Northern Institute for Cancer Research CANCER RESEARCH UK RESEARCH

Northern Institute for Cancer Research CANCER RESEARCH UK RESEARCH H O D G K I N ’ S L Y M P H O M A M Y E L O M A 1 Contents Research at Newcastle University ............................................................................................................................................................................................ 3 An Introduction to the Institute ............................................................................................................................................................................................ 4-8 Prof Alan Boddy - Pharmacological Studies of Drugs Used in the Treatment of Cancer ................................................................................................ 9-10 Prof Hilary Calvert - Translational Cancer Research within the Clinical Trials Unit ........................................................................................................ 11-12 Dr Steven Clifford - Paediatric Neuro-Oncology Research: Genomics to Improved Therapies...................................................................................... 13-14 Prof Nicola Curtin - Drug Development – Pre-clinical Pharmacology ............................................................................................................................ 15-16 Dr Barbara Durkacz - The Roles of DNA Damage-Activated Repair and Signalling Enzymes in Chemotherapeutic Response .................................. 17-18 Dr Richard Edmondson - Understanding the Role and Effects of Hormones in Ovarian Cancer .................................................................................. 19-20 Prof Roger Griffin - Drug Development – Medicinal Chemistry ...................................................................................................................................... 21-22 Prof Andy Hall - The Biology of Relapsed Childhood Leukaemia .................................................................................................................................. 23-24 Dr Ian Hardcastle - Drug Development – Medicinal Chemistry ...................................................................................................................................... 25-26 Mr Rakesh Heer - Human Prostate Epithelial Differentiation.......................................................................................................................................... 27-28 Dr Julie Irving - Optimisation of Chemotherapy in Childhood Acute Lymphoblastic Leukaemia.................................................................................. 29-30 Prof Hing Leung - Prostate Cancer Research .................................................................................................................................................................... 31-32 Dr John Lunec - Molecular Oncology & Developmental Therapeutics ............................................................................................................................ 33-34 Dr Ross Maxwell - Oncology Functional Imaging Research ............................................................................................................................................ 35-36 Dr Felicity EB May - Structure and Function of Trefoil Proteins in Normal and Malignant Cells................................................................................... 37-38 Prof Herbie Newell - Drug Development – Pre-clinical Pharmacology ........................................................................................................................ 39-40 Dr Ruth Plummer - Early Clinical Trials, Melanoma and Sarcoma .................................................................................................................................. 41-42 Dr Christopher Redfern - Cell and Molecular Biology of Retinoids ................................................................................................................................ 43-44 Dr Helen Reeves - The Molecular Pathogenesis of Gastrointestinal Cancers - The Search for Key Biomarkers and Therapeutic Targets ................ 45-46 Prof Craig Robson - Molecular Analysis of Prostate Cancer ............................................................................................................................................ 47-48 Dr Mike Tilby - Intra - Cellular Pharmacology of DNA-Reactive Anticancer Drugs ........................................................................................................ 49-50 Dr Debbie Tweddle - The Role of MYCN and p53 in Neuroblastoma ............................................................................................................................ 51-52 Dr Gareth Veal - Translational and Clinical Pharmacology Studies in Paediatric Oncology .......................................................................................... 