The Urothelial Transcriptomic Response to Interferon Gamma: Implications for Bladder Cancer Prognosis and Immunotherapy.

Interferon gamma (IFNγ) is central to the inflammatory immune response, such as that entrained by BCG immunotherapy for bladder cancer. However, immune-mediated tumour cell killing is subject to modulation by immunoinhibitory "checkpoint" receptors such as PD-L1. We investigated the effects of IFNγ on barrier-forming in vitro-differentiated normal human urothelium using mRNA-sequencing, and showed canonical upregulation of MHC class I/II and de novo expression of the T cell tropic CXCL9-11 chemokines. Normal urothelium constitutively expressed immunoinhibitory B7 family member VSIR (VISTA), while CD274 (PD-L1) expression was induced/upregulated by IFNγ. We generated a urothelial IFNγ response gene signature. When applied to the unsupervised clustering of non-muscle-invasive bladder cancers, the IFNγ-signature predicted longer recurrence-free survival. In muscle-invasive cancers, the IFNγ-signature split the basal/squamous consensus subtype, with significantly worse overall survival when weak or absent. This study offers novel insights into strategies to enhance immunotherapy via the IFNγ and VISTA/PD-L1 nexus.

LPCAT4 Knockdown Alters Barrier Integrity and Cellular Bioenergetics in Human Urothelium.

Urothelium is a transitional, stratified epithelium that lines the lower urinary tract, providing a tight barrier to urine whilst retaining the capacity to stretch and rapidly resolve damage. The role of glycerophospholipids in urothelial barrier function is largely unknown, despite their importance in membrane structural integrity, protein complex assembly, and the master regulatory role of PPARγ in urothelial differentiation. We performed lipidomic and transcriptomic characterisation of urothelial differentiation, revealing a metabolic switch signature from fatty acid synthesis to lipid remodelling, including 5-fold upregulation of LPCAT4. LPCAT4 knockdown urothelial cultures exhibited an impaired proliferation rate but developed elevated trans-epithelial electrical resistances upon differentiation, associated with a reduced and delayed capacity to restitute barrier function after wounding. Specific reduction in 18:1 PC fatty acyl chains upon knockdown was consistent with LPCAT4 specificity, but was unlikely to elicit broad barrier function changes. However, transcriptomic analysis of LPCAT4 knockdown supported an LPC-induced reduction in DAG availability, predicted to limit PKC activity, and TSPO abundance, predicted to limit endogenous ATP. These phenotypes were confirmed by PKC and TSPO inhibition. Together, these data suggest an integral role for lipid mediators in urothelial barrier function and highlight the strength of combined lipidomic and transcriptomic analyses for characterising tissue homeostasis.

Barrier-Forming Potential of Epithelial Cells from the Exstrophic Bladder.

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

Induction of APOBEC3-mediated genomic damage in urothelium implicates BK polyomavirus (BKPyV) as a hit-and-run driver for bladder cancer.

Limited understanding of bladder cancer aetiopathology hampers progress in reducing incidence. Mutational signatures show the anti-viral apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) enzymes are responsible for the preponderance of mutations in bladder tumour genomes, but no causative viral agent has been identified. BK polyomavirus (BKPyV) is a common childhood infection that remains latent in the adult kidney, where reactivation leads to viruria. This study provides missing mechanistic evidence linking reactivated BKPyV-infection to bladder cancer risk. We used a mitotically-quiescent, functionally-differentiated model of normal human urothelium to examine BKPyV-infection. BKPyV-infection led to significantly elevated APOBEC3A and APOBEC3B protein, increased deaminase activity and greater numbers of apurinic/apyrimidinic sites in the host urothelial genome. BKPyV Large T antigen (LT-Ag) stimulated re-entry from G0 into the cell cycle through inhibition of retinoblastoma protein and activation of EZH2, E2F1 and FOXM1, with cells arresting in G2. The single-stranded DNA displacement loops formed in urothelial cells during BKPyV-infection interacted with LT-Ag to provide a substrate for APOBEC3-activity. Addition of interferon gamma (IFNγ) to infected urothelium suppressed expression of the viral genome. These results support reactivated BKPyV infections in adults as a risk factor for bladder cancer in immune-insufficient populations.

Surface Shave: Revealing the Apical-Restricted Uroglycome.

