Prostate cancer (PC) is characterised by dependence upon androgen receptor (AR) as its driving oncogene. When organ-confined, radical treatment can be curative, however there is no cure for advanced, castration-resistant prostate cancer (CRPC). There is therefore a need to better understand the biology of PC, and how influencing AR can modify disease progression. Estrogen is essential for prostate carcinogenesis with evidence from epidemiological, in vitro, human tissue and animal studies. Most suggests that estrogen receptor beta (ERβ) is tumour-suppressive, but trials of ERβ-selective agents have not improved clinical outcomes. ERβ has also been implicated as an oncogene, therefore its role remains unclear. Additional evidence suggests interplay between ERβ and AR, the mechanisms of which are uncertain. The study hypothesis ‘ERβ is an important modulator of prostate carcinogenesis’ was developed to establish whether targeting ERβ could affect PC progression. Much of the confusion around ERβ stems from use of inadequately validated antibodies and cell line models. The first phase of this work was to test ERβ antibodies using an ERβ-inducible cell system. Eight ERβ antibodies were assessed by multiple techniques, showing that commonly used antibodies are either non-specific or only specific in one modality. Two reliable antibodies were identified. Next, cell lines previously used to study ERβ were assessed using validated antibodies and independent approaches. No ERβ expression was detected; an important finding that casts doubt on previously published ERβ biology. Subsequently, a PC cell line with inducible ERβ expression (LNCaP-ERβ) was developed and validated to enable controlled experiments on the effects of ERβ on proliferation, gene expression and ERβ/AR genomic cross-talk. Phase three of this work focused on ERβ biology in PC and its relationship to AR. Interrogation of clinical datasets showed that greater ERβ expression associated with favourable prognosis. Gene expression data from men treated with androgen deprivation therapy revealed that AR represses ERβ. This was confirmed in vitro. The LNCaP-ERβ cell line was treated with androgen and/or ERβ-selective estrogen. Activated ERβ in the presence of androgen-stimulated AR inhibited cell proliferation and down-regulated androgen-dependent genes. Genome-wide mapping of ERβ binding sites reveals that ERβ antagonises AR through competition for shared DNA binding sites. In conclusion, ERβ expression is down-regulated by AR during malignant transformation of prostate epithelium. We reveal an antagonistic relationship between ERβ and AR whereby sustaining or replacing ERβ may inhibit tumour growth through down-regulation of AR-target genes. In future, an ERβ-selective compound may be used to slow or abrogate PC progression.

Cadherin-based cell-cell contacts are prominent sites for phosphotyrosine signalling, being enriched in tyrosine-phosphorylated proteins, tyrosine kinases and phosphatases. The functional interplay between cadherin adhesion and tyrosine kinase signalling, however, is complex and incompletely understood. In my thesis I have tested the hypothesis that c-Src contributes positively to cadherin biology by functioning as part of an adhesion activated cell-signalling pathway. I found that c-Src is active at both established and reforming cell-cell contacts, and c-Src can be activated by homophilic ligation of the adhesion receptor. However, c-Src has a biphasic impact on cadherin function, exerting a positive supportive role at lower signal strengths, but inhibiting function at high signal strengths. Inhibiting c-Src under circumstances when it is activated by cadherin adhesion decreased several measures of cadherin function. This suggests that the cadherin-activated c-Src signalling pathway serves positively to support cadherin function, while quantitative changes in signal strength may result in qualitative differences in functional outcome. Finally, my data implicated PI3-kinase signalling and cortactin as potential targets for cadherin-activated c-Src signalling. By inhibiting protein tyrosine phosphatases with pervanadate, I found that tyrosine phosphatase activity and not just protein binding was required to stimulate Src activity in response to cadherin ligation. I identified the tyrosine phosphatase RPTPα as a possible regulator of cadherin-activated Src signalling. RPTPα localises to cell-cell adhesions and it is found in a complex with E-cadherin and c-Src. Furthermore, knockdown of RPTPα disrupted the integrity of cadherin-based contacts and the activity of Src at these cell-cell contacts. This suggests that in response to cadherin-homophilic ligation PTP activity is required to stimulate Src signalling. Finally, I identified a novel pathway by which aberrant growth factor signalling could be downregulating cadherin function and promoting the invasion of epithelial cells. Stimulating cells with high levels of EGF revealed that aberrant epidermal growth factor signalling could disrupt cadherin-activated cell signalling. The integrity of cadherin-based contacts and the activity of Src at the cell-cell contacts were both disrupted in the presence of high levels of EGF. Analysis of E-cadherin and RPTPα immunoprecipitates suggested that activation of cadherin-bound EGFR might disrupt Src activation by displacing E-cadherin-RPTPα binding. Finally, analysing the subcellular distribution of these proteins revealed that, in response to high levels of EGF, E-cadherin, β-catenin, EGFR and pEGFR are internalised together in phospho-cortactin-rich endosomal-like structures. Therefore I propose that E-cadherin adhesion activates a cell-signalling pathway involving c-Src that functions to dynamically regulate the actin cytoskeleton and to maintain the adhesive strength of cell-cell adhesions. Perturbation of cadherin-activated Src signalling downregulates cadherin function and promotes the disassembly of cell-cell adhesive contacts. The concept of a cadherin-activated Src signalling pathway provides a new way to think about cadherin biology. Instead of merely functioning as passive glue holding two cells together, E-cadherin functions as an adhesion-activated signalling receptor. Dysregulation of E-cadherin-activated Src signalling and downregulation of cell-cell adhesions could be a mechanism promoting the invasion and metastasis of epithelial tumours.

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