Determining the functional order of Wnt signalling components
Lead Research Organisation:
CARDIFF UNIVERSITY
Department Name: School of Biosciences
Abstract
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.
People |
ORCID iD |
Trevor Dale (Principal Investigator) |
Publications
Carotenuto M
(2014)
H-Prune through GSK-3ß interaction sustains canonical WNT/ß-catenin signaling enhancing cancer progression in NSCLC.
in Oncotarget
Dale T
(2015)
A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease.
in Nature chemical biology
De Groot RE
(2014)
Huwe1-mediated ubiquitylation of dishevelled defines a negative feedback loop in the Wnt signaling pathway.
in Science signaling
Dietrich L
(2017)
Cell Permeable Stapled Peptide Inhibitor of Wnt Signaling that Targets ß-Catenin Protein-Protein Interactions.
in Cell chemical biology
Ewan K
(2010)
A useful approach to identify novel small-molecule inhibitors of Wnt-dependent transcription.
in Cancer research
Ewan KB
(2008)
The potential for targeting oncogenic WNT/beta-catenin signaling in therapy.
in Current drug targets
Forde J
(2007)
Glycogen synthase kinase 3: A key regulator of cellular fate
in Cellular and Molecular Life Sciences
Freeman J
(2008)
Investigating h-Prune activation of Wnt signalling in breast cancer
in Breast Cancer Research
Freeman J
(2015)
Additional fie 3: Table S2.
Freeman J
(2015)
A functional connectome: regulation of Wnt/TCF-dependent transcription by pairs of pathway activators.
in Molecular cancer
Lloyd-Lewis B
(2013)
Toward a quantitative understanding of the Wnt/ß-catenin pathway through simulation and experiment.
in Wiley interdisciplinary reviews. Systems biology and medicine
Mallinger A
(2015)
Discovery of potent, orally bioavailable, small-molecule inhibitors of WNT signaling from a cell-based pathway screen.
in Journal of medicinal chemistry
Perrins RD
(2011)
Doing more with less: a method for low total mass, affinity measurement using variable-length nanotethers.
in Analytical chemistry
Wolkenhauer O
(2010)
Systems biologists seek fuller integration of systems biology approaches in new cancer research programs.
in Cancer research
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 |
Description | Nanotether has developed nanotechnology that aims to speed up drug discovery. |
Year Established | 2012 |
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. |
Website | http://www.nanotether.co.uk |