This study aimed to investigate the highly differentiated urothelial apical surface glycome. The functions of the mammalian urothelium, lining the majority of the urinary tract and providing a barrier against toxins in urine, are dependent on the correct differentiation of urothelial cells, relying on protein expression, modification, and complex assembly to regulate the formation of multiple differentiated cell layers. Protein glycosylation, a poorly studied aspect of urothelial differentiation, contributes to the apical glycome and is implicated in the development of urothelial diseases. To enable surface glycome characterization, we developed a method to collect tissue apical surface N- and O-glycans. A simple, novel device using basic laboratory supplies was developed for enzymatic shaving of the luminal bladder urothelial surface, with subsequent release and mass spectrometric analysis of apical surface O- and N-glycans, the first normal mammalian urothelial N-glycome to be defined. Trypsinization of superficial glycoproteins was tracked using immunolabeling of the apically expressed uroplakin 3a protein to optimize enzymatic release, without compromising the integrity of the superficial urothelial layer. The approach developed for releasing apical tissue surface glycans allowed for comparison with the N-glycome of the total porcine bladder urothelial cells and thus identification of apical surface glycans as candidates implicated in the urothelial barrier function. Data are available in MassIve: MSV000087851.

Translation of mechanical strain to a scalable biomanufacturing process for acellular matrix production from full thickness porcine bladders.

Bladder acellular matrix has promising applications in urological and other reconstructive surgery as it represents a naturally compliant, non-immunogenic and highly tissue-integrative material. As the bladder fills and distends, the loosely-coiled bundles of collagen fibres in the wall become extended and orientate parallel to the lumen, resulting in a physical thinning of the muscular wall. This accommodating property can be exploited to achieve complete decellularisation of the full-thickness bladder wall by immersing the distended bladder through a series of hypotonic buffers, detergents and nucleases, but the process is empirical, idiosyncratic and does not lend itself to manufacturing scale up. In this study we have taken a mechanical engineering approach to determine the relationship between porcine bladder size and capacity, to define the biaxial deformation state of the tissue during decellularisation and to apply these principles to the design and testing of a scalable novel laser-printed flat-bed apparatus in order to achieve reproducible and full-thickness bladder tissue decellularisation. We demonstrate how the procedure can be applied reproducibly to fresh, frozen or twice-frozen bladders to render8×8 cm2patches of DNA-free acellular matrix suitable for surgical applications.

Augmentation of the insufficient tissue bed for surgical repair of hypospadias using acellular matrix grafts: A proof of concept study.

Acellular matrices produced by tissue decellularisation are reported to have tissue integrative properties. We examined the potential for incorporating acellular matrix grafts during procedures where there is an inadequate natural tissue bed to support an enduring surgical repair. Hypospadias is a common congenital defect requiring surgery, but associated with long-term complications due to deficiencies in the quality and quantity of the host tissue bed at the repair site. Biomaterials were implanted as single on-lay grafts in a peri-urethral position in male pigs. Two acellular tissue matrices were compared: full-thickness porcine acellular bladder matrix (PABM) and commercially-sourced cross-linked acellular matrix from porcine dermis (Permacol™). Anatomical and immunohistological outcomes were assessed 3 months post-surgery. There were no complications and surgical sites underwent full cosmetic repair. PABM grafts were fully incorporated, whilst Permacol™ grafts remained palpable. Immunohistochemical analysis indicated a non-inflammatory, remodelling-type response to both biomaterials. PABM implants showed extensive stromal cell infiltration and neovascularisation, with a significantly higher density of cells (p < 0.001) than Permacol™, which showed poor cellularisation and partial encapsulation. This study supports the anti-inflammatory and tissue-integrative nature of non-crosslinked acellular matrices and provides proof-of-principle for incorporating acellular matrices during surgical procedures, such as in primary complex hypospadias repair.

Procarcinogen Activation and Mutational Signatures Model the Initiation of Carcinogenesis in Human Urothelial Tissues In Vitro.

