Molecular Mechanisms of Cell Death
Programme Leader: Marion MacFarlane
Summary of Research Interests
Apoptotic cell death is a key regulator of tissue homeostasis. Consequently, a failure to kill aberrant cells is a major contributory factor in the development of cancer. The development of therapeutic agents with high apoptotic efficacy and low toxic side-effects is thus of high priority. In this context, our current research is primarily focussed on investigating the mechanisms underlying the differential sensitivity of normal and transformed cells to TNF-related apoptosis-inducing ligand (TRAIL). Unlike other members of the TNF family, TRAIL is considered to be unique as it selectively activates the ‘extrinsic’ death pathway in tumour cells while showing low toxicity to normal tissues.
TRAIL-induced ligation of its cognate death receptors (TRAIL-R1/R2), results in formation of a ‘death-inducing signalling complex’ (DISC), and initiation of the extrinsic cell death pathway (Fig 1). TRAIL-induced apoptosis can also be regulated by ‘inhibitory’ molecules, such as c-FLIP, which reportedly compete with caspase-8 for binding to FADD and thereby abrogate DISC activation. Despite promising results for TRAIL as a potential cancer therapeutic in some solid tumours, in collaboration with Profs. Dyer and Cohen within the Toxicology Unit we found that primary lymphoid malignancies are resistant to TRAIL. Thus, in order to better exploit the potent anti-tumour activity of TRAIL there is a need to understand the molecular basis of this phenomenon. In this respect, we have already established that primary lymphoid tumour cells exhibit impaired TRAIL DISC activation, but significantly these tumour cells can be sensitised to soluble forms of TRAIL that specifically signal via TRAIL-R1, but not TRAIL-R2. Our more recent findings in tumour cell lines suggests that other primary tumours, including those of epithelial origin, may also exhibit preferential apoptotic signalling via TRAIL-R1. The refractoriness of certain tumour cells to TRAIL is likely due to different molecular mechanisms, including: low expression/impaired activation of TRAIL-R1/-R2, high levels of intracellular inhibitors (eg. c-FLIP or IAPs), or concomitant TRAIL-induced activation of pro-survival pathways (eg. NF-κB). It is also highly likely that, as yet unidentified, proteins may specifically modulate TRAIL-R1/TRAIL-R2.
The Receptor-mediated Signalling and Cell Death Group is primarily focussed on unravelling the molecular mechanisms that determine death or survival of cells following exposure to members of the TNF family of death-inducing ligands. In this context our most significant advance has been the first in vitro reconstitution of the CD95 Death-Inducing Signalling Complex (DISC). Using only three core components, namely purified CD95, FADD and procaspase-8, our lab recently delineated a critical switch in DISC catalytic activity that determines CD95 signalling for death or survival (Fig 2).
These novel findings not only have major implications in terms of death receptor activation per se, but are also important in the context of human disease with several diseases of the immune system already linked to defects in death receptor signalling outcome (e.g. ALPS). Our current work is focussed on extending the CD95 DISC model to examine other members of the TNF death receptor family including the selectively toxic and potential cancer therapeutic, TRAIL. We are also using RNA interference (RNAi) screening approaches to identify potentially novel regulators of TRAIL signalling in tumour cells, and in collaboration with Dr K Cain (MRC-Protein Profiling Group) both proteomics and cell metabolism-based approaches are being employed to investigate factors that modulate the sensitivity of tumour cells to TRAIL/mechanisms of resistance. In collaboration with Prof. R Walker (Dept. of Cancer Studies & Molecular Medicine), our translational work is focussed on investigating the potential use of TRAIL, as wel as receptor-selective TRAIL ligands developed within the MRC Toxicology Unit and patented by MRC-Technology, in the treatment of breast cancer using an ex-vivo primary breast tumour tissue explant model.Such knowledge should provide valuable new insights into the fundamental mechanisms that regulate cellular resistance/sensitivity at the level of the DISC, as well as a better understanding of the critical factors that determine activation of the ‘extrinsic’ apoptotic cell death pathway per se.
Currently our research is focussed on 3 key areas:
- Molecular characterization of the DISC using both in vitro models and cell biology approaches.
- Identification of apical modulators of TRAIL Receptor Signalling using ‘forward genetics’ screens and purification of TRAIL receptor signalling complexes using a variety of affinity-based approaches.
- Translational studies to determine TRAIL efficacy and selective targeting of tumour over normal cells using receptor-specific forms of TRAIL in primary tumour models, namely Breast Cancer and CLL