The down-regulated tumour suppressor Wnt inhibitory factor 1 (WIF1) regulates non-canonical Wnt signalling in pituitary adenomas (PA)

The Wnt developmental pathway has been implicated in tumour growth and development in a number of tissues. Most frequently, the canonical Wnt pathway acting through the nuclear translocation of B-catenin has been the key player in driving tumour growth. However, non-canonical Wnt pathways, namely the Wnt-Calcium signalling and the Wnt-planar polarity pathways have also been found de-regulated in a number of cancers. In this study, microarray analysis on locally resected PA revealed strong down-regulation Wnt pathway antagonists, namely the Wnt inhibitory factor 1 (WIF1) and the secreted frizzled-related proteins 2 and 4 (SFRP2 and 4). These results have been confirmed by qPCR and shown that WIF1 is under – expressed in all tumour types while SFRP’s tend to be repressed in functional PAs. The aim of this study was to functionally assess the role of WIF1 in PA in relation to the different Wnt signalling pathways using two established cell lines, the rat sommatotroph/lactrotroph GH3 and prolactinoma MMQ cell lines in the presence of known canonical and non-canonical Wnt ligands, Wnt3, Wnt4 and Wnt5a. WIF1 over-expression reduced significantly GH3 and MMQ cell proliferation using a fluorescent-based Alamar Blue assay, both in the absence and presence of Wnt ligands. However, both Wnt ligands and lithium chloride, an established canonical Wnt pathway activator, failed to activate β-catenin driven transcription using the TOP/FOP flash luciferase system. In fact, canonical Wnt signalling appears to be completely absent in GH3 and MMQ cells. In order to study the influence of Wnt ligands and their inhibitor, WIF1, on other non-canonical Wnt pathways, the Wnt-Calcium signalling pathway was chosen, owing to the important role that calcium signalling plays in regulating hormone release from these cells. Using the FluoForte Calcium assay (Enzo Biologicals, US) to assess free calcium in real-time in the chosen cell lines, we studied the effect of the Wnt ligands in the absence and presence of the WIF1 inhibitor. Wnt ligands activated calcium release with variable potentials with WIF1 displaying an inhibitory but selective role on this effect. Real-time PCR of targets of the canonical and non-canonical Wnt pathways is also being undertaken together with analysis of growth hormone and prolactin secretion from both cell lines. Preliminary data reveals that the Wnt agonists may activate the Wnt-Calcium signalling pathway and WIF1 could play a role in PA by inhibiting specific aspects of this pathway.peer-reviewe