Disparity between genome-wide mutations in bladder and other cancers where smoking is a risk factor raises questions about carcinogenesis in different epithelia. To develop an experimental model of bladder carcinogenesis, we clonally expanded in vitro differentiated normal human urothelial (NHU) cells following exposure to an exemplar procarcinogen and used whole-genome DNA sequencing to derive mutational signatures. Benzo[a]pyrene (BaP) was activated by endogenous cytochrome P450 (cytochrome P450 family 1 subfamily A member 1 [CYP1A1]) to create genomically modified NHU cells. Comparison with the Catalogue of Somatic Mutations in Cancer (COSMIC) showed that mutations induced by BaP in NHU cells were similar to smoking-associated signatures in bladder and other cancers, including single- and doublet-base substitution signatures characterised by C > A transversions (COSMIC_SBS4 and COSMIC_DBS2, respectively), and an insertion/deletion signature of C deletions in homopolymer regions (COSMIC ID3). Our study provides the first direct evidence that BaP is activated locally in the urothelium, initiating the well-described smoking-associated mutational signatures. An absence of other common bladder cancer (BLCA)-associated genomic signatures points strongly to other primary causes of BLCA, which the new experimental approach described here is well placed to investigate. Mutational signatures ignore whether genes are affected, but tissue-specific drivers (KMT2D, KMT2C, and CDKN1A) were significantly overmutated in this model, providing insight on the emergent selection pressures. PATIENT SUMMARY: In a carefully controlled laboratory setting, we exposed normal human urothelial tissues to a procarcinogen (benzo[a]pyrene) found in cigarette smoke. We show that the urothelial tissues activated the carcinogen and led to mutations forming across the genome in a characteristic pattern. This particular "mutational signature" is found in bladder tumours and other smoking-induced cancers (eg, lung); however, our study highlights that there are other unknown mutational processes in bladder cancer that is not the direct result of smoke carcinogens, and this will require further investigation.

Loss of Janus Associated Kinase 1 Alters Urothelial Cell Function and Facilitates the Development of Bladder Cancer.

Inherited Primary Immunodeficiency (PID) disorders are associated with increased risk of malignancy that may relate to impaired antitumor immune responses or a direct role for PID germline mutations in tumorigenesis. We recently identified germline loss of function mutations in Janus Associated Kinase 1 (JAK1) causing primary immunodeficiency characterized by infections and associated with early onset, fatal high-grade bladder carcinoma. Somatic mutations in JAK1, required for immune cell signaling in response to interferon gamma (IFNγ), have been associated with several non-hematopoietic and hematopoietic cancer cell types but pathogenic mechanisms remain largely unexplored. Here we demonstrate that JAK1 is required for the intrinsic IFNγ response of urothelial cells impacting immunogenicity and cell survival. Specifically, JAK1-deficient urothelial cells showed reduced surface expression of major histocompatibility complex class II (MHC II), intercellular adhesion molecule-1 (ICAM-1) and programmed death-ligand-1 (PD-L1) after IFNγ stimulation and were resistant to IFNγ-induced apoptosis and lymphocyte-mediated killing. In addition, we identify a previously unknown role for IFNγ signaling in modulating urothelial differentiation. Together, our findings support a role for urothelial cell JAK1 in immune surveillance and development of bladder cancer. Our results have implications for patients with rare JAK1 PID and, more broadly, inform development of biomarker and targeted therapies for urothelial carcinoma.

Specificity of the Metallothionein-1 Response by Cadmium-Exposed Normal Human Urothelial Cells.

Occupational and environmental exposure to cadmium is associated with the development of urothelial cancer. The metallothionein (MT) family of genes encodes proteins that sequester metal ions and modulate physiological processes, including zinc homeostasis. Little is known about the selectivity of expression of the different MT isoforms. Here, we examined the effect of cadmium exposure on MT gene and isoform expression by normal human urothelial (NHU) cell cultures. Baseline and cadmium-induced MT gene expression was characterized by next-generation sequencing and RT-PCR; protein expression was assessed by Western blotting using isoform-specific antibodies. Expression of the zinc transporter-1 (SLC30A1) gene was also assessed. NHU cells displayed transcription of MT-2A, but neither MT-3 nor MT-4 genes. Most striking was a highly inducer-specific expression of MT-1 genes, with cadmium inducing transcription of MT-1A, MT-1G, MT-1H, and MT-1M. Whereas MT-1G was also induced by zinc and nickel ions and MT-1H by iron, both MT-1A and MT-1M were highly cadmium-specific, which was confirmed for protein using isoform-specific antibodies. Protein but not transcript endured post-exposure, probably reflecting sequestration. SLC30A1 transcription was also affected by cadmium ion exposure, potentially reflecting perturbation of intracellular zinc homeostasis. We conclude that human urothelium displays a highly inductive profile of MT-1 gene expression, with two isoforms identified as highly specific to cadmium, providing candidate transcript and long-lived protein biomarkers of cadmium exposure.

Differential transcription factor expression by human epithelial cells of buccal and urothelial derivation.

