carcinoma [27]. However, in other cell types, such as mammary epithelial cells [10,28,29], as well as melanoma and breast cancer cell lines [11], E-cadherin-mediated cell-cell contacts have been shown to suppress proliferation signals that are driven by RTKs, particularly from the EGFR pathway. These contradictory findings prompted us to study the precise influence of E-cadherin on downstream growth-promoting cell signalling pathways in a normal epithelial cell system. At low density, NHU cells grown in physiological calcium exhibited transiently-higher growth rates than cultures grown in low calcium, which was attributable to activation of the PI3K/ AKT pathway. That this was the result of functional E-cadherin cell contacts, rather than being attributable to the increase in calcium alone, was indicated by the confinement of AKT activation to juxtaposed NHU cells and the loss of the effect in E-cadherin-null cultures. These observations are supported by the findings of Liu and co-workers, who showed that E-cadherin- mediated contacts were responsible for promoting proliferation in mammary epithelial cells cultured at low densities [30], as well as a study reporting that induction of cell proliferation by cell:cell contacts in endothelial and smooth muscle cells was associated with PI3K/AKT activation [31]. Our study was originally instigated on the basis of in vitro/in virtuo comparison with an agent-based computational model of epithelial cell interactions [17,18,19]. The most recent version of the model incorporates a mathematical model of juxtacrine EGFR/ERK activation to explore the importance of cell-cell contact in regulating ERK activity and NHU cell proliferation [20]. The modelling results predicted that transient cell contacts occurring in low calcium conditions would result in rapid but transient ERK activation. Simulated ‘‘virtual immunoblotting’’ data for a population of cells suggested that transient contacts resulted in a high overall phospho-ERK signal. By comparison, slowly-formed contacts stabilised by the formation of E-cadherin junctions in physiological calcium conditions resulted in sustained ERK activation, but the overall amount of phospho-ERK for the total population was less than for cultures maintained in low calcium concentrations [20]. The present experimental work confirmed the model’s prediction that in physiological calcium conditions, the amount of ERK phosphorylation for the population is less than for an equivalent culture grown in low calcium conditions. By extending our study to the AKT pathway, we have also now provided an explanation for the increase in proliferation rate observed previously in NHU cell cultures grown in physiological calcium [17]. As E-cadherin contacts suppress EGFR/ERK signalling in urothelial cells, the implication is that E-cadherin loss may directly contribute to the activation of RTKs frequently reported in carcinomas [32], including those of the bladder ([33] and references therein). Engineering NHU cells with functionally- inactivated E-cadherin enabled us to specifically probe the importance of E-cadherin-associated cell contacts in cell prolifer- ation. Expression of the dominant-negative H-2Kd-E-cad mutant down-regulated endogenous E-cadherin by blocking its localisa- tion to sites of cell-cell contact and abrogating the recruitment of a-catenin and b-catenin. The H-2Kd-E-cad chimera interacted with endogenous protein to recruit it to perinuclear, Golgi/late- endosome compartments, reminiscent of the situation with normal keratinocytes [22], but not immortalised HaCaT cells, where surface localisation was reported [34]. Inactivation of E-cadherin abolished calcium-mediated growth restraints, allowed de-repres- sion of EGFR/ERK, diminished the influence of PI3-K/AKT in NHU cell proliferation at low density and ‘‘released’’ b-catenin to permit its re-localisation to the nucleus. The b-catenin knock- E-Cadherin Growth Regulation down experiments revealed a mutually-exclusive relationship between PI3-K and b-catenin signalling, as well as demonstrating that active b-catenin has a repressor role on E-cadherin expression. As a result of the loss of functional E-cadherin, the de-repression of EGFR/ERK and constitutive activation of b- catenin/TCF signalling (discussed below) allowed NHU cells to resume their high proliferative potential even in physiological calcium conditions. Unlike previous studies that have suggested no constitutive Wnt/b-catenin signalling in NHU cells [35], we believe that our work provides the first evidence for an activated Wnt signalling pathway in urothelial cells, as assessed by TCF/LEF reporter assay in conditions where E-cadherin signalling was suppressed (low calcium medium or expression of the H-2Kd-E-cad chimera). Various reports give credence to a role for Wnt signalling in bladder cancer, including epigenetic loss of Wnt inhibitory components, such as WIF-1, DKK subunits and secreted frizzled-related proteins (sFRP) [36,37], hypermethylation of the APC locus [38] and loss of E-cadherin expression via CDH1 locus hypermethylation or mutation [39]. This emergent evidence in combination with our findings indicates that the down-regulation of E-cadherin is permissive for Wnt signalling in urothelial cells. It is well-established that E-cadherin plays a critical role in epithelial carcinogenesis due to its importance in regulating cell- cell adhesion [40]. E-cadherin expression is frequently lost in aggressive epithelial cancers, including those of the bladder, prostate, lung and pancreas [41]. In the case of bladder cancer, loss of E-cadherin expression is a common event [42], which occurs in advanced tumours as a result of CDH1 locus hypermethylation or mutation [39] and there is evidence for a strong association between hypermethylation of the E-cadherin locus with high disease-progression risk [43]. Because restoration of its expression often leads to growth-retardation, reduction of invasive properties [44] and/or reversion of the tumour phenotype from malignant to benign [45,46], E-cadherin has been generally termed a metastasis suppressor [47]. Sensitivity to the EGFR inhibitor, cetuximab, has been shown to require intact E-cadherin expression and silencing of E-cadherin by RNA interference was reported to reduce responsiveness to EGFR inhibition in previously sensitive bladder cancer cell lines [48]. Although no biological evidence was provided, the authors proposed that the requirement for E-cadherin to achieve increased growth inhibition by cetuximab might indicate a mechanism of E-cadherin-mediated EGFR activation. We believe that our observation of E-cadherin- mediated down-regulation of EGFR/ERK is not contradictory and may provide a mechanistic explanation for these findings. Our results imply that a reduced response to EGFR inhibitor by E- cadherin-negative cells [48] may, at least in part, be due to high amounts of EGFR-driven ERK activation resulting from loss of E- cadherin function, thus reducing the efficacy of cetuximab treatment. More importantly, loss of E-cadherin may result in activation of additional growth-promoting signalling pathways normally attenuated by E-cadherin-mediated contacts, such as activation of Wnt and b-catenin/TCF signalling; such signals would not be attenuated by cetuximab-mediated EGFR inhibition. In summary, following a functional inactivation approach our studies provide, for the first time, molecular evidence that E- cadherin engagement can dictate the activity of several mitogenic signalling pathways, as it differentially regulates EGFR/ERK, PI3- K/AKT and b-catenin/TCF. Studies using normal epithelial cell cultures [49], including our own, clearly suggest that E-cadherin loss-of-function may provide a growth advantage. This indicates that its role in carcinogenesis is not limited to metastatic spread, PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13621