Lisa A. Kirkwood

Deregulation of Rab and Rab effector genes in bladder cancer

Growing evidence indicates that Rab GTPases, key regulators of intracellular transport in eukaryotic cells, play an important role in cancer. We analysed the deregulation at the transcriptional level of the genes encoding Rab proteins and Rab-interacting proteins in bladder cancer pathogenesis, distinguishing between the two main progression pathways so far identified in bladder cancer: the Ta pathway characterized by a high frequency of FGFR3 mutation and the carcinoma in situ pathway where no or infrequent FGFR3 mutations have been identified. A systematic literature search identified 61 genes encoding Rab proteins and 223 genes encoding Rab-interacting proteins. Transcriptomic data were obtained for normal urothelium samples and for two independent bladder cancer data sets corresponding to 152 and 75 tumors. Gene deregulation was analysed with the SAM (significant analysis of microarray) test or the binomial test. Overall, 30 genes were down-regulated, and 13 were up-regulated in the tumor samples. Five of these deregulated genes (LEPRE1, MICAL2, RAB23, STXBP1, SYTL1) were specifically deregulated in FGFR3-non-mutated muscle-invasive tumors. No gene encoding a Rab or Rab-interacting protein was found to be specifically deregulated in FGFR3-mutated tumors. Cluster analysis showed that the RAB27 gene cluster (comprising the genes encoding RAB27 and its interacting partners) was deregulated and that this deregulation was associated with both pathways of bladder cancer pathogenesis. Finally, we found that the expression of KIF20A and ZWINT was associated with that of proliferation markers and that the expression of MLPH, MYO5B, RAB11A, RAB11FIP1, RAB20 and SYTL2 was associated with that of urothelial cell differentiation markers. This systematic analysis of Rab and Rab effector gene deregulation in bladder cancer, taking relevant tumor subgroups into account, provides insight into the possible roles of Rab proteins and their effectors in bladder cancer pathogenesis. This approach is applicable to other group of genes and types of cancer.

Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium

In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca²⁺. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca²⁺ and in a scratch repair assay. The results confirmed the functional expression of P2Y₄ receptors and excluded nonexpressed receptors/channels (P2X₁, P2X₃, P2X₆, P2Y₆, P2Y₁₁, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X₂, P2X₄, P2Y₁, P2Y₂, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca²⁺ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting.

Differential Expression of Oct4 Variants and Pseudogenes in Normal Urothelium and Urothelial Cancer

The transcription factor octamer-binding protein 4 (Oct4; encoded by POU5F1) has a key role in maintaining embryonic stem cell pluripotency during early embryonic development and it is required for generation of induced pluripotent stem cells. Controversy exists concerning Oct4 expression in somatic tissues, with reports that Oct4 is expressed in normal and in neoplastic urothelium carrying implications for a bladder cancer stem cell phenotype. Here, we show that the pluripotency-associated Oct4A transcript was absent from cultures of highly regenerative normal human urothelial cells and from low-grade to high-grade urothelial carcinoma cell lines, whereas alternatively spliced variants and transcribed pseudogenes were expressed in abundance. Immunolabeling and immunoblotting studies confirmed the absence of Oct4A in normal and neoplastic urothelial cells and tissues, but indicated the presence of alternative isoforms or potentially translated pseudogenes. The stable forced expression of Oct4A in normal human urothelial cells in vitro profoundly inhibited growth and affected morphology, but protein expression was rapidly down-regulated. Our findings demonstrate that pluripotency-associated isoform Oct4A is not expressed by normal or malignant human urothelium and therefore is unlikely to play a role in a cancer stem cell phenotype. However, our findings also indicate that urothelium expresses a variety of other Oct4 splice-variant isoforms and transcribed pseudogenes that warrant further study.

