The role of Endo180 in prostate cancer cell migration and metastasis

Expression of the transmembrane type I receptor Endo180 (CD280, MRC2, uPARAP) has been associated with fibrosis, breast cancer, glioma and head and neck cancer. In this thesis I have evaluated the role of Endo180 in prostate cancer pathology with a particular focus on its link to increased tumour cell migration and metastasis. The aims of my project were as follows: (a) to ascertain the co-evolution of Endo180, its interaction partners and domain function in silico; (b) to design a non-radioactive assay to enable the study of Endo180 phosphorylation in vitro; (c) to establish of a multi-cell line model as a clinically relevant experimental tool to investigate the relevance of Endo180 expression with prostate cancer progression; and (d) to conduct the first full clinicopathological evaluation of Endo180 in prostate cancer patients. The findings presented confirm that the MRC2 gene, which encodes Endo180, is conserved throughout all known vertebrate genomes and that functional amino acid residues within the receptor are conserved across a wide range of species. A putative urokinase-plasminogen activator uPA-binding motif located in the C-type lectin domain-4 (CTLD-4) of the receptor was present in all mammals but no other species. Three-dimensional modelling revealed that the motif is accessible for predicted high-affinity binding between the two molecules. To investigate the biological consequence of the interaction between Endo180 and uPA, site directed mutagenesis was used to generate Endo180 lacking uPA-binding capacity. 2-D SDS-PAGE, immunoprecipitation and band-shift assays failed to detect Endo180 phosphorylation in fibroblasts; a finding that may reflect redundancy of this post-translational modification in stromal versus epithelial cells. A high-throughput screening approach was taken to identify factors that promote epithelial-to-mesenchymal transition (EMT) like responses in prostate cell lines derived from normal prostate epithelium (RWPE-1), benign hyperplasia (RCN170, RCN165), primary prostate cancer (RC92a, RC58T) and secondary metastatic prostate cancer (LNCaP, DU145 and PC3). With the exception of LNCaP cells all lines expressed low to intermediate levels of Endo180. The Endo180-transcriptional regulatory factor, TGF-β1, increased Vimentin expression in RC92a cells. The invasive properties of this panel of cell lines were assessed using a modified organotypic invasion assay in the absence and presence of TGF-β1. Multiple tissue microarray analysis revealed an increase of 26% in the five-year survival rate of patients with negative Endo180 expression (p = 0.02) and 46% in patients with negative Endo180 expression and low Gleason grade (p = 0.015). This data is the first to confirm a major clinical impact of Endo180 expression in any disease state. Future work should now be focused on the mechanisms through which Endo180 can promote prostate cancer progression and its application as a biomarker. The mechanistic studies will benefit from the clinically relevant in vitro models established as part of this thesis; and will help pinpoint the precise molecular interactions that could be potential therapeutic targets.Open Acces