Fearnhead Group

Caspases are cysteine proteases that play key roles in the regulation of programmed cell death and inflammation. More recently caspases have also been implicated in the control of cellular differentiation.

The Caspase Laboratory is a research group led by Dr Howard Fearnhead and is a part of the Apoptosis Cluster.  The Laboratory investigates the role of caspase activation in cancer cell apoptosis.
Overview
All our cells carry a genetic program that enables a cell to quickly and cleanly kill itself, a form of cell death called apoptosis. This program is found in all metazoans examined and plays important roles at different stages of an organism's life. Apoptosis is activated at various stages during normal embryonic development, removing unwanted cells and so sculpting our tissue and organs. It is also activated in bacterially or virally infected cells and serves to limit the success of these pathogens. Apoptosis is also a stress response, acting to remove damaged cells that can no longer serve a purpose or that pose a threat to the organism as a whole. A failure in the regulation of apoptosis is associated with auto-immune diseases and degenerative diseases (too much apoptosis) and cancer (too little apoptosis).

Programmed cell death or apoptosis is of fundamental importance to cancer as it both limits tumorigenesis and is also triggered by many cancer chemotherapeutics. Importantly, cancer cells often acquire mutations that compromise the apoptotic process, allowing these cells both to escape normal growth constraints and to become resistant to many anti-cancer drugs, resulting in the emergence of drug-resistant malignancies. Thus discovering how apoptosis is regulated and why it fails in cancer is central both to understanding cancer progression and developing new therapies to counter chemo-resistant cancers.

Biochemical and genetic studies in a range of model systems have identified the key components of the apoptotic machinery. One gene family that contributes to the commitment to apoptosis and execution of the death program encodes a family of cysteine proteases called caspases. Caspases are expressed in cells as inactive zymogens and are activated at the onset of apoptosis. In many cases (but not all) this is sufficient to kill the cell. Different types of apoptotic signals initiate apoptosis by activating different 'initiator' caspases. These 'initiator' caspases then activate a common set of 'effector' caspases by proteolysis. Ultimately, it is these effector caspases that produce the apoptotic phenotype by cleaving a wide range of intracellular substrates;  hundreds of substrates have been identified so far.  Caspases also play non-apoptotic roles, being important in the regulation of inflammation. More recently, caspases normally associated with inducing cell death have also been implicated in the induction of differentiation of stem and progenitor cells.  How caspase mediated-proteolysis is regulated to induce differentiation but not death in these cases is unclear.
 
Plants and fungi also appear to use proteases to induce programmed cell death.  In these cases, the proteases are "metacaspases", which share some sequence similarity with caspases.  However, the molecular mechanisms of metacaspase activity are less well understood and so far there are relatively few metacaspase substrates identified.                                                                                                                                         
Research Themes
Drug-repurposing

Much cancer research is focussed on a molecular understanding of cancer cell biology with the aim of developing tailored therapies that are designed to target tumours displaying specific molecular lesions.  The power of this approach for treating breast cancer is demonstrated by Trastuzumab and anti-estrogen therapies, designed to target tumours that are HER2 or ER positive respectively.  Despite the success of these targeted treatments for a subset of patients, many other patients do not benefit and efforts to identify new therapies to either complement or replace existing chemotherapy continue. The difficulty is that the estimated time and cost of discovering and developing a new drug is ~15 years and hundreds of millions of dollars. The time involved means many will die before a therapy becomes available whilst the high development cost increases the cost of the therapy and can render it  unaffordable for many.

Repurposing existing drugs for new uses provides an approach that can address both problems.  Repurposing is attractive as a wealth of preclinical and clinical data are already available, greatly reducing the time and money required to bring a candidate to clinical trial.  This accelerated development translates into better, cheaper patient care, faster.  We have therefore been screening the Johns Hopkins Clinical Compound Library of ~1,400 approved compounds using drug-resistant breast cancer cells and the Core Screening Laboratory at NUI Galway. 
 
