Determining the functional order of Wnt signalling components

Lead Research Organisation: Cardiff University
Department Name: School of Biosciences


During development, groups of cells communicate with each other using protein messengers that are secreted from one cell and bind to receptors on neighbouring cells. One important family of protein messengers are called Wnts (pronounced wints). When the message is received, the responding cell switches on a number of genes in its nucleus that in turn bring about changes to processes such as growth and cell type. The connections inside the cell that transmit the Wnt signal from the cell surface to the nucleus are very important since they control how the signal is amplified and aimed at the right genes. The proposed research will use a method to the order in which connecting Wnt signalling components function inside the cell. The basic idea is to use a very sensitive cells that have been engineered to send out a burst of light when the Wnt signal is active in the nucleus. We then use a new method called RNAi to remove proteins that might be Wnt connecting components. If the protein IS required, the burst of light will be lost. This question is asked again and again for many genes using robots to speed up the process. We can ask whether and how many genes are required for Wnt signalling Once we know which proteins are involved in the Wnt pathway, we can study how they work and how they control animal development.

Technical Summary

Wnt signalling controls a range of developmental processes including patterning, differentiation and cell proliferation. In cells responding to Wnt ligands, target gene transcription is regulated by a complex containing beta-catenin and the DNA binding factor TCF. Previous studies have identified up to 250 genes that may be involved in Wnt signalling in different systems. We propose to identify components of the Wnt signalling pathway in human 293 cells using an RNAi based screen. RNAi is a sequence-specific method of reducing target protein levels that has recently been developed for use in mammalian cells. RNAi screening studies of other signalling pathways have identified many novel components. We have optimised screening techniques in a highly sensitive TCF-reporter cell line and can immediately carry out a 96-well format screen of a 180 gene subset of putative Wnt signalling components. In collaboration with GE Biosciences (formerly Amersham; Cardiff), we will establish further Wnt signalling assays using a new machine capable of high throughput confocal laser microscopy. The combination of these cutting edge techniques will be used to establish the functional order of Wnt signalling components by genetic epistasis. At a technical level, this work should establish a platform of expertise for further high throughput RNAi/Cell line based screens in the UK. At the scientific level, this work will order novel and existing Wnt components with respect to each other and should identify new branches to the existing molecular Wnt pathways. Results from the screens (including microscopic images of RNAi treated cells) will be stored in a form that is compatible with gene-specific databases and will be accessible to the scientific community through a web-accessible relational database.


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Description A genome-wide RNA interference screen to identified novel Wnt pathway regulators using an endoribonuclease prepared esiRNA library and a HEK293 derived cell line carrying a TCF-luciferase reporter. Primary screen hits included known 'core' Wnt pathway genes like APC, Axin, b-catenin and TCF7. Comparison with Wnt pathway regulators identified in recent genome-scale screens in HeLa and DLD-1 cancer cell lines uncovered a small shared set of regulators that were selectively enriched for 'core' pathway components. Based on comparisons of primary and validated hit identification rates, we argue that the majority of 'non-core' Wnt pathway regulators have cell-specific functions. Morpholino-oligonucleotide mediated depletion of novel regulators induced Wnt-dependent phenotypes in zebrafish embryos that were consistent with tissue-specific roles. These findings underscore the importance of cell context when considering therapies for diseases involving deregulation of the Wnt pathway.
Exploitation Route Alterations of the expression of the identified gene products may be linked to dysregulation of the Wnt pathway in disease. The genes and reagents that target them can be used as biomarkers of pathway activation and response to therapeutic molecules that target distinct branches of a Wnt network.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description Findings have been important in developing small molecule inhibitors of the Wnt signalling pathway as anti-cancer therapeutics. The work is also important for understanding the network structure of the pathway.
First Year Of Impact 2012
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

Company Name Nanotether Discovery Science 
Description NDS is developing an innovative new bio-chip technology to quantify biochemical binding parameters for the pharmaceutical and biotechnology markets. NDS technology miniaturises 'test tubes' in arrays, allowing assays that are cheaper, faster and of higher quality than competitive approaches. The Merck Serono collaboration was highly successful and one compound from the screen is entering preclinical development. The total value of the funding to Cardiff University and the Institute of Cancer Research from Merck Serono over the last 5 years has been in excess of £7M. Interactions with both Merck Serono and GE Healthcare (Cardiff) were also central to the establishment of a startup company (Nanotether Discovery Science; £2.3M Venture Capital) that builds on links between high throughput screening and drug screening. 
Year Established 2013 
Impact The BBSRC grant was central to the establishment of a major drug discovery programme with Merck Serono and in the development of a novel technology for in vitro drug discovery - via contacts made during the BBSRC funded interaction. The new technology was commercialised through a TSB grant with GE Healthcare and subsequently through the establishment of a new startup company (NDS Ltd) which is funded by £2.3M of Venture Capital and currently employs 7 full time staff.