53-54 Dr Mark Verrill - Breast Cancer & Sarcoma Research ...................................................................................................................................................... 55-56 Prof Josef Vormoor - Cellular Biology of Childhood ALL and Ewing’s Sarcoma and Phase I/II Studies on New Agents for Children with Cancer .. 57-58 Prof Bruce R Westley - Oestrogen Responsive Breast Cancer ........................................................................................................................................ 59-60 List of Sponsors .................................................................................................................................................................................................................. 61-62 Staff List.............................................................................................................................................................................................................................. 63-64 How to Find Us .................................................................................................................................................................................................................. 65-66 2 Research at Newcastle University Mission Statement: To be a world class research intensive University, to deliver teaching of the highest quality and to play a leading role in the economic, social and cultural development in the North-East of England. Introduction As one of the UK's leading universities, Newcastle University’s reputation rests on the quality of its teaching and research. Currently there are 14,000 undergraduate and 4,000 postgraduate students, and over 4,500 members of staff. A wealth of world-class, subject-based research is carried out in 27 Schools spread over three Faculties: Humanities and Social Sciences; Medical Sciences and Science, Agriculture and Engineering. Currently, there are a total of 12 Research Institutes and 12 Research Centres which bring together Research Assessment Exercise (RAE) grade 5/5* interdisciplinary researchers across School and Faculty boundaries, enabling a pooling of knowledge and the creation of a rich and dynamic environment for research. These include the Northern Institute for Cancer Research, Institute for Ageing and Health, Institute of Cellular Medicine, Institute of Cell and Molecular Biosciences, Institute of Human Genetics, Institute of Neuroscience, and the Centre for Stem Cell Biology and Developmental Genetics. to create 'Science Central', an ambitious science, business and education complex in Newcastle city centre. Science Central will be the hub of Science City and will bring together University researchers with businesses and other interested partner organisations, such as the NHS. New, high-tech companies would emerge, existing companies would have access to cutting edge technologies, making them globally competitive and hospitals would benefit from the latest medical discoveries. The North East has already pioneered one industrial revolution and believes that history can be repeated, not with coal and heavy engineering but with the growth industries of the 21st century, such as biotechnology, molecular engineering and nanotechnology. Facilities for Research At Newcastle, there are first-rate IT, scientific, technical and advisory services available which are provided centrally. These include: three on-site University libraries with one dedicated to serving the Faculty of Medicine; the Information System and Services providing fully networked computer services that reaches all corners of the University; a number of scientific facilities providing a variety of imaging, analytical and molecular biology services, used to support research activities; a dedicated Clinical Research Centre which provides practical support and patient care facilities for clinical research projects. Newcastle – A Science City Newcastle Science City is a unique integration of science and technology research and development, and its application, with spatial planning and urban redevelopment of the local area. Newcastle University has formed a partnership with Newcastle City Council and One NorthEast, the regional development agency, Figure 1. University of Newcastle upon Tyne (Photographer: Graeme Peacock and used with kind permission from www.visitNewcastleGateshead.com). 