Caspases and Differentiation
The caspases comprise a family of cysteine proteases and some of the family are responsible for causing apoptosis.  Of the apoptotic caspases, caspases-3,-6 and -7 are classed as ?effectors? and cleave a wide range of intracellular proteins and responsible for causing many of the changes typical of apoptosis.   Effectors are expressed as zymogens and are activated when cleaved by ?initiator? caspases such as -2, -8 and -9.  Initiators are also expressed as zymogens but, in contrast to effectors, are activated by their recruitment into large multi-protein complexes.  Different apoptotic signals activate different initiators, but all the initiators activate a common set of effectors.  Thus, during apoptosis very different pro-apoptotic signals are all integrated and amplified through a proteolytic cascade of initiator and effector caspase activity.  Implicit in this model is that the activity of initiator and effector caspases is synonymous with apoptosis and that once effectors are active an irreversible choice has been made.

Not all caspases are involved in apoptosis; caspases-1, -4 and -5 are involved in inflammatory responses where they are important for the proteolytic maturation of cytokines.  The specificity of caspases for a particular process is defined by the selective cleavage of substrate; inflammatory caspases preferentially bind and cleave cytokines, while apoptotic caspases cleave a different set of proteins.  It is therefore remarkable that the apoptotic effector caspase, caspase-3 is not only required for the differentiation of muscle progenitor cells into myofibers but apparently is sufficient for this differentiation.

To date there is no explanation that reconciles the apoptotic activity of caspase-3 with its role in differentiation.  The explanation of how a cell containing active caspase-3 retains the ability to choose a fate other than apoptosis is likely to be critical for our understanding of how cells make life-and-death decisions.  It may also have important implications beyond muscle biology; mutations that allow cells to avoid apoptosis contribute to tumourigenesis and uncovering a mechanism through which cells survive despite the presence of active caspase-3 may reveal new pathways important in cancer cell biology.
 
Publication:
 
A non-apoptotic role for caspase-9 in muscle differentiation. Murray TV, McMahon JM, Howley BA, Stanley A, Ritter T, Mohr A, Zwacka R, Fearnhead HO. J Cell Sci. 2008 Nov 15;121(Pt 22):3786-93.
 
Putative Serine proteases and apoptosis
Many chemotherapeutics exert their anti-cancer activity through the induction of programmed cell death or apoptosis. Much recent research into the mechanisms of apoptosis has identified new therapeutic targets within the apoptotic machinery and is driving the development of novel chemotherapeutics. Proteases, most notably the caspase cysteine proteases, are critical in the induction of apoptosis. However, roles for serine proteases have also been suggested based, in part, on the ability of protease inhibitors like N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) to block apoptosis. Despite these data, the relevant target for TPCK has not been identified. Identifying this target is the first step in assessing whether it represents a valid target for cancer chemotherapy. The research aims to identify TPCK targets by a biochemical approach and then to use a genetic approach to test the roles of these candidates in apoptosis.
 
Publication:
 

TPCK-induced apoptosis and labelling of the largest subunit of RNA polymerase II in Jurkat cells. Fabian Z, O'Brien P, Pajęcka K, Fearnhead HO. Apoptosis 2009 Oct;14(10):1154-64.

TPCK targets elements of mitotic spindle and induces cell cycle arrest in prometaphase. Fabian Z, Fearnhead HO. Biochem Biophys Res Commun. 2010 May 14;395(4):458-64.

MicroRNAs, apoptosis and cancer
Breast cancers are phenotypically diverse, presumably reflecting a spectrum of distinct molecular defects in processes controlling cell proliferation and survival. This complexity makes breast cancer progression hard to predict and treatment difficult to manage. Currently, clinicians rely heavily on the status of two receptors, the estrogen receptor and HER2/neu receptor for clinical decisions relating to prognosis and treatment. These markers, while enormously valuable, do not adequately define different types of breast cancers or predict their sensitivity to therapy. Improving breast cancer treatment will require identification of more molecules that control the proliferation and survival of breast cancer cells.
 
In recent years, many instances of post-transcriptional control of eukaryotic protein expression by non-coding micro RNA (miRNA) molecules have been identified. miRNAs are small (~21-25 nucleotide), highly conserved RNAs which bind specifically to the 3'-untranslated regions of target messenger RNAs (mRNAs) and prevent their translation. Importantly, in mammalian cells this process can reduce protein levels without decreasing mRNA levels. These alterations would therefore not be detected by conventional RNA microarray analysis, as these provide estimates only of mRNA abundance without reference to translation. Recent reports have indicated that miRNAs are key regulators of fundamental biological processes including cell proliferation and cell death, development and stem cell differentiation. Several recent high impact publications have demonstrated that determining relative miRNA levels is a powerful technique which has been instrumental in leading to an understanding of aspects of the regulation of important biological processes.

