Prof Trevor Dale - PhD
Screening for Genes and Small Molecules that Modulate Wnt/β-catenin Signalling
The Wnt/β-catenin pathway is activated in a wide range of tumours. Cell-based screening is an efficient way of identifying novel Wnt/β-catenin regulators. We have used high throughput cell based screening to identify novel proteins and small molecules that regulate the pathway. The novel proteins and small molecules are initially used as molecular tools to further characterise the Wnt pathway. This has enabled us to demonstrate that the Wnt pathway behaves like a molecular network. Some of the small molecules are now being developed as candidate therapeutics for colorectal and breast cancer in a major collaboration with Merck Serono.
Figure 1: Screening for modulators of the Wnt pathway.
A: Strategy of screening. Loss of function (siRNA) and gain of function (cDNA) genome-scale screens have been carried out in reporter cell lines using TCF-dependent reporter activity and b-catenin abundance/ location as readouts. Chemical libraries were screened for small-molecule inhibitors of Wnt/β-catenin signalling. This led to a large-scale drug discovery project in collaboration with Merck-Serono, the Institute of Cancer Research and Cancer Research Technology.
B: The 7dF3 TCF reporter line. The HEK293 based cells contain the TCF reporter and an inducible upstream Wnt regulator (A Dishevelled-oestrogen receptor fusion protein Dsh-ER). In this cell line, Estradiol (E2) activates TCF-dependent transcription ~12X. The GSK-3 inhibitor Li+ activates TCF-reporter activity 11,000X. (Data shown from Ewan et al. 2010,)
Three dimensional primary culture systems are more relevant for predicting utility of possible therapeutic agents for use in vivo than 2D culture of established cell lines. Developing medium-throughput organoid culture systems to test Wnt pathway inhibitors is an important research direction for the laboratory. Culture of both normal tissue and tumour organoids are being developed.
Figure 2: Development of a small intestinal organoid culture system
A: Maintenance of the small intestinal epithelium by the stem cell niche. Differentiated cells only live a week in the small intestine, so structures known as crypts comprised of stem and proliferative cells continually replenish the intestine with new cells. These cells migrate from the stem cell niche, through the proliferative zone of the crypt and differentiate into mature cells upon entering the villus. A gradient of Wnt/β-catenin signalling, highest in the stem cell niche, regulates cell proliferation and differentiation.
B: Tet-O-ΔN89-β-catenin mouse line. We developed a mouse line to express oncogenic β-catenin (ΔN89-β-catenin) in all cell types conditionally for global Wnt/β-catenin pathway activation. Expression of ΔN89-β-catenin is induced by Doxycycline, which acts as a ‘molecular switch’. This induces hyperplasia of the crypt structures in the small intestine due to block of cellular differentiation.
C: Intestinal crypt culture: Organoid stained for a reporter of Wnt signalling (Axin2-lacZ). The organoid consists of an epithelium surrounding a central cavity. The blue-stained outpocketings are equivalent to the crypts and the unstained epithelium of the central body is equivalent to the villus. (Data shown from Jarde et al., 2013)
Axin1’s Role in Liver Tumour Development
Mutations in genes encoding proteins in the Wnt signalling pathway, including CTNNB1 (β-catenin gene) and the GSK-3 binding protein AXIN1, are found in more than 50% of human hepatocellular carcinomas (HCCs). A murine model was developed to conditionally disrupt the function of the Axin1 and Axin2 genes in the liver. Livers lacking Axin1 showed greater cell proliferation and developed liver tumours that matched the subtype of human liver cancer in which Axin mutations are found. Surprisingly, the changes observed following Axin loss were different from those that are characteristic of Wnt pathway activation suggesting that Axin may repress liver cancer through a novel molecular pathway.
Figure 3: Tumours in two mouse livers that are deficient in Axin1 gene function. Axin1 was disrupted in the livers one year before dissection. The tumour boundaries are indicated with dashed white lines. (data shown from Feng et al. 2013,)
Biochemistry and Structure of Components of the Wnt/β-catenin Signalling Pathway
We are particularly interested in studying how β-catenin turnover is altered following Wnt ligand binding at the cell surface and following oncogenic mutations. Both Wnt ligands and oncogenic changes stabilise β-catenin and activate β-catenin/TCF-dependent transcription. Work is aimed at understanding how these changes alter the composition and interactions between β-catenin turnover complex components such as APC, Axin and CK1.
Figure 4: Biochemistry of the Wnt signalling pathway.
A: Pathway: In the absence of a Wnt signal, the β-catenin turnover complex enhances β-catenin degradation. In the presence of Wnt ligands, the function of the β-catenin turnover complex is blocked leading to the accumulation of β-catenin, which then translocates to the nucleus and acts as a co-transcription factor with members of the TCF DNA binding protein family. Mutations to Wnt, Axin, APC, β-catenin and TCF family members have been shown to induce tumours and activate TCF-dependent transcription. Over one hundred additional regulators that are not shown in this linear diagram comprise a Wnt signalling network.
B: GSK-3/Axin interaction: Work in collaboration with Laurence Pearl (University of Surrey) focused on the kinase GSK-3 that plays a central role in targeting β-catenin for degradation within the β-catenin turnover complex. We determined the structure of GSK-3 and a complex between GSK-3 and Axin. These studies have provided important insights into the mechanisms underlying GSK-3 substrate recognition and regulation. (Data from Dajani et al., 2003)
High throughput screening for protein interactions
The slowest step in many biochemical assays is the production and purification of sufficient protein for quantitative assays. In collaboration with Professors Adrian Harwood and Paola Borri, we have developed a novel technique termed ‘Nanotether’ that could break this biochemical bottleneck.
The idea of behind the technology is to tether two biomolecules to the ends of flexible (DNA) tethers such that they can interact in a nano-scale volume. Arrayed spots of interacting molecules containing as few as 1 million molecules are analysed by FRET to measure the proportion of tethered biomolecules.
The key advantages of the technology are:
- Tethered arrays of molecule pairs are easily assembled by DNA hybridisation.
- Hybridisation concentrates the interacting molecules near the surface while the length of the tethers control the effective concentration (low nM-uM range).
- High concentrations (> 10uM) can be generated from low masses of protein - this should be compatible with techniques such as in vitro translation.
This technology is now being commercially developed in a Cardiff University spin-out company. The first application area will be high throughput protein kinase binding assays. See www.nanotether.co.uk (Proof of concept data can be found in Perrins et al. 2011.)
Wnt/β-catenin Signalling and Mammary Development and Tumourigenesis
The mammary gland undergoes numerous developmental processes postnatally, from the elongation of the ductal tree-like structure to the pregnancy-induced development of the lobulo-alveolar units that make milk. Mammary epithelial stem cells have been suggested to be central to the control of enormous tissue expansion and remodelling during these phases of mammary development. The Wnt signalling pathway plays a critical role in these biological steps and is suggested to be involved in the maintenance of the stem cell population. It has also been implicated in certain types of breast cancer.
Figure 5: Wnt signalling in normal development and cancer.
A: Wnts regulate normal development. In the mammary gland, some Wnt family members are involved in the control of lobular development.
B: Wnt ligand as mammary oncogene. The prototype member of the Wnt family (Wnt-1) was originally identified as a mammary oncogene and causes dramatic pre-cancerous changes in the mammary epithelium.