Prof Alan Clarke - PhD
The principal interest of my laboratory lies in understanding the molecular basis of cancer, with a specific focus on genes involved in the very early stages of divergence from normality. The genes that control these key early events are often regarded as gatekeepers to the disease process and represent new potential targets for therapeutic intervention.
ES cells being injected into a blastocyst.
The approach we have adopted relies heavily upon the use of transgenic models, as we believe that, despite the many advantages and uses of in vitro experimentation, gene function can only be truly understood in an in vivo context. Although we make great use of existing models, we are also aware of considerable difficulties in their use and a parallel goal has therefore been to enhance and refine these systems using Cre-lox technology to render targeted genes conditional (switchable).
Apc deficient intestines.
The primary focus of the laboratory is upon colorectal cancer, although we also have a strong interest in similar genotype –phenotype relationships in the mammary gland. In the intestine, we are investigating the mechanisms by which intestinal epithelial cells regulate normal homeostasis and through which they progress to neoplasia. Although the crypt/villus axis of the small intestine is well characterized in terms of its cell biology, the precise biological mechanisms which govern the physiology of the normal and diseased epithelium remain unclear. To investigate this fundamental relationship within the normal and diseased intestine, we have previously used a series of direct in vivo approaches using gene targeted murine strains (eg Sansom et al 2004).
The effect of C-Myc upon Apc deficiency (see Sansom 2007).
These experiments have begun to reveal genetic hierarchies within the intestine, relating to elements of the Wnt, PI3-kinase, Jak-Stat, TGF-beta?and Ras pathways. These analyses repeatedly point to deregulation of the Wnt pathway as the key mediator of neoplasia in the gut. Thus, immediately following loss of the Wnt regulator Apc, we observe a ‘crypt progenitor’ phenotype which mirrors early stage lesions. This phenotype is characterized by the upregulation of several candidate stem cell markers, an experimental platform we are currently trying to use in a BBSRC funded project to derive cultures enriched for intestinal stem cells. Our studies have also identified the Wnt pathway as a regulator of normal intestinal homeostasis, and indeed we have observed a requirement for the Wnt pathway in crypt repopulation following injury. Layered onto this genetic analysis, we have also begun to experimentally address the role played by epigenetics in the intestine using a series of murine strains targeted for the methyl binding domain (MBD) genes- required to interpret the methylation signals on DNA. A consistent feature of these models is their impact upon the Wnt pathway, either directly or indirectly (Sansom et al 2003).
Invasive adenocarcinoma as a result of combined deficiency of Pten and Apc.
Thus activation of K-Ras and Pten augments the phenotype of Wnt deregulation, and deletion of either Mbd2 or the related protein Kaiso represses the Wnt pathway and indeed represses tumorigenesis. Transcriptome analysis of these strains has identified many novel potential therapeutic candidates for colorectal cancer and also candidate intestinal stem cell markers, a selection of which we are currently characterizing, both in terms of their potential to modify stem cell fate and tumorigenesis.
Current Grant Support
- Cancer Research UK
- Prostate Cancer Research Centre
- Prostate Cancer UK
- Welsh Assembly Government (NISCHR)
Adrian Bird - Edinburgh University, Scotland
Douglas Winton - Cambridge, UK
Hans Clevers - Utrecht, Netherlands
Owen Sansom - Beatson Institute, Glasgow
Postgraduate Research Students
Mr Matthew Jefferies