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.

The human urothelial tight junction: claudin 3 and the ZO-1α+ switch.

OBJECTIVE: Tight junctions are multicomponent structures, with claudin proteins defining paracellular permeability. Claudin 3 is a candidate for the exceptional "tightness" of human urothelium, being localised to the terminal tight junction (TJ) of superficial cells. Our aim was to determine whether claudin 3 plays an instigating and/or a functional role in the urothelial TJ. MATERIALS AND METHODS: Normal human urothelial (NHU) cells maintained as non-immortalised cell lines were retrovirally-transduced to over-express or silence claudin 3 expression. Stable sublines induced to stratify or differentiate were assessed for TJ formation by immunocytochemistry and transepithelial electrical resistance (TER). Expression of claudin 3, ZO-1 and ZO-1α+ was examined in native urothelium by immunohistochemistry. RESULTS: Claudin 3 expression was associated with differentiation and development of a tight barrier and along with ZO-1 and ZO-1α+ was localised to the apical tight junction in native urothelium. Knockdown of claudin 3 inhibited formation of a tight barrier in three independent cell lines, however, overexpression of claudin 3 was not sufficient to induce tight barrier development in the absence of differentiation. A differentiation-dependent induction of the ZO-1α+ isoform was found to coincide with barrier formation. Whereas claudin 3 overexpression did not induce the switch to co-expression of ZO-1α-/ZO-1α+, claudin 3 knockdown decreased localisation of ZO-1 to the TJ and resulted in compromised barrier function. CONCLUSIONS: Urothelial cytodifferentiation is accompanied by induction of claudin 3 which is essential for the development of a terminal TJ. A coordinated switch to the ZO-1α+ isotype was also observed and for the first time may indicate that ZO-1α+ is involved in the structural assembly and function of the urothelial terminal TJ.

Differentiation-associated reprogramming of the transforming growth factor ß receptor pathway establishes the circuitry for epithelial autocrine/paracrine repair.

Transforming growth factor (TGF) ß has diverse and sometimes paradoxical effects on cell proliferation and differentiation, presumably reflecting a fundamental but incompletely-understood role in regulating tissue homeostasis. It is generally considered that downstream activity is modulated at the ligand:receptor axis, but microarray analysis of proliferative versus differentiating normal human bladder epithelial cell cultures identified unexpected transcriptional changes in key components of the canonical TGFß R/activin signalling pathway associated with cytodifferentiation. Changes included upregulation of the transcriptional modulator SMAD3 and downregulation of inhibitory modulators SMURF2 and SMAD7. Functional analysis of the signalling pathway revealed that non-differentiated normal human urothelial cells responded in paracrine mode to TGFß by growth inhibition, and that exogenous TGFß inhibited rather than promoted differentiation. By contrast, in differentiated cell cultures, SMAD3 was activated upon scratch-wounding and was involved in promoting tissue repair. Exogenous TGFß enhanced the repair and resulted in hyperplastic scarring, indicating a feedback loop implicit in an autocrine pathway. Thus, the machinery for autocrine activation of the SMAD3-mediated TGFßR pathway is established during urothelial differentiation, but signalling occurs only in response to a trigger, such as wounding. Our study demonstrates that the circuitry of the TGFßR pathway is defined transcriptionally within a tissue-specific differentiation programme. The findings provide evidence for re-evaluating the role of TGFßR signalling in epithelial homeostasis as an autocrine-regulated pathway that suppresses differentiation and promotes tissue repair. This provides a new paradigm to help unravel the apparently diverse and paradoxical effect of TGFß signalling on cell proliferation and differentiation.

Toll-like receptor responses of normal human urothelial cells to bacterial flagellin and lipopolysaccharide.