Differential Regulation of Growth-Promoting Signalling Pathways by E-Cadherin

BACKGROUND Despite the well-documented association between loss of E-cadherin and carcinogenesis, as well as the link between restoration of its expression and suppression of proliferation in carcinoma cells, the ability of E-cadherin to modulate growth-promoting cell signalling in normal epithelial cells is less well understood and frequently contradictory. The potential for E-cadherin to co-ordinate different proliferation-associated signalling pathways has yet to be fully explored. METHODOLOGY/PRINCIPAL FINDINGS Using a normal human urothelial (NHU) cell culture system and following a calcium-switch approach, we demonstrate that the stability of NHU cell-cell contacts differentially regulates the Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-Regulated Kinase (ERK) and Phosphatidylinositol 3-Kinase (PI3-K)/AKT pathways. We show that stable cell contacts down-modulate the EGFR/ERK pathway, whilst inducing PI3-K/AKT activity, which transiently enhances cell growth at low density. Functional inactivation of E-cadherin interferes with the capacity of NHU cells to form stable calcium-mediated contacts, attenuates E-cadherin-mediated PI3-K/AKT induction and enhances NHU cell proliferation by allowing de-repression of the EGFR/ERK pathway and constitutive activation of β-catenin-TCF signalling. CONCLUSIONS/SIGNIFICANCE Our findings provide evidence that E-cadherin can differentially and concurrently regulate specific growth-related signalling pathways in a context-specific fashion, with direct, functional consequences for cell proliferation and population growth. Our observations not only reveal a novel, complex role for E-cadherin in normal epithelial cell homeostasis and tissue regeneration, but also provide the basis for a more complete understanding of the consequences of E-cadherin loss on malignant transformation.

A novel bidirectional positive-feedback loop between Wnt-β-catenin and EGFR-ERK plays a role in context-specific modulation of epithelial tissue regeneration.

ABSTRACT By operating as both a subunit of the cadherin complex and a key component of Wnt signalling, &bgr;-catenin acts as the lynchpin between cell–cell contact and transcriptional regulation of proliferation, coordinating epithelial tissue homeostasis and regeneration. The integration of multiple growth-regulatory inputs with &bgr;-catenin signalling has been observed in cancer-derived cells, yet the existence of pathway crosstalk in normal cells is unknown. Using a highly regenerative normal human epithelial culture system that displays contact inhibition, we demonstrate that the receptor tyrosine kinase (RTK)-driven MAPK and Wnt–&bgr;-catenin signalling axes form a bidirectional positive-feedback loop to drive cellular proliferation. We show that &bgr;-catenin both drives and is regulated by proliferative signalling cues, and its downregulation coincides with the switch from proliferation to contact-inhibited quiescence. We reveal a novel contextual interrelationship whereby positive and negative feedback between three major signalling pathways – EGFR–ERK, PI3K–AKT and Wnt–&bgr;-catenin – enable autocrine-regulated tissue homeostasis as an emergent property of physical interactions between cells. Our work has direct implications for normal epithelial tissue homeostasis and provides insight as to how dysregulation of these pathways could drive excessive and sustained cellular growth in disease.

Immortalisation of Normal Human Urothelial Cells Compromises Differentiation Capacity

