Dissecting the mechanism of Wnt signal transduction using chemical probes

The Wnt ligand signaling pathway is considered a key therapeutic target in conditions including cancer, heart disease, arthritis, schizophrenia and Alzheimer's disease. Mutations to 'core' Wnt signaling components including b-catenin, APC and Axin inappropriately activate Wnt signaling in cancers of the colon and liver. Many further cancers show signs of inappropriate Wnt pathway activation (Eg. stabilized nuclear b-catenin in 50% of breast cancers), although the mechanisms leading to pathway activation remain unclear. Similar uncertainty surrounds the mechanism(s) that lead to pathogenic Wnt pathway deregulation in other diseases. To develop drug-like small molecules against the Wnt pathway, we have established a consortium of groups that have expertise ranging from medicinal chemistry, Wnt signal transduction and cell biology through to mouse models of deregulated Wnt signalling in colon cancer and clinical trials of metastatic disease. In previous work, we screened a library of 70,000 small molecule drug like compounds in a cell based assay for inhibitors of Wnt/TCF-dependent transcription. Following a series of deconvolution assays, we identified 4 small molecule inhibitors that blocked tumour cell growth and appeared to operate at distinct points in the Wnt signal transduction cascade. Using biotinylated small molecule analogues of one series of active compounds, we have identified the FET oncogene family as a probable molecular target of the Wnt-inhibitory activity. Members of the FET gene family has been shown to be fused to transcription factors and are the primary initiating oncogene in a range of human sarcomas (Eg EWS in Ewings Sarcoma), but few links have previously been established between EWS gene family members and Wnt signaling. As part of the CASE studentship, we now propose to study the mechanism by which the compounds inhibit EWS family function in the context of the Wnt signaling. This work will involve overlapping biochemical and molecular approaches to identify the molecular process by which the FET family regulates Wnt signaling and the mechanism by which the anti-Wnt signaling drug alters EWS/Wnt function. At the biochemical level, we will study the physical interaction between FET family members and components of the Wnt signalling pathway, since published reports suggest a direct interaction between the FET family member, Fus and the Wnt signaling proteins, b-catenin and the TCF. At the cell biological level, we will study how alterations to FET family member expression alter Wnt signaling. This work will rely on our ability to alter the expression of EWS and Wnt signaling components through RNAi gene depletion and cDNA gene overexpression. Both of these techniques are well established within the laboratory and preliminary data has shown that RNAi reduction of EWS and Fus expression lowers Wnt/TCF-dependent signaling. In the longer-term, deletion analysis will be used to identify regions of the EWS family members that are required for function in the Wnt pathway and drug binding leading to the identification of the mechanism of drug action. The output of the work will be a molecular understanding of the role of EWS family members in Wnt signaling. This work will directly contribute to the development of a series of putative anti-cancer drugs and will generate high quality information on the basic biology of Wnt-EWS pathway interactions that will contribute to the field of signal transduction.