2006 IRISH SCIENTIST YEAR BOOK

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Queen's University Belfast
Patrick Johnston
Centre for Cancer Research & Cell Biology

The Centre for Cancer Research & Cell Biology (CCRCB), which was officially launched in October 2004, is an interdisciplinary research centre with scientific teams from Biomedical Science, Clinical Medicine (Oncology, Haematology, Pathology and Surgery), Chemistry, Immunology, Virology, Mathematics, Engineering and Radiobiology. The development of the CCRCB, adjacent to the Northern Ireland Clinical Cancer Centre on the Belfast City Hospital campus, has created a comprehensive cancer centre linking basic research with clinical trials and clinical application. The �65 million Northern Ireland Clinical Cancer Centre, which opened in March 2006, provides a modern state of the art clinical facility entirely devoted to the diagnosis and treatment of cancer. The CCRCB has been formed as a result of a �25 million investment by the University, part of a �45 million investment in Life Sciences.

The primary objective of the CCRCB, under the leadership of Professor Patrick Johnston, has been "to create a world-class Centre for Cancer Research and Cell Biology at QUB that will be recognised for its excellence by the international scientific community". CCRCB will ensure that Northern Ireland contributes in a strategically important way to future UK and international research initiatives in Cancer as well as Infection & Immunity and Medicinal Chemistry. A key element of this project has been the provision of a new building with state of the art laboratories and enhanced key enabling technologies. The 4,500m 2 building, which is due for completion in April 2007, has been designed primarily as a generic research facility, and will bring key research groups into a single location. With the new CCRCB we have created the first comprehensive cancer centre in Ireland � promoting international, high quality research programmes linking the University, the Health Service and other funders of cancer research.


Figure 1: During a recent visit to the Centre (left to right) Ms Margaret Grant, Senior Clinical Trial Co-ordinator, Birmingham CRCTU; Ms Sarah Bathers, Assistant Director, Operational Issues, Birmingham CRCTU; Dr Richard Wilson, Director, NICCTU; Dr Cindy Billingham, Assistant Director, Research Methodology, Birmingham CRCTU; Dr Melanie Morris, Manager, NICCTU; Mr Paul Mason, Information Technology, Birmingham CRCTU; and Sr Ruth Boyd, Senior Nurse Co-ordinator, NICCTU.

A number of significant interdisciplinary research projects are already underway � these include the MRC Cancer Grid Project along with colleagues from Cambridge, Oxford, Birmingham and University College London (UCL). In addition the Centre has been successfully appointed as a National Clinical Trials Unit by Cancer Research UK and is in the process of moving towards managing national clinical trials in partnership with the Cancer Research UK Clinical Trials Unit (CRCTU) based in Birmingham (Figure 1). Cancer Clinical trials are already coordinated within the Centre by the Northern Ireland Cancer Clinical Trials Unit (NICCTU) with 48 clinical trial protocols currently open for accrual. These trials include first-in-human (FIH) phase I through to phase IV trials along with translational, genetic epidemiology, quality of life and other studies. The trials portfolio concentrates on haematological malignancies and major solid tumour sites including cancers of the breast, ovary, gastrointestinal tract and lung, with increasing activity in genitourinary cancers and radiation oncology.

The Centre has also recently been selected as one of the Experimental Cancer Medicine Centres for the UK and through that mechanism there are a number of ongoing projects in collaboration with colleagues in Cambridge, UCL, University of Newcastle and the Institute for Cancer Research. The Centre's aims in experimental medicine are to drive the development of phase I and II trials, to facilitate the translation of pre-clinical ideas into the clinical arena and back, and to co-ordinate the collection of clinical samples. Two Cancer Research UK first-in-human (FIH) phase I trials, the first such studies in cancer on the island of Ireland, have recently been completed in Belfast and have led to the development of phase II/III programmes. Another exciting FIH phase I study exploring nano-technology drug delivery has just opened and will add to our ever increasing activity in early clinical trials.

Emanating from the Support Programme for University Research (SPUR) initiative and the University's strategic plans for the medical Faculty, the CCRCB has gone through a period of significant expansion with recruitment of a number of senior academics, from the United States, Canada and Europe. A key appointment in July 2006 to the Chair in Cancer Research & Cell Biology has been Professor Dennis McCance (Figure 2), from the University of Rochester Medical Center.