PURPOSE: We determined toll-like receptor expression in normal human urothelium and functional responses in normal human urothelial cell cultures to bacterial lipopolysaccharide via toll-like receptor-4 and to flagellin via toll-like receptor-5. MATERIALS AND METHODS: Toll-like receptor protein expression was examined immunohistochemically. Toll-like receptor transcript expression was determined in freshly isolated urothelium, and in proliferating and differentiated normal human urothelial cultured cells. Lipopolysaccharide binding was assessed by flow cytometry. Functional responses of proliferating and differentiated normal human urothelial cells to lipopolysaccharide and flagellin were determined by interleukin-6 and 8 secretion, and transcription factor activation. Polymyxin B and siRNA were used to confirm the specificity of toll-like receptor-4 and 5 responses, respectively. Western blot detection of phosphorylated IκB was used to confirm toll-like receptor-4 results. RESULTS: Human urothelium expressed transcripts for toll-like receptor-4 and 5. Although bladder cancer derived T24 cells responded to lipopolysaccharide, there was no lipopolysaccharide binding to normal human urothelial cells and no functional response of proliferative or differentiated normal human urothelial cells even in the presence of exogenous CD14 and MD-2 accessory proteins. In contrast, flagellin evoked a toll-like receptor-5 mediated response in proliferating but not in differentiated normal human urothelial cells, which was abrogated by toll-like receptor-5 specific siRNA. CONCLUSIONS: Results suggest that human urothelium may mediate a host response to uropathogenic Escherichia coli through the detection of flagellin. The absent constitutive toll-like receptor-4 response may reflect an adaptation of urothelium toward sustaining barrier function and limiting inflammation to soluble bacterial products.

Differentiation potential of urothelium from patients with benign bladder dysfunction.

OBJECTIVE: To develop a novel in vitro approach to test the hypothesis that failure of urothelial differentiation underlies the aetiopathology of interstitial cystitis (IC), where there is evidence of compromised urinary barrier function, as benign dysfunctional bladder disease encompass several poorly understood clinically defined conditions, including IC, idiopathic detrusor overactivity (IDO) and stress urinary incontinence (SUI). MATERIALS AND METHODS: Biopsy-derived urothelial cells from dysfunctional bladder biopsies were propagated as finite cell lines and examined for their capacity to differentiate in vitro, as assessed by the acquisition of a transitional cell morphology, a switch from a cytokeratin (CK)13(lo)/CK14(hi) to a CK13(hi)/CK14(lo) phenotype, expression of claudin 3, 4 and 5 proteins, and induction of uroplakin gene transcription. RESULTS: Two of 12 SUI cell lines showed early senescent changes in culture and were not characterized further; one of seven IC, one of five IDO and a further three SUI cell lines had some evidence of senescence at passage 3. Of the seven IC-derived cell lines, four showed a near normal range of differentiation-associated responses, but the remainder showed little or no response. Most IDO cell lines (four of five) showed a normal differentiation response, but at least three of the 10 SUI cell lines showed some compromise of differentiation potential. CONCLUSION: This study supports the existence of a subset of patients with IC in whom a failure of urothelial cytodifferentiation might contribute to the disease, and provides a novel platform for investigating the cell biology of urothelium from SUI and other benign dysfunctional conditions.

CD40-mediated death and cytokine secretion in colorectal cancer: a potential target for inflammatory tumour cell killing.

CD40, a member of the tumour necrosis factor family, is expressed in a variety of epithelial cells. Although soluble CD40 agonists are growth-inhibitory, membrane-presented CD40 ligand (CD40L) induces extensive apoptosis in carcinoma cells. This study investigated whether CD40 is expressed in human colorectal carcinoma (CRC) cells and explored the functional consequences of CD40 ligation. CD40 expression in a panel of CRC lines was assessed by flow cytometry and in resected human CRCs by immunohistochemistry. CRC cells were treated in vitro with soluble CD40 agonists or cocultured with fibroblasts expressing membrane-bound CD40 ligand. Apoptosis was determined by flow cytometry using Annexin V/propidium iodide labelling and by a DNA fragmentation assay. Cytokine secretion induced by CD40 ligation was quantified by a multiplex-bead array approach. We show that CD40 is expressed in a proportion of established CRC lines in culture and that receptor expression is functional. Activation of CD40 by membrane-presented CD40L, but not soluble agonists, causes high levels of death in CD40-positive CRC cells and induces secretion of proinflammatory cytokines. In agreement with our in vitro observations, immunohistochemical studies demonstrated that CD40 is highly expressed in a proportion of colorectal cancer specimens. The high level of susceptibility of CRC cells to CD40-killing combined with the ability of CD40 to induce concomitant secretion of proinflammatory cytokines suggest that CD40 ligation may represent a novel mechanism for elimination of CRC cells and render CD40 a promising therapeutic target for the eradication of colorectal tumours.