BACKGROUND The development of urothelial malignancy is not solely a consequence of loss of proliferation constraints but also involves loss of cellular differentiation, defined histopathologically as grade. Although tumour grade is an independent prognostic marker for urothelial carcinoma (UC), the molecular events underpinning the loss of urothelial differentiation are poorly understood. OBJECTIVE To examine the effect of gene alterations implicated in UC development on the ability of human urothelial cells to undergo molecular differentiation and form a functional urothelial barrier. DESIGN, SETTING, AND PARTICIPANTS Laboratory study. INTERVENTION Normal human urothelial (NHU) cell cultures were transduced with recombinant retroviruses to produce stable sublines overexpressing wild-type or oncogenic mutated fibroblast growth factor receptor 3 or human telomerase reverse transcriptase (hTERT). Previously generated NHU sublines carrying dominant-negative CDK4 and p53 mutant genes or immortalised with the human papillomavirus 16 E6 oncoprotein were included. MEASUREMENTS The activity of introduced transgenes was demonstrated by comparing phenotypes of transgene-expressing and isogenic control NHU cells. Modified and control sublines were compared for changes in generational potential (life span) and capacity to respond to differentiation-inducing signals by transcript expression of uroplakins 2 and 3. The ability to form a barrier epithelium was assessed by measuring the transepithelial electrical resistance. RESULTS AND LIMITATIONS By contrast to tumour suppressor loss of function or oncogene overactivation, hTERT overexpression alone led to life span extension and immortalisation. The hTERT immortalised cells carried no gross genomic alterations but became progressively insensitive to differentiation signals and lost the ability to form an epithelial barrier. Further characterisation of hTERT cells revealed a downregulation of p16 cyclin-dependent kinase inhibitor expression and loss of responsiveness to peroxisome proliferator-activated receptor γ, providing mechanistic explanations for the subjugation of senescence constraints and the abrogation of differentiation capability, respectively. Although immortalised urothelial cell lines without karyotypic aberrations may be generated, such cell lines are compromised in terms of differentiation and functional capacity. CONCLUSIONS Overexpression of hTERT promotes development of an immortalised differentiation-insensitive urothelial cell phenotype. Although such cells offer a useful insight into the grade/stage paradigm of UC, they have limited value for investigating normal urothelial cell/tissue biology and physiology.

Aquaporin Expression Contributes to Human Transurothelial Permeability In Vitro and Is Modulated by NaCl

It is generally considered that the bladder is impervious and stores urine in unmodified form on account of the barrier imposed by the highly-specialised uro-epithelial lining. However, recent evidence, including demonstration of aquaporin (AQP) expression by human urothelium, suggests that urothelium may be able to modify urine content. Here we have we applied functional assays to an in vitro-differentiated normal human urothelial cell culture system and examined both whether AQP expression was responsive to changes in osmolality, and the effects of blocking AQP channels on water and urea transport. AQP3 expression was up-regulated by increased osmolality, but only in response to NaCl. A small but similar effect was seen with AQP9, but not AQP4 or AQP7. Differentiated urothelium revealed significant barrier function (mean TER 3862 Ω.cm2), with mean diffusive water and urea permeability coefficients of 6.33×10−5 and 2.45×10−5 cm/s, respectively. AQP blockade with mercuric chloride resulted in decreased water and urea flux. The diffusive permeability of urothelial cell sheets remained constant following conditioning in hyperosmotic NaCl, but there was a significant increase in water and urea flux across an osmotic gradient. Taken collectively with evidence emerging from studies in other species, our results support an active role for human urothelium in sensing and responding to hypertonic salt concentrations through alterations in AQP protein expression, with AQP channels providing a mechanism for modifying urine composition. These observations challenge the traditional concept of an impermeable bladder epithelium and suggest that the urothelium may play a modulatory role in water and salt homeostasis.

Differentiation-Associated Reprogramming of the Transforming Growth Factor β Receptor Pathway Establishes the Circuitry for Epithelial Autocrine/Paracrine Repair

Transforming growth factor (TGF) β 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 TGFβ 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 TGFβ by growth inhibition, and that exogenous TGFβ 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 TGFβ 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 TGFβR 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 TGFβR pathway is defined transcriptionally within a tissue-specific differentiation programme. The findings provide evidence for re-evaluating the role of TGFβR 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 TGFβ signalling on cell proliferation and differentiation.