Genomic approaches have been employed to compare gene expression profiles in normal and cancer cells, but these expression patterns do not allow good discrimination between the two cell types. More recently, the patterns of microRNA expression in normal and cancer cells have been compared and observed to differentiate between non-tumour cell and tumour cell. Subsequently, specific miRNAs were found to correlate with the stage of breast cancer. Moreover, some of these miRNAs regulate genes that control cancer cell proliferation and survival, providing a mechanistic underpinning for the correlative studies.

The strategy is to use clinical cancer samples in a genomics-based approach to identify miRNAs associated with aspects of cancer biology. More specifically to identify miRNAs associated with tumour stage and grade, with sensitivity or resistance to particular treatments or with a particular prognosis. The identification of these miRNAs will drive both basic research through collaborations inside and outside of NUIG and also translational research aimed at developing diagnostic tools for cancer. The Breast Cancer Research group headed by Professor of Surgery, Michael Kerins, maintains The Breast Cancer Tissue Bank. This consists of matched non-tumour and tumour frozen tissue specimens from 200 patients and the associated detailed clinical histories. This tissue bank and the accompanying clinical histories is an enormously valuable, and so far untapped, resource for Breast cancer research.

Facilitated by a collaboration with Dr Vladimir Benes of EMBL, the Caspase Laboartory has identified several miRNAs that correlate with HER2 status in late stage breast cancers.Tthese candiadtea miRNAs are now under investiagtion to establish whether they play a role in HER2 signalling or are controlled by HER2.
 
Publication:
 
The therapeutic potential of targeting microRNAs in cancer.  Howley, BV and Fearnhead, HO. 2010 Cellscience Vol.6, Number 3, January 27th 2010, available: http://www.cellscience.com/journal/journalindex.asp
 
Key Publications
1.

Autophagosomal IkappaBalpha degradation plays a role in the long term control of tumor necrosis factor-alpha-induced NF-kappaB activity. Colleran A, Ryan A, O'Gorman A, Mureau C, Liptrot C, Dockery P, Fearnhead H, Egan LJ. J Biol Chem. 2011 Mar 31.

 

TPCK targets elements of mitotic spindle and induces cell cycle arrest in prometaphase. Fabian Z, Fearnhead HO. Biochem Biophys Res Commun. 2010 May 14;395(4):458-64.

 

The therapeutic potential of targeting microRNAs in cancer.  Howley, BV and Fearnhead, HO. 2010 Cellscience Vol.6, Number 3, January 27th 2010, available: http://www.cellscience.com/journal/journalindex.asp

 

p53-mediated induction of Noxa and p53AIP1 requires NFkappaB. O'Prey J, Crighton D, Martin AG, Vousden KH, Fearnhead HO, Ryan KM.Cell Cycle. 2010 Mar 1;9(5):947-52.

 

TPCK-induced apoptosis and labelling of the largest subunit of RNA polymerase II in Jurkat cells. Fabian Z, O'Brien P, Pajęcka K, Fearnhead HO. Apoptosis 2009 Oct;14(10):1154-64.

 

The Apaf-1*procaspase-9 apoptosome complex functions as a proteolytic-based molecular timer. Malladi S, Challa-Malladi M, Fearnhead HO, Bratton SB. EMBO J. 2009 Jul 8;28(13):1916-25.

 

Activation of p73 and induction of Noxa by DNA damage requires NF-kappa B. Martin AG, Trama J, Crighton D, Ryan KM, Fearnhead  HO. Aging Volume 1 Number 3, March, 2009 pp 275-349

 

A non-apoptotic role for caspase-9 in muscle differentiation. Murray TV, McMahon JM, Howley BA, Stanley A, Ritter T, Mohr A, Zwacka R, Fearnhead HO. J Cell Sci. 2008 Nov 15;121(Pt 22):3786-93.

         

Caspases as therapeutic targets.

Howley B, Fearnhead HO. J Cell Mol Med. 2008 Feb 24.

