Dr Richard Clarkson
- Sylwebydd y cyfryngau
As animals grow and develop their tissues are re-organised in a highly ordered way. Central to this remodeling is the balance between apoptosis and cell survival/proliferation signals that ultimately determines the composition and size of tissues. Appropriate regulation of these signals is important not only for the efficient removal of supernumerary, potentially harmful, cells but also for the maintenance of pluripotent cells (eg. stem cells) necessary for tissue expansion. The consequences of errors in these cell death/survival signals can be severe, and may lead to major developmental defects, immune diseases or cancer.
My lab focuses on how the aberrant regulation of these processes might contribute to breast cancer. Using the mouse mammary gland as our principal model, we identified a number of candidate genes that appear to play a role in the re-organisation of different cell types during tissue morphogenesis. We have since found that some of these genes have disease-modifying effects on the establishment or progression of breast cancers, either by altering the proportion of tumour initiating cells (cancer stem cells) within the tumour or by directly affecting tumour malignancy – such as promoting the migration of cancer cells to other sites within the body.
Work in our laboratory is directed at using conditional gene technology to regulate these disease modifiers in order to establish whether altering their expression could have beneficial effects on the development and progression of breast cancers. Our ongoing studies include efforts to identify novel therapeutic and diagnostic strategies that target these novel disease modifiers. This has led to the development of an experimental pharmacological agent that potently suppresses the spread of tumour cells in pre-clinical models of metastatic breast cancer.
My lab looks at how genes that have demonstrable or presumptive roles in the removal / reorganization of supernumerary cells from adult mammary tissues could play a role in breast cancer.
Traditionally we have focused on identifying gene products that control apoptosis in mouse mammary tissues and have more recently begun to ask whether these factors contribute to the aberrant regulation of tissue homeostasis which is indicative of breast cancer. To do this, we have used conditional transgenics to modify gene activity in normal and cancer cells in vitro and in vivo, and have observed the effects of these changes on mammary tumour behaviour.
We have combined cell culture and in vivo models of mammary epithelial cell apoptosis to look at global gene expression profiles (using microarrays) of regressing tissues and to determine the role of specific transcription factors in these processes (click on Publications tab for list of relevant papers). We have focused on murine post-lactational mammary involution, a period of the normal mammary gland pregnancy cycle characterized by dramatic remodeling of the tissue architecture preceded by a wave of epithelial cell destruction and clearance. We hope that by understanding the transcriptional basis of mammary tissue remodelling in a physiologically normal context, this knowledge may be applied to the study of the same pathways in diseases affecting tissue homeostasis, such as breast cancer.
Response of mammary epithelial cells in vitro, to the deletion of neighbouring cells by apoptosis. Confluent mammary epithelial cells (KIM-2) were induced to die by serum withdrawal over a period of 48 hours. Cell populations at 0 hrs, 24hrs and 48hrs were stained for vimentin (green - a marker of cell motility and trans-differentiation) and histone H3 (red - a marker of cell division). Epithelial cells were observed to repopulate the spaces vacated by apoptotic cells, in a manner resembling wound healing processes in vitro.
Much of our current work stems from two microarray-based studies of mammary involution and epithelial apoptosis (Clarkson 2000; 2003; 2006). Thus in a global analysis of the mammary transcriptome during the pregnancy cycle we deduced from gene expression profiles that two distinct cell-death pathways were sequentially activated during involution, the first characterized by the activation of members of the TNF superfamily, a cytokine activated pathway implicated in extrinsic (death receptor mediated) apoptosis and the second associated with remodeling enzymes. We also identified a possible molecular link between these two phases of involution, involving the transition from LIF-STAT3 to OSM-STAT1 signalling in mammary epithelial cells (Tiffen, 2008).
Representation of gene ontologies from an affymetrix microarray analysis of mouse mammary gland during the pregnancy cycle. Genes were grouped according to their expression profile and further subdivided according to their known molecular function (GO terms). See Clarkson et al (2003)
However, in a recent study using a conditional inhibitor of caspase activity (baculovirus p35 protein) in murine mammary tissues, we provided evidence to support the proposal that apoptosis was redundant during mammary involution (Kreuzaler, 2011). Despite this, caspase dependent pathways appear to have an unexpected role in neoplastic tissues. These observations have important implications for therapeutic strategies that recruit pro-apoptotic mechanisms.