3 An Introduction to the Institute The Northern Institute for Cancer Research (NICR) is the focus of research excellence in cancer studies in Newcastle University. The mission of the NICR is to improve the management of cancer by using molecular genetic information derived from human cancers to identify novel therapeutic targets, developing drugs that exploit these novel targets and innovative clinical evaluation of these drugs. Molecular genetic approaches developed by the NICR will also allow disease stratification, dose optimisation and treatment individualisation. The improvement of treatment outcomes for patients with cancer lies at the heart of our endeavour. Cancer is a disease of uncontrolled cellular growth. Understanding how cancer cells escape the mechanisms regulating normal tissue growth and how we can re-impose control or eliminate cancer cells from the body requires a multidisciplinary approach. We seek to understand molecular interactions and exploit these as possible drug targets. Our teams of molecular and cellular biologists, chemists and clinical researchers work in collaboration with stem-cell biologists, cell signalling specialists and nanotechnologists across the University to identify the vulnerabilities of cancer cells and to probe the effects of novel drugs. Advances at molecular and cellular levels are translated into clinical practice aided by a strong framework of interaction with medical and paediatric oncologists in Newcastle hospitals, with extensive outreach nationally and internationally. A full range of laboratory and clinical disciplines needed to undertake research in contemporary experimental cancer medicine are available in the NICR, placing Newcastle in a leading position to drive the development of molecular oncology for patient benefit. The research environment has been greatly strengthened through the construction of the Paul O’Gorman Building, bringing together the full range of scientific disciplines. Completed in 2004, this building houses 120 staff and the facilities required for contemporary genomic, cell biology, pharmacological and clinically-related research. The location of the Paul O’Gorman Building as part of the Medical and Dental School complex facilitates extensive interactions between the Institute and other academic biomedical science units of Newcastle University. Clinical research is conducted at Newcastle General Hospital, the Royal Victoria Infirmary, Queen Elizabeth Hospital (Gateshead) and the Freeman Hospital and there are strong links between clinical staff, both ward and laboratory based, and basic scientists. This enables a smooth and rapid translation of research results into clinical practice and has permitted the development of new drugs from ‘bench to bedside’. In addition, the research-intensive environment of the NICR makes an important contribution to training future generations of cancer specialists and researchers. 4 The Newcastle Magnetic Resonance (MR) Centre (Figure 1) was formally opened on 6th March 2006 by Prof. Colin Blakemore. Funding for a more powerful MR Imaging and Spectroscopy system to complement existing equipment has been provided by Cancer Research UK and the Science Research Investment Fund - Round 3 (SRIF3) for installation early in 2007. In parallel, the establishment of a Positron Emission Tomography (PET) Centre on the same site is being planned by Newcastle University, in collaboration with the Newcastle upon Tyne Hospitals NHS Trust, in response to a Department of Health initiative for the provision of PET facilities across the UK. The complementary use of PET will ensure that multiple functional imaging approaches are available to exploit the targets being studied by NICR researchers. Research groups Figure 1: Philips 3 Telsa 3 MR system at Newcastle Magentic Resonance Centre. Research collaborations within the Institute and across the University are facilitated by integrated research groups, both formal and informal. In this way, the whole range of relevant expertise available within Newcastle is harnessed towards the goal of improving the care and treatment of cancer patients. The Drug Development group employs a rational approach to target compound design, utilising known small-molecule ‘leads’ combined with information regarding macromolecular interactions, to enable structure-based drug design. Combinatorial chemistry is also used to determine structureactivity relationships and optimise biological activity for lead compounds. The medicinal chemistry and anticancer drugdesign aspects of the programme are conducted in collaboration with Professor Bernard T Golding in the School of Natural Sciences. Research by the group is based on two sites, with biological studies conducted in the Paul O’Gorman Building, and medicinal chemistry in the Wolfson-CR UK Laboratory (Bedson Building) of the School of Natural Sciences, (Figure 2). Techniques for the non-invasive imaging of cancer are increasing in resolution and sensitivity, and have an increasingly important role in anti-cancer drug development. The molecular imaging of biological targets in preclinical and clinical studies is now possible in Newcastle thanks to the establishment of a comprehensive functional imaging centre on the Newcastle General Hospital campus. 