 
Identification of an inhibitor of caspase activation from heart extracts; ATP blocks apoptosome formation (2006) Samali A, O'mahoney M, Reeve J, Logue S, Szegezdi E, McMahon J, Fearnhead HO. Apoptosis. Vol 3, 465-74.

Intracellular Nucleotides Act as Critical Prosurvival Factors by Binding to Cytochrome C and Inhibiting Apoptosome (2006) Dhyan Chandra, Shawn B. Bratton, Maria D. Person, Yanan Tian, Angel G. Martin, Mary Ayres, Howard O.Fearnhead, Varsha Gandhi, and Dean G. Tang Cell, Vol 125, 1333-1346.

Small molecule inhibitors of Apaf-1-related caspase- 3/-9 activation that control mitochondrial-dependent apoptosis. (2005) Malet G, Martin AG, Orzaez M, Vicent MJ, Masip I, Sanclimens G, Ferrer-Montiel A, Mingarro I, Messeguer A, Fearnhead HO, Perez-Paya E. Cell Death Differ. Dec 9.

Apo cytochrome c inhibits caspases by preventing apoptosome formation. (2004) Angel G. Martin, Jack Nguyen, Jim Wells and Howard O. Fearnhead. Biochem. Biophys. Res. Commun. 2;319(3):944-50
 
Assay for ubiquitin Ligase activity: High Throughput screen for inhibitors of MDM2. (2004) Davydov I.V., Woods D., Safiran Y.J., Oberoi P., Fearnhead H.O., Fang S., Jensen J., Weissman A.M. , Kenten J.H. and Vousden K.H. Journal of Biomolecular Screening. Dec 9 (8), 695-703.

Getting Back on Track, or What to Do When Apoptosis Is De-Railed: Recoupling Oncogenes to the Apoptotic Machinery. (2004) Howard O. Fearnhead. Cancer Biol Ther. Jan;3(1):21-8. Review
 
Apocytochrome c blocks caspase-9 activation and Bax-induced apoptosis (2002) Angel G. Martin and Howard O. Fearnhead. J. Biol. Chem. 52 50834-50841.

Molecular Cloning of ILP-2, a novel member of the Inhibitor of Apoptosis Protein family (2001) B.W.M Richter, S.S. Mir, L.J. Eiben, J. Lewis, S. Berkey Reffey, A. Frattini, L. Tian, S. Frank, R. J. Youle, D. L. Nelson, L. D. Notarangelo, P. Vezzoni, H.O. Fearnhead and C. S. Duckett. Mol. Cell Biol. 13 4292-4301.

Cell-free systems to study apoptosis (2001) H O. Fearnhead. In Methods in Cell Biology (L Schwartz and J Ashwell, Eds). Academic Press, San Diego. 66 167-185.

Oncogene-dependent caspase activation is mediated by caspase-9 (1998) H. O.Fearnhead, J. Rodriguez, E. Govek, W. Guo, R. Kobayashi, G. Hannon and Y. A. Lazebnik. Proc. Natl. Acad. Sci. USA 95 13664-13669.

Oncogene-dependent apoptosis in extracts from drug-resistant cells. (1997) H.O. Fearnhead, M.E. McCurrach, J. O'Neill, K. Zhang, S.W. Lowe and Y.A. Lazebnik. Genes and Development 11 1266-1276.

Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells. (1997) L. Faleiro, R. Kobayashi, H.O. Fearnhead and Y.A. Lazebnik. EMBO J. 16 2271-2281.

A pre-existing protease is a common effector of thymocyte apoptosis mediated by diverse stimuli. (1995) H.O. Fearnhead, A.J. Rivett, D. Dinsdale and G.M. Cohen FEBS Lett. 357 242-246.

DNA degradation and proteolysis in thymocyte apoptosis. (1995) H.O. Fearnhead, M. MacFarlane, D. Dinsdale and G.M. Cohen Toxicology Lett. 82-83 135-141.

An interleukin-1 beta-converting enzyme-like protease is a common mediator of apoptosis in thymocytes. (1995) H. O. Fearnhead, D. Dinsdale, G.M. Cohen. FEBS Lett 375 283-288.

An ICE-like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytic THP.1 cells. (1995) H. Zhu, H.O. Fearnhead, G.M. Cohen FEBS Lett 374 303-308.