Figure 2: Newly appointed Professor in Cancer Research & Cell Biology, Dennis McCance


A brief outline of Professor McCance's research is given below:


Role of the retinoblastoma protein (pRb105) and AKT in epithelial cell cycle control and differentiation

The research interests of the laboratory centre on the role of pRb105 in keratinocyte differentiation and the pathways controlled by this protein. The pRb105 pathway is disrupted in a number of cancers and the role during the cell cycle has been extensively studied. However, its role in normal cell differentiation is unclear. Human papillomaviruses (HPV) are small DNA tumor viruses causally associated with cervical cancer. The early gene product E7 from high-risk HPV is considered the major transforming protein expressed by the virus. Although many functions have been described for E7 in disrupting normal cellular processes, we have recently determined a new cellular target in primary human keratinocytes (HFK), namely, the serine/threonine kinase AKT. Expression of HPV type-16 E7 in HFK caused inhibition of differentiation, hyper-proliferation, and upregulation of AKT activity in organotypic raft cultures. The ability of E7 to up-regulate AKT activity is dependent on it binding to and inactivating the pRb105 family of proteins. Furthermore, we show that knocking down pRb105 alone, with short-hairpin RNAs (shRNAs), is sufficient to up-regulate AKT activity in differentiated keratinocytes (Figure 3). Upregulation of AKT activity and loss of pRb105 was also observed in HPV-positive cervical high grade squamous intraepithelial lesions (HSIL), when compared to normal cervical tissue. Together, these data provide evidence linking inactivation of pRb105 by E7 in the upregulation of AKT activity during cervical cancer progression. Development of AKT inhibitors may therefore help in the treatment of cancers associated with HPV infection.


Figure 3: Knock-down of Rb is sufficient to upregulate AKT activity in HFK. (A) Primary HFK were transduced with retrovirus expressing 2 different short-hairpin RNAs (shRNA) targeting Rb (shRb1 and shRb2), as well as a shRNA targeting no known gene (shScram) as control. Western blot analysis was performed to measure the amount of knockdown of Rb. The relative knockdown of Rb, determined by densitometry analysis, was approximately 70% and 80% for shRb 1 and shRb2 respectively. The levels of Rb-family members p107 and p130 are unaltered. p120 RasGAP and actin are loading controls. (B) The cells were then differentiated on organotypic raft cultures for 14 days. Cells were pulsed with 20 �M BrdU 12 hours prior to harvesting rafts. The rafts were then fixed, paraffin-embedded, sectioned and stained for BrdU (red), K1 (green), P-AKT (green), and DAPI (blue) to stain nuclei. Hemotoxylin and eosin (H&E) stained sections are also shown for all cell lines to examine morphology.

AKT kinase phosphorylates a number of substrates, although what substrates are the target of AKT during keratinocyte differentiation are unknown. Using a proteomics/mass spectrometry approach we are investigating novel targets of AKT during differentiation. Elucidation of such substrates may help in identifying targets for therapeutic interventions.


Role of p63 isoforms in keratinocyte differentiation

There are 6 isoforms of the p63 family and they have high sequence homology to both the p53- and p73- family of proteins. Some of the p63 isoforms are up-regulated in epithelial cancers and this inappropriate expression is thought to play a role in malignant conversion. Using mice null for the p63 family, two groups have shown that p63 is required for skin development, although the role of each family member is unclear. Using RNAi technology we are investigating the role of each family member in keratinocyte differentiation.


Genomic instability in keratinocytes caused by HPV-16 E6 and E7

The oncoproteins, E6 and E7 from HPV-16, cause polyploidy in primary human keratinocytes within 48 hours of expression. The polyploidy cells remain stable for many passages but eventually aneuploidy cells appear. We are investigating the cellular alterations which lead to polyploidy and aneuploidy by a variety of methods, including live imaging of cells, in collaboration with Dr Conly Rieder, Wadsworth Center, Albany, NY.

For further information, please refer to the CCRCB web site: www.qub.ac.uk/ccrcb or contact:
Professor Patrick Johnston,
Scientific Director, Centre for Cancer Research & Cell Biology,
Queen's University Belfast, University Floor, Belfast City Hospital, Belfast BT9 7AB
Tel: 028 9026 3911.