5 A joint clinical trials facility has been developed at the Northern Centre for Cancer Treatment, which can be used by all the Medical and Clinical Oncology NHS staff, allowing investigative and new drug studies to be carried out alongside multicentre Phase III trials. Phase I trials are conducted on drugs, including those sourced from the NICR in-house program. Newcastle is recognised as a key centre for Phase I trials and is a Cancer Research UK/Department of Health Centre for Experimental Cancer Medicine. Established academic strengths in Paediatric Oncology from across the Faculty are brought together under the umbrella of the Childhood Cancer Research Group (CCRG). The CCRG encompasses major research efforts in leukaemia, neuroblastoma and paediatric brain tumours, aimed at the identification of paediatric targets for optimising current therapies and developing new therapeutic approaches. Leukaemia studies are supported by a Programme Grant from the Leukaemia Research Fund. The group has close links with the United Kingdom Children’s Cancer Study Group (UKCCSG) and International Society for Paediatric Oncology (SIOP), as well as extensive collaborations with other leading national and international research centres. Newcastle is a UKCCSG national centre for clinical trials, pharmacology, biological studies and pathological review, and one of the phase I/II centres accredited by the European Consortium for Innovative Therapies for Children with Cancer (ITCC). Figure 2: A medicinal chemistry laboratory within the Wolfson-CR UK Laboratory within the Bedson Building of Newcastle University. The Clinical & Translational Research Group promotes the practical exploitation of research using innovative designs for clinical trials and early clinical studies and contributes significantly to national and international Phase III studies in common disease types. The range of expertise and research activities of the group facilitates correlative molecular pathology studies as a method of target identification, as well as identifying patients who are potential candidates for translational studies. Importantly, the Paul O’Gorman Building houses a ‘Good Clinical Laboratory Practice’-compliant facility for performing pharmacokinetic studies on novel agents. 6 The Breast Cancer Research group utilises expertise in pharmacology and pharmacokinetics to understand the biology and role of a variety of proteins in breast cancer and to take key proteins forward as potential clinical response markers and explore their use as future targets for treatment. The Urological Oncology Research Group consists of scientists based in the NICR and other schools/institutes of Newcastle University, together with clinicians based at the Freeman Hospital Clinical Department of Urology. The group has a strong international track record for translational research in the areas of prostate and bladder cancer as well as functional urology. The range of expertise of the Breast Cancer and Urological Oncology Research Groups is complemented by the Gynaecological and Gastroenterology Cancer Research Groups, multi-disciplinary groups of surgeons, oncologists and scientists who meet regularly to develop cancer research projects and approve the adoption of national studies. Research income In addition to an annual budget of £1.2M from Cancer Research UK (CRUK), research grants generated by the members of the NICR over the past five years are currently as follows: CRUK project grants/studentships, £5.88M; Leukaemia Research Fund (LRF) programme/project grants, £2.5M; research councils, £1.44M; Wellcome Trust, £373,528; NHS and European Community, £2.7M; other international, national and local medical charities, £7.5M; industry, £2.8M. Collaborations with pharmaceutical and bioscience companies include those with Agouron Pharmaceuticals (now Pfizer), Amgen, AstraZeneca, Astex Therapeutics, Boehringer Ingelheim, Bristol Myers Squibb, Cell Therapeutics Inc, Cyclacel, Daiichi, Eli Lilly Co, Enzacta, GlaxoSmithKline, Janssen, Kudos Pharmaceuticals, Novartis, Human Genome Sciences, Methylgene, Nanocarrier, Novocastra, NovusPharma, Pharmamar, Roche, Schering AG, Schering-Plough, SmithKline Beecham Taxolog and Tularik. Figure 3: A research laboratory within the Paul O’Gorman Building of Newcastle University. 7 Department of Health 11% Research Councils EU 4% 1% LRF 9% The NICR thus provides a high level of practical and intellectual resource, with a unique emphasis on linking basic research to improvements in cancer diagnosis and treatment. As one of the major research institutes at Newcastle University, with strong links to the Institutes of Ageing and Health, Human Genetics, Neurosciences, Stem Cell Biology and Regenerative Medicine, Cell and Molecular Biosciences, and Nanoscale Science and Technology, the NICR puts Newcastle University at the cutting edge of cancer research at an international level. Prof Andy Hall Scientific Director Other Medical Charities 27% Newcastle Pharmaceutical Healthcare Trust Company 1% 9% CRUK 38% Prof Hilary Calvert Clinical Director Figure 4: NICR research