Identification of transcription factors expressed by differentiated cells is informative not only of tissue-specific pathways, but to help identify master regulators for cellular reprogramming. If applied, such an approach could generate healthy autologous tissue-specific cells for clinical use where cells from the homologous tissue are unavailable due to disease. Normal human epithelial cells of buccal and urothelial derivation maintained in identical culture conditions that lacked significant instructive or permissive signaling cues were found to display inherent similarities and differences of phenotype. Investigation of transcription factors implicated in driving urothelial-type differentiation revealed buccal epithelial cells to have minimal or absent expression of PPARG, GATA3 and FOXA1 genes. Retroviral overexpression of protein coding sequences for GATA3 or PPARy1 in buccal epithelial cells resulted in nuclear immunolocalisation of the respective proteins, with both transductions also inducing expression of the urothelial differentiation-associated claudin 3 tight junction protein. PPARG1 overexpression alone entrained expression of nuclear FOXA1 and GATA3 proteins, providing objective evidence of its upstream positioning in a transcription factor network and identifying it as a candidate factor for urothelial-type transdifferentiation or reprogramming.

Implications of TERT promoter mutations and telomerase activity in urothelial carcinogenesis.

Telomerase activity imparts eukaryotic cells with unlimited proliferation capacity, one of the cancer hallmarks. Over 90% of human urothelial carcinoma of the bladder (UCB) tumours are positive for telomerase activity. Telomerase activation can occur through several mechanisms. Mutations in the core promoter region of the human telomerase reverse transcriptase gene (TERT) cause telomerase reactivation in 60-80% of UCBs, whereas the prevalence of these mutations is lower in urothelial cancers of other origins. TERT promoter mutations are the most frequent genetic alteration across all stages of UCB, indicating a strong selection pressure during neoplastic transformation. TERT promoter mutations could arise during regeneration of normal urothelium and, owing to consequential telomerase reactivation, might be the basis of UCB initiation, which represents a new model of urothelial cancer origination. In the future, TERT promoter mutations and telomerase activity might have diagnostic and therapeutic applications in UCB.

Differentiation-associated urothelial cytochrome P450 oxidoreductase predicates the xenobiotic-metabolizing activity of "luminal" muscle-invasive bladder cancers.

Extra-hepatic metabolism of xenobiotics by epithelial tissues has evolved as a self-defence mechanism but has potential to contribute to the local activation of carcinogens. Bladder epithelium (urothelium) is bathed in excreted urinary toxicants and pro-carcinogens. This study reveals how differentiation affects cytochrome P450 (CYP) activity and the role of NADPH:P450 oxidoreductase (POR). CYP1A1 and CYP1B1 transcripts were inducible in normal human urothelial (NHU) cells maintained in both undifferentiated and functional barrier-forming differentiated states in vitro. However, ethoxyresorufin O-deethylation (EROD) activity, the generation of reactive BaP metabolites and BaP-DNA adducts, were predominantly detected in differentiated NHU cell cultures. This gain-of-function was attributable to the expression of POR, an essential electron donor for all CYPs, which was significantly upregulated as part of urothelial differentiation. Immunohistology of muscle-invasive bladder cancer (MIBC) revealed significant overall suppression of POR expression. Stratification of MIBC biopsies into "luminal" and "basal" groups, based on GATA3 and cytokeratin 5/6 labeling, showed POR over-expression by a subgroup of the differentiated luminal tumors. In bladder cancer cell lines, CYP1-activity was undetectable/low in basal PORlo T24 and SCaBER cells and higher in the luminal POR over-expressing RT4 and RT112 cells than in differentiated NHU cells, indicating that CYP-function is related to differentiation status in bladder cancers. This study establishes POR as a predictive biomarker of metabolic potential. This has implications in bladder carcinogenesis for the hepatic versus local activation of carcinogens and as a functional predictor of the potential for MIBC to respond to prodrug therapies.

Publisher Correction: Genome-wide linkage and association study implicates the 10q26 region as a major genetic contributor to primary nonsyndromic vesicoureteric reflux.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Genome-wide linkage and association study implicates the 10q26 region as a major genetic contributor to primary nonsyndromic vesicoureteric reflux.

Vesicoureteric reflux (VUR) is the commonest urological anomaly in children. Despite treatment improvements, associated renal lesions - congenital dysplasia, acquired scarring or both - are a common cause of childhood hypertension and renal failure. Primary VUR is familial, with transmission rate and sibling risk both approaching 50%, and appears highly genetically heterogeneous. It is often associated with other developmental anomalies of the urinary tract, emphasising its etiology as a disorder of urogenital tract development. We conducted a genome-wide linkage and association study in three European populations to search for loci predisposing to VUR. Family-based association analysis of 1098 parent-affected-child trios and case/control association analysis of 1147 cases and 3789 controls did not reveal any compelling associations, but parametric linkage analysis of 460 families (1062 affected individuals) under a dominant model identified a single region, on 10q26, that showed strong linkage (HLOD = 4.90; ZLRLOD = 4.39) to VUR. The ~9Mb region contains 69 genes, including some good biological candidates. Resequencing this region in selected individuals did not clearly implicate any gene but FOXI2, FANK1 and GLRX3 remain candidates for further investigation. This, the largest genetic study of VUR to date, highlights the 10q26 region as a major genetic contributor to VUR in European populations.