PPARgamma-regulated tight junction development during human urothelial cytodifferentiation.

Urothelial barrier function is maintained by apical membrane plaques and intercellular tight junctions (TJ). Little is known about the composition and regulation of TJ expression in human urothelium. In this study, we have characterised the expression of TJ components in situ and their regulation in an in vitro model of differentiating normal human urothelial (NHU) cells. In normal ureteric urothelium in situ, there was a differentiation-associated profile of claudins 3, 4, 5, 7, ZO1 and occludin proteins. Proliferating NHU cells in vitro expressed predominantly claudin 1 protein and transcripts for claudins 1-5 and 7. Following induction of differentiation by pharmacological activation of PPARgamma and blockade of EGFR, there was de novo expression of claudin 3 mRNA and protein and downregulation of claudin 2 transcription. There was also a massive increase in expression of claudin 4 and 5 proteins which was due to inhibition of proteasomal degradation of claudin 4 and consequential stabilisation of the claudin 5 heterodimerisation partner. NHU cell differentiation was accompanied by relocalisation of TJ proteins to intercellular junctions. The differentiation-associated development of TJ formation in vitro reflected the stage-related TJ expression seen in situ. This was distinct from changes in TJ composition of NHU cells mediated by increasing the calcium concentration of the medium. Our results imply a role for PPARgamma and EGFR signalling pathways in regulating TJ formation in NHU cells and support the hypothesis that TJ development is an integral part of the urothelial differentiation programme.

Autocrine regulation of human urothelial cell proliferation and migration during regenerative responses in vitro.

Regeneration of the urothelium is rapid and effective in order to maintain a barrier to urine following tissue injury. Whereas normal human urothelial (NHU) cells are mitotically quiescent and G0 arrested in situ, they rapidly enter the cell cycle upon seeding in primary culture and show reversible growth arrest at confluency. We have used this as a model to investigate the role of EGF receptor signaling in urothelial regeneration and wound-healing. Transcripts for HER-1, HER-2, and HER-3 were expressed by quiescent human urothelium in situ. Expression of HER-1 was upregulated in proliferating cultures, whereas HER-2 and HER-3 were more associated with a growth-arrested phenotype. NHU cells could be propagated in the absence of exogenous EGF, but autocrine signaling through HER-1 via the MAPK and PI3-kinase pathways was essential for proliferation and migration during urothelial wound repair. HB-EGF was expressed by urothelium in situ and HB-EGF, epiregulin, TGF-alpha, and amphiregulin were expressed by proliferating NHU cells. Urothelial wound repair in vitro was attenuated by neutralizing antibodies against HER-1 ligands, particularly amphiregulin. By contrast, the same ligands applied exogenously promoted migration, but inhibited proliferation, implying that HER-1 ligands provoke differential effects in NHU cells depending upon whether they are presented as soluble or juxtacrine ligands. We conclude that proliferation and migration during wound healing in NHU cells are mediated through an EGFR autocrine signalling loop and our results implicate amphiregulin as a key mediator.

Effects of loss of p53 and p16 function on life span and survival of human urothelial cells.

Human urothelial cell carcinoma evolves via the accumulation of numerous genetic alterations, with loss of p53 and p16 function representing important stages in the development of superficial lesions and their progression to malignant disease. To investigate the effects of disabling either or both proteins in otherwise normal human urothelial cells, we performed retroviral transductions with a dominant negative p53 miniprotein and/or mutant cyclin-dependent kinase 4 (CDK4R24C) in 3 independent cell lines. Although cells with disabled p53 function showed a higher proliferation rate, inactivation of neither p53 nor p16 function resulted in any extension of life span and the double-transductants failed to flourish, demonstrating that further genetic alterations are required to attain an immortalised phenotype. However, CDK4R24C transductants showed a marked increase in apoptotic susceptibility to membrane-presented CD40 ligand, being intermediate between normal cells (nonsusceptible) and transformed cells (highly susceptible). By contrast, loss of p53 function alone only slightly increased the apoptotic susceptibility of urothelial cells. These results demonstrate that loss of p16 function, while insufficient to immortalise urothelial cells, nevertheless renders the cells more vulnerable to apoptosis induced by CD40 ligation.

