojha More articles by this author Shrawan Singh More articles by this author Arup K Mandal More articles by this author Vivekanand Jha More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP68-04 CLOCK GENES REGULATE CIRCADIAN RHYTHM OF VNUT EXPRESSION, CONNEXIN26 EXPRESSION AND STRETCH-EVOKED ATP RELEASE IN THE CULTURED UROTHELIAL CELLS

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology II1 Apr 2016MP68-04 CLOCK GENES REGULATE CIRCADIAN RHYTHM OF VNUT EXPRESSION, CONNEXIN26 EXPRESSION AND STRETCH-EVOKED ATP RELEASE IN THE CULTURED UROTHELIAL CELLS Tatsuya Ihara, Satoru Kira, Tatsuya Miyamoto, Norifumi Sawada, Hiroshi Nakagomi, Takahiko Mitsui, Hideki Kobayashi, Mitsuharu Yoshiyama, Masayuki Takeda, Yuki Nakamura, Atsuhito Nakao, Eiji Shigetomi, Yohichi Shinozaki, and Shuichi Koizumi Tatsuya IharaTatsuya Ihara More articles by this author , Satoru KiraSatoru Kira More articles by this author , Tatsuya MiyamotoTatsuya Miyamoto More articles by this author , Norifumi SawadaNorifumi Sawada More articles by this author , Hiroshi NakagomiHiroshi Nakagomi More articles by this author , Takahiko MitsuiTakahiko Mitsui More articles by this author , Hideki KobayashiHideki Kobayashi More articles by this author , Mitsuharu YoshiyamaMitsuharu Yoshiyama More articles by this author , Masayuki TakedaMasayuki Takeda More articles by this author , Yuki NakamuraYuki Nakamura More articles by this author , Atsuhito NakaoAtsuhito Nakao More articles by this author , Eiji ShigetomiEiji Shigetomi More articles by this author , Yohichi ShinozakiYohichi Shinozaki More articles by this author , and Shuichi KoizumiShuichi Koizumi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1341AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Clock genes exist in almost all the cells and organs, and the products of Per, Cry, Bmal and Clock have the most important role to regulate circadian rhythm as representative clock genes. It has been known that lower urinary tract function is also regulated by clock genes. We previously reported that the Clock mutant mouse (ClockΔ19/Δ19) showed phenotype of nocturia (NOC) and nocturnal polyuria (NP). We hypothesis that clock genes regulate circadian rhythm of perception of the bladder fullness through the circadian expression of the ATP transmitters such as VNUT and Connexin26 in urothelium, which release ATP after mechanical stretch stimulation and send signals of urine perception to the CNS. In this study, we investigated the circadian rhythm of urine perception in mouse primary urothelial cell cultures (MPUCC), measuring the gene and protein expression of VNUT, Connexin26, and the ATP release after stretch stimulation. METHODS MPUCC isolated from C57BL/6 mice (WT) and C57BL/6 ClockΔ19/Δ19 were used. To reset and synchronize the gene expression rhythms in each cells, 50% horse serum shock (HSS) were added for 2hrs. Samples were collected every 4hrs from 12hrs later after HSS. That time was defined as 0. The expression of major clock genes, VNUT, and Connexin26 in MPUCC was investigated by RT-PCR and Western blot. Released ATP after stretch stimulation was quantified using luciferin-luciferase bioluminescence assay. Statistical analyses were done by one-way ANOVA. RESULTS In addition to major clock genes (e.g. Per2 and Bmal1), the expressions of mRNA and proteins of VNUT and Connexin26, showed circadian rhythm in WT. Meanwhile, these circadian rhythm were disrupted in ClockΔ19/Δ19 mice. ATP release after stretch stimulation showed circadian rhythm in WT, but ClockΔ19/Δ19lost this circadian rhythm. These oscillation changes were almost same as circadian rhythm of VNUT- and Connexin26-mRNA (Fig.1), which indicated that CLOCK could regulate the perception of bladderfullness. CONCLUSIONS Clock genes regulate circadian rhythm of perception of the bladder fullness, and disruption of clock genes could induce NOC and NP. Redressing of abnormalities of clock genes can lead to novel treatment of NOC/NP. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e890 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Tatsuya Ihara More articles by this author Satoru Kira More articles by this author Tatsuya Miyamoto More articles by this author Norifumi Sawada More articles by this author Hiroshi Nakagomi More articles by this author Takahiko Mitsui More articles by this author Hideki Kobayashi More articles by this author Mitsuharu Yoshiyama More articles by this author Masayuki Takeda More articles by this author Yuki Nakamura More articles by this author Atsuhito Nakao More articles by this author Eiji Shigetomi More articles by this author Yohichi Shinozaki More articles by this author Shuichi Koizumi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...