income - 2006 Research output The primary research outputs of the NICR are new patents, papers in published journals and the entry of patients into clinical trials of new drugs. Drug development research has resulted in 15 patents being filed on novel cancer therapeutics, there are active adult and paediatric Phase I/II Early Clinical Trial programmes, and strong surgical research programmes allowing the acquisition of tumour tissue and establishment of the Newcastle Central Tissue Resource within the NICR in full concordance with current legislation. The NICR has a substantial record of regular publication in the scientific literature, particularly general science and biological science journals (including Nature, Journal of Biological Chemistry, Journal of Cell Science, Nucleic Acids Research, Gene) and the main specialist cancer journals (including the Journal of the National Cancer Institute, Cancer Research, Journal of Clinical Oncology, Oncogene, Clinical Cancer Research). 8 Prof Alan Boddy We perform pharmacological studies in adult and paediatric patients with cancer to understand the processes that determine drug concentrations in plasma and in the tumour. In the case of novel drugs this information is essential in guiding dose escalation and evaluating drug-target interaction. It is important to incorporate new technologies and an increased understanding of genetic and other influences to optimize the use of established drugs. NICR Team: Gordon Taylor, Julieann Sludden, Melanie Griffin, Julie Errington, Michael Cole, Johanne Lee, Sophie Rowbotham, Girish Chinaswammy, Simon Pridgeon and in close collaboration with Gareth Veal, Mike Tilby, Steve Clifford, Ruth Plummer, Mark Verrill and Josef Vormoor. Introduction We perform studies of the mechanisms of action of drugs used in the treatment of cancer in both adults and children. In addition, we investigate the pathways by which these drugs enter the body, distribute to tumour and other compartments, and how they are metabolized and eliminated from the body. The overall aims are to use this mechanistic information to optimize the treatment of cancer in each individual patient and to use pharmacological principles to guide the development of novel treatments. In order to perform these studies, our group has been exploring novel technologies for drug analysis, incorporating pharmacogenetic variables into clinical studies and developing methods to understand the mechanism of action of drugs as they interact with their targets in cancer patients. Figure 1: Plasma concentration-time data for actinomycin D in children with cancer. These are the first ever data on this drug in children, using a novel LCMS method developed in our group. Novel analytical technologies Liquid Chromatography/Mass Spectrometry (LCMS) Over the last 5 years we have built up a strong background in the analytical applications of LCMS and currently we have 3 instruments providing tandem mass-spectrometry analysis. These instruments have allowed us to obtain previously unattainable data, including the first ever pharmacokinetic data on actinomycin D (Figure 1). Recent advances in related instrumentation, coupled with advances in chromatographic technology, promise to permit further advances in terms of metabolite identification and the determination of pharmacodynamic endpoints. Accelerator Mass Spectrometry (AMS) AMS is a technique for measuring very low concentrations of drug in plasma and tissues. Because very low doses of drug can be used, it is possible to perform pharmacological studies in cancer patients without toxicity and to measure drug in tissues at very low concentrations. We have recently completed the first UK study to use the technique of AMS to investigate 9 Pharmacological Studies of Drugs Used in the Treatment of Cancer the pharmacology of a chemotherapeutic agent. Further collaborations with Xceleron, a York-based company which has pioneered the use of AMS, are already in place. Pharmacogenetic studies In the last few years, our knowledge of the genetic influences that may underlie variation in clinical response has increased enormously. We are currently undertaking a number of studies in adults and children aimed at understanding the influence of specific genetic polymorphisms on the pharmacology of individual drugs. We are building on the important observation of a relationship between clinical outcome and the clearance and metabolism of cyclophosphamide in children. A follow-up study incorporating pharmacogenetic endpoints is now underway, as are similar studies in breast cancer and in neuroblastoma in children. chemotherapeutic agents. Such pharmacodynamic endpoints have formed an integral part of many of our clinical studies over the last 10 years. Techniques such as the comet assay and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS)-based methods have been used to investigate the interaction of drugs with DNA. The mechanism of