Role of PPARgamma and EGFR signalling in the urothelial terminal differentiation programme.

Recently, considerable interest has focused on the ability of activated peroxisome proliferator-activated receptor gamma (PPARgamma) to promote cytodifferentiation in adipocytes and some carcinoma cells; however, the role of PPARgamma in normal epithelial cytodifferentiation is unknown. Using uroplakin (UP) gene expression as a specific correlate of terminal urothelial cytodifferentiation, we investigated the differentiation-inducing effects of PPARgamma activation in normal human urothelial (NHU) cells grown as finite cell lines in monoculture. Two high-affinity activators of PPARgamma, troglitazone (TZ) and rosiglitazone (RZ) induced the expression of mRNA for UPII and UPIb and, to a lesser extent, UPIa. The specificity of the effect was shown by pretreating cells with a PPARgamma antagonist, GW9662, which attenuated the TZ-induced response in a dose-specific manner. The PPARgamma-mediated effect on UP gene expression was maximal when there was concurrent inhibition of autocrine-activated epidermal growth factor receptor (EGFR) signalling through either the phosphatidylinositol 3-kinase or extracellular signal-regulated kinase (ERK) pathways. The use of a specific EGFR tyrosine kinase inhibitor, PD153035, correlated with PPARgamma dephosphorylation and translocation to the nucleus, indicating a mechanism for regulating the balance between proliferation and differentiation. This is the first identification of specific factors involved in regulating differentiation-associated gene changes in urothelium and the first unambiguous evidence of a role for PPARgamma signalling in the terminal differentiation programme of a normal epithelium.

The effects of malignant transformation on susceptibility of human urothelial cells to CD40-mediated apoptosis.

BACKGROUND: The tumor necrosis factor (TNF) superfamily of ligands and receptors mediates immune cell survival. Some members possess a death domain, a protein motif that functions to transmit apoptotic signals, whereas others, such as CD40, do not. CD40 is expressed by both normal and malignant epithelial cells. To investigate the functional significance of this expression, we studied the effects of ligation of CD40, Fas, and TNF receptors (TNFRs) on the proliferation and survival of normal and malignant human urothelial cells and urothelial cells with disabled p53 function. METHODS: Normal and malignant human urothelial cells were cultured with soluble TNF family agonists (CD40 ligand [CD40L], TNF-alpha, anti-Fas antibody, or cocultured with mouse fibroblasts stably transfected with plasmids that caused the cells to constitutively express CD40L or CD32; cell proliferation was estimated by an [(3)H]thymidine incorporation assay, and apoptosis was determined by Annexin V staining and by a DNA fragmentation assay. Messenger RNA levels for CD40 and potential downstream effector molecules were quantified by polymerase chain reaction-based and ribonuclease protection assays, respectively, and nuclear factor (NF) kappaB nuclear translocation was detected by immunofluorescence. All statistical tests were two-sided. RESULTS: Soluble trimeric CD40L inhibited the growth of normal and malignant urothelial cells but did not induce apoptosis. Cell surface-presented CD40L induced massive apoptosis in CD40-positive transitional cell carcinoma cells but not in normal urothelial cells. Normal cells underwent CD40L-mediated apoptosis only in the presence of other TNFR agonists. An agonistic anti-CD40 antibody presented on the surface of CD32-transfected fibroblasts also induced apoptosis in transitional cell carcinoma cells and in normal urothelial cells. Apoptotic responses of tumor (but not normal) cells to soluble agonists were enhanced by blocking protein synthesis. Karyotypically normal urothelial cells with disabled p53 function underwent apoptosis during coculture with CD40L-expressing fibroblasts alone but were not additionally sensitive to additional TNFR agonists. CONCLUSIONS: Susceptibility to CD40 ligation-induced apoptosis may be a novel mechanism for eliminating neoplastically transformed urothelial cells. Loss of CD40 expression may be an important adaptive mechanism for transitional cell carcinoma development and progression.