Mechanisms of Cell Death, with Particular Reference to Apoptosis. (1995) Gerald M. Cohen, Marion MacFarlane, Howard, O. Fearnhead, Xiao-ming Sun and David Dinsdale. In Molecular and Cellular Mechanisms of Toxicity ( F. deMatteis and L.L. Smith, Eds). CRC Press, Boca Raton.

Dexamethasone and etoposide induce apoptosis in rat thymocytes from different phases of the cell cycle. (1994) H.O. Fearnhead, M. Chwalinski, R.T. Snowden, M.G. Ormerod, G.M. Cohen. Biochem Pharmacol 48 1073-1079.
 
Cdc2 activation is not required for thymocyte apoptosis. (1994) C. Norbury, M. MacFarlane, H. Fearnhead, G.M. Cohen Biochem Biophys Res Commun 1994 202 1400-1406.

Formation of large molecular weight fragments of DNA is a key committed step of apoptosis in thymocytes. (1994) Cohen G.M, X.M.Sun, H. O. Fearnhead, M. MacFarlane, D.G. Brown, R.T. Snowden, D. Dinsdale J Immunol 153 507-516.
 
Increased membrane permeability of apoptotic thymocytes: a flow cytometric study. (1993) M.G. Ormerod, X.M. Sun, R.T. Snowden, R. Davies, H.O. Fearnhead, G.M. Cohen. Cytometry 14 595-602.
 
Researchers
Past
 
Post-docs
 
Jill McMahon
Stephen Elliman
Ananya Gupta
Zsolt Fabian
 

Post-graduate Students
 
Thomas Murray
Breege Howley
Phillipe O'Brien

 
Present


Post-graduate Students
David Monaghan
Funding
                           
Collaborators
  • Professor Michael Kerin, Department of Surgery, NUI, Galway
  • Professor Frank Giles, Clinical Research Facility, NUI Galway
  • Professor Laurence Egan, Pharmacology and Therapeutics, NUI Galway
  • Dr Grace McCormack, Zoology, NUI Galway
  • Dr Timothy Gant, MRC Toxicology Unit, University of Leicester, UK
Group Leader
Bio-Sketch
In 1991 Dr Fearnhead completed a BSc. in Pharmacology and Toxicology at the London School of Pharmacy, before being awarded a PhD. in 1995 by the University of Leicester for work perfomed at the MRC-Toxicology Centre. From '95 to '99 Dr Fearnhead was a post-doctoral fellow at Cold Spring Harbor Laboratory, NY before moving to the National Cancer Institute in Maryland as a principal investigator. In 2004 Dr Fearnhead moved to the National Centre of Biomedical Engineering Science (NCBES), National University of Ireland, Galway and in 2006 was appointed a lecturer in Pharmacology and Therapeutics (http://www.nuigalway.ie/pharmacology).
                                                                                                                                                   
 Contact details  
 Mail: Room 213 NCBES,                                                       
Orbsen Building
NUI, Galway
Galway
 Email:  howard.fearnhead@nuigalway.ie
 Telephone:   (+353) 91 495240
 Fax:   (+353) 91 494596
      
   
Training & Supervision
    Supervising Ph.D students
    Three Phd students have completed their studies within the group:
     
    Dr Thomas Murray (now post-doc at Kings College London, UK)
    Dr Phillipe O'Brien (now post-doc at University of Michigan, USA)
    Dr Breege Howley
     
    Supervising MSc research projects
    Each year 2-4 MSc students complete their research projects within  the group
     
    Lecturing in Pharmacology and Therapeutics to undergraduate Pharmacology, nursing and medical students
     
    Lecture series:
    Pharmacology of the Endocrine System (10 lectures)
    Antibacterials (5)
    Chemotherapy (10)
    Molecular Pharmacology (10)
    Introduction to Toxicology (5)
    Molecular Mechanisms in Toxicology (5)
    Advanced Toxicology (5)
     
    Practical classes:
    Cardiovascular Pharmacology Practicals (2 sessions)
    Endocrine Pharmacology Practicals (2)
    Molecular Pharmacology Practicals (3)
     
    Training Guidelines
    Training guidelines are now decribed by the Graduate Studies Office (http://www.nuigalway.ie/graduatestudies/)
    Protocols