Abstract 3126: Intercellular communication in the urothelium: Evidence for a switch in gap junction regulation during differentiation

Cells have a requirement for intercellular communication in order to maintain normal tissue polarity and homeostasis. Knowledge of the signaling molecules transferred through specific gap junctions is limited; however aberrant expression of connexins, the protein subunits of gap junctions, has been linked to many disorders including neoplastic transformation of brain and colorectal tissues. A number of reports have documented changes in connexin expression associated with bladder cancer. The aims of this study were 1) to define the connexins expressed by the epithelial lining of the human urinary tract (urothelium) in situ and in vitro; 2) to determine if expressed connexins assemble into functional gap junctions and 3) the contribution of specific connexins to urothelial tissue physiology. Established culture systems for normal human urothelial (NHU) cells were used to replicate proliferative (regenerative) or differentiated cell phenotypes. RT-PCR was used to screen normal urothelial tissue and NHU cell cultures for expression of connexin genes. Immunochemical techniques were employed to determine connexin expression and localization in situ and in vitro. Dye-transfer techniques were used on cultured cells to confirm that assembled gap junctions supported functional cell-cell communication. Of the 20 human connexin genes, six were expressed in situ and by differentiated cultures, whilst two were expressed only in proliferative culture conditions. NHU cells were capable of dye transfer in both proliferative and differentiated cultures, indicating the presence of functional gap junctions. Immunofluorescence microscopy demonstrated that the switch in medium from a low (0.09mM) to near physiological (2mM) calcium concentration resulted in increased assembly of gap junctions containing connexin-43. Connexin-32 was only expressed by differentiated cultures, as shown by RT-PCR and immunochemistry, where it was localized to intercellular borders indicating a functional role. This data represents the first systematic characterization of connexin family expression in the urothelium and provides evidence that urothelial cells alter their connexin expression upon differentiation to produce specific channels. Connexin-32 has been reported to be involved in terminal differentiation of human mammary gland and future work will focus on characterizing the role of connexins in defining the differentiated urothelial phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2011-3126

Abstract 4020: Wnt and EGFR crosstalk: Evidence for a bi-directional feedback loop important in maintaining the proliferative phenotype of normal urothelial cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction The urothelial lining of the bladder is normally mitotically quiescent, but has a high capacity for self-renewal in response to injury. This regenerative ability serves to maintain urinary barrier function during normal homeostasis and offers a primary target for deregulation in cancer. Previous work in our laboratory has implicated autocrine signaling through EGFR activation but the role of other regulatory pathways remains poorly understood. There is some evidence that Wnt signaling may play a role in bladder cancer by promoting cell proliferation through β-catenin. In the absence of Wnt signaling, β-catenin is sequestered by the destruction complex and is targeted for degradation via the proteosome. When the Wnt signaling cascade is activated, the destruction complex is inactivated, allowing β-catenin to translocate to the nucleus, interact with specific transcription factors (TCF/LEF) and drive proliferation. The aim of this study was to assess whether Wnt signalling is functional in normal human urothelial (NHU) cells, and analyze the effects of Wnt and EGFR downstream signaling and potential crosstalk on proliferation. Materials and Methods NHU cells were grown as finite lifespan cell lines in proliferating monolayer cultures. Destruction complex antagonists were used to activate the Wnt pathway. Proliferation assays, immunofluorescence labeling, and TOPFLASH luciferase reporter assays were used to assess NHU responses. Pathway crosstalk was analyzed using shRNA targeted to β-catenin, as well as antagonists of the EGFR signaling pathways. Results Pharmacological inhibition of the destruction complex or activation of the Wnt pathway with exogenous Wnt3a ligand led to nuclear accumulation of β-catenin and an increase in TCF/LEF transcription factor activity. These effects were only apparent when autocrine EGFR signaling was blocked. Knockdown of β-catenin led to significant reduction in phospho-ERK and a modest increase in phospho-AKT. Conclusion Wnt signalling can be activated in NHU cells, verifying the presence of a functional pathway. Activation of the pathway is however masked by EGFR signaling, indicating pathway crosstalk. The effect of β-catenin knockdown on phospho-ERK indicated a bi-directional, positive feedback loop between the Wnt and EGFR signaling cascades in NHU cells. This work identifies the Wnt signaling pathway as an important signaling cascade in regulating the regenerative phenotype of NHU cells and indicates at least two pathways of regulation that may be circumvented in urothelial carcinogenesis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4020.