H&E sections of tissue from involuting mammary glands in which the caspase inhibitor p35 has been induced (by addition of doxocycline) or not induced. Sections are labeled with an antibody to cleaved caspase 3, demonstrating the persistance of luminal bodies in the p35+ve tissues, despite a marked reduction in caspase activity (arrows).
The identification of TNF-related signaling components in the mammary gland has led us to investigate the role of TNFRsf signaling in the homeostasis of normal and neoplastic mammary tissues (Piggott, 2011). Using conditional transgenics to inhibit the endogenous TNFRsf suppressor c–FLIP, we have demonstrated a hypersensitivity of tumour cells, but not normal mammary epithelial cells, to the cytotoxic effects of the TNFsf ligand TRAIL. Central to this hypersensitivity is the complete elimination of cancer stem cell activity within tumour cell populations, irrespective of the breast cancer subtype targeted or whether the tumour cells had previously undergone treatment.
Tumour spheres (colonies of cells in suspension culture formed by a single cancer stem cell) are destroyed by the combined effects of c-FLIP inhibition (by siRNA) and TRAIL ligand.
We now aim to focus on the potential for this combined c-FLIP/TRAIL treatment to be used as a therapeutic strategy in the clinic, by assessing the tumour efficacy of targeting c-FLIP inhibition long-term in pre-existing tumours in vivo.
In a separate microarray study, we used conditionally active forms of two STAT transcription factors, STAT3 and STAT5 to identify the genes responsible for their known roles in mammary cell apoptosis and differentiation respectively (Clarkson, 2006). This has lead to the identification of a number of gene targets that are likely to play important roles in the maintenance of tissue homeostasis in the mammary gland, one of which, Bcl3, is the subject of ongoing studies within our lab due to its surprising role in disease progression in vivo.
We are currently using state-of-the-art molecular modeling strategies in collaboration with Andrea Brancale and Andrew Westwell of the Cardiff School of Pharmacy, to design, synthesise and characterize novel inhibitors of Bcl3 that could be used to modify disease in breast cancer models. We are also looking at the role of Bcl3-NF-kB complexes in the aetiology of disease processes. Using conditional transgenic techniques we are specifically altering the equilibrium of NF-kB subunits bound to Bcl3 in target cells to determine the role of canonical and non-canonical NF-kB pathways dependent on Bcl3 in mammary tumours.
Model of (Bcl-3)-(p50) complex with DNA
Our models of breast cancer are also being put to use to test novel therapeutic and diagnostic agents under development through collaborative projects with researchers in the Schools of Biosciences, Chemistry and Medicine within the University of Cardiff. These include studies of natural plant extracts with tumour specific properties, and novel PET/SPECT imaging agents for the early identification of metastatic tumours in vivo.
Future work will continue to focus on demonstrating the specific roles of the gene products identified to date in the development of breast cancer, and through our collaborations we aim to develop novel therapeutic strategies that target these new pathways.
Current grant support
- Breast Cancer Campaign
- Cancer Research Wales
- Sêr Cymru - National Research Network
- Tiziana Pharmaceuticals (Bcl3i drug development)
- Sian Griffiths Memorial Fund
- Andrew Westwell, Andrea Brancale – School of Pharmacy, Cardiff University
- Ladislav Andera – Institute of Molecular Genetics, Czech Academy of Sciences
- Peter Edwards – Department of Chemistry, Cardiff University
- Stephen Paisey, Chris Marshall - Wales Research and Diagnostic Positron Emission Tomography Imaging Centre (PETIC), Medical School, Cardiff University
- Matthias Eberl – Dept Infection, Immunity and Biochemistry, Cardiff School of Medicine
- Julia Gee – School of Pharmacy, Cardiff University
- Peter Barrett-Lee - Medical Director, Velindre Cancer Centre, Cardiff
- Dr Philippa Young - Consultant Radiologist, Cardiff Breast Clinic