Targeting Deficiencies in the TLR5 Mediated Vaginal Response to Treat Female Recurrent Urinary Tract Infection.

The identification of the host defence peptides as target effectors in the innate defence of the uro-genital tract creates new translational possibilities for immunomodulatory therapies, specifically vaginal therapies to treat women suffering from rUTI, particularly those carrying the TLR5_C1174T SNP. Urinary tract infections (UTIs) are a microbial disease reported worldwide. Women are particularly susceptible with many suffering debilitating recurrent (r) infections. Treatment is by antibiotics, but such therapy is linked to antibiotic resistance and re-infection. This study explored the innate protective mechanisms of the urogenital tract with the aim of boosting such defences therapeutically. Modelling UTIs in vitro, human vaginal and bladder epithelial cells were challenged with uropathogenic Escherichia coli (CFT073) and microbial PAMPs including flagellin, LPS and peptidoglycan. Flagellin functioning via the TLR5/NFκB pathway was identified as the key UPEC virulence factor causing a significant increase (P < 0.05) in the production of the host-defence peptide (HDP), BD2. BD2-depleted urine samples from bladder infected mice supported increased UPEC growth, strengthening the significance of the HDPs in protecting the urogenital tissues from infection. Clinically, vaginal-douche BD2 concentrations were reduced (p < 0.05) in women suffering rUTIs, compared to age-matched healthy controls with concentrations further decreased (p < 0.05) in a TLR5392Stop SNP rUTI subgroup. Topical vaginal estrogen treatment increased (p < 0.001) BD2 concentrations in all women, including those carrying the SNP. These data identify therapeutic and antibiotic sparing roles for vaginal immunomodulatory agents that specifically target HDP induction, facilitate bacterial killing and disrupt the UPEC infection cycle.

Heterarchy of transcription factors driving basal and luminal cell phenotypes in human urothelium.

Cell differentiation is affected by complex networks of transcription factors that co-ordinate re-organisation of the chromatin landscape. The hierarchies of these relationships can be difficult to dissect. During in vitro differentiation of normal human uro-epithelial cells, formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and RNA-seq was used to identify alterations in chromatin accessibility and gene expression changes following activation of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) as a differentiation-initiating event. Regions of chromatin identified by FAIRE-seq, as having altered accessibility during differentiation, were found to be enriched with sequence-specific binding motifs for transcription factors predicted to be involved in driving basal and differentiated urothelial cell phenotypes, including forkhead box A1 (FOXA1), P63, GRHL2, CTCF and GATA-binding protein 3 (GATA3). In addition, co-occurrence of GATA3 motifs was observed within subsets of differentiation-specific peaks containing P63 or FOXA1. Changes in abundance of GRHL2, GATA3 and P63 were observed in immunoblots of chromatin-enriched extracts. Transient siRNA knockdown of P63 revealed that P63 favoured a basal-like phenotype by inhibiting differentiation and promoting expression of basal marker genes. GATA3 siRNA prevented differentiation-associated downregulation of P63 protein and transcript, and demonstrated positive feedback of GATA3 on PPARG transcript, but showed no effect on FOXA1 transcript or protein expression. This approach indicates that as a transcriptionally regulated programme, urothelial differentiation operates as a heterarchy, wherein GATA3 is able to co-operate with FOXA1 to drive expression of luminal marker genes, but that P63 has potential to transrepress expression of the same genes.

Characterization and classification of adherent cells in monolayer culture using automated tracking and evolutionary algorithms.

This paper presents a novel method for tracking and characterizing adherent cells in monolayer culture. A system of cell tracking employing computer vision techniques was applied to time-lapse videos of replicate normal human uro-epithelial cell cultures exposed to different concentrations of adenosine triphosphate (ATP) and a selective purinergic P2X antagonist (PPADS), acquired over a 24h period. Subsequent analysis following feature extraction demonstrated the ability of the technique to successfully separate the modulated classes of cell using evolutionary algorithms. Specifically, a Cartesian Genetic Program (CGP) network was evolved that identified average migration speed, in-contact angular velocity, cohesivity and average cell clump size as the principal features contributing to the separation. Our approach not only provides non-biased and parsimonious insight into modulated class behaviours, but can be extracted as mathematical formulae for the parameterization of computational models.