action of antimetabolites has been demonstrated with the LCMS analysis (Figure 2) and with PET. We have made further advances in integrating quantitative pharmacodynamic endpoints such as real-time RTPCR and Western blotting into early clinical trials. These techniques present novel problems in method validation, according to the principles of the EU directive on Clinical Trials. We have successfully addressed these issues employing the principles established for more conventional analytical methods. In addition to our clinical work, we are also involved in preclinical development in a number of areas, including novel treatments for bladder and ovarian cancer, as well as paediatric cancers. Veal GJ, Cole M, Errington J, Parry A, Hale J, Pearson ADJ, Howe K, Chisholm JC, Beane C, Brennan B, Waters F, Glaser A, Hemsworth S, McDowell H, Wright Y, Pritchard-Jones K, Pinkerton R, Jenner G, Nicholson J, Elsworth AM, Boddy AV. “Pharmacokinetics of Dactinomycin in a Pediatric Patient Population – A United Kingdom Children’s Cancer Study Group study.” Clinical Cancer Research (2005) 11: 5893-5899. Yule SM, Price L, McMahon AD, Pearson ADJ, Boddy AV. “Cyclophosphamide metabolism in children with non-Hodgkin’s lymphoma.” Clinical Cancer Research (2004) 10: 455-460. Willits IA, Price L, Parry A, Tilby MJ, Ford D, Cholerton S, Pearson ADJ, Boddy AV “Pharmacokinetics and metabolism of ifosfamide in relation to DNA damage assessed by the COMET assay in children with cancer” British Journal of Cancer (2005) 92: 1626-1635. Pharmacodynamic endpoints We are increasingly able to perform studies in patients to identify and quantify the pharmacological effects of Figure 2: Elevation of plasma deoxyuridine as a pharmacodynamic marker for the inhibition of thymidylate synthase in a Phase I study of pemetrexed. Boddy AV, Plummer ER, Todd R, Sludden J, Griffin M, Robson L, Cassidy J, Bissett D, Bernareggi A, Verrill M, Calvert AH. “A phase I and pharmacokinetic study of paclitaxel poliglumex (PPX, XYOTAX™), investigating both every 3-weekly and every 2-weekly schedules”. Clinical Cancer Research (2005) 11: 7834-7840. 10 Prof Hilary Calvert The Clinical Trials Unit (CTU) based at the Northern Centre for Cancer Treatment (NCCT) at Newcastle General Hospital enables new chemotherapy agents to undergo the translation from the research laboratory into clinical applications, through the use of clinical trials. I have an extensive involvement with clinical trials involving new chemotherapies within the CTU, and an added interest in ovarian cancer. NICR Team: Sandy Beare, and in close collaboration with Herbie Newell, Roger Griffin, Nicola Curtin, Alan Boddy, Ruth Plummer, Richard Edmondson, John Lunec, Ian Hardcastle, Bruce Westley, Mark Verrill, Josef Vormoor and clinical staff based at the NCCT. which has four clinical trial co-ordinators, a team of research nurses and a clinical team directed by myself. Over the past year, translational cancer research has included: • An extensive Phase I/II/pharmacological clinical trials portfolio with 23 studies active or closed in the past year and 5 additional studies in active follow-up; • Completion of the first-in-class Phase I/pharmacodynamic study of the PARP inhibitor AG140699 given with temozolomide, demonstrating excellent tolerability, and clinical activity; • Completion of the first Phase I combination trial using a novel binary MCRM (modified continual assessment methodology) technique developed in Newcastle; • Completion of the first Phase II combination trial of temozolomide in combination with a PARP inhibitor in melanoma; • Completion of a review of a randomised translational research study investigating the role of mutations in a specific gene (p53) in determining the response to carboplatin or paclitaxel in first-line ovarian cancer (GOKEROC). Introduction Studies on the mechanism of action of drugs are often described as “translational research”, although this term is broader in that it includes studies of features of the molecular biology of human cancers that can be used to predict the response to treatment, select the correct treatment or to identify a potential way of designing a new drug. The revolution in drug discovery (driven by genomic and proteomic techniques coupled with massive improvements in drug development technology) means that there is an exponentially increasing demand for early clinical trials. A Phase I clinical trial is the first time a drug is given to humans. Phase I trials of drugs for cancer are usually performed on patients for whom no established treatment is available. The primary objective is to establish the optimum dose of the drug for subsequent testing in phase II and III, but patient safety is a prime concern and very careful protocols are used to minimise the risks of serious side effects. At the same time as establishing the optimum dose, it is also important to find out if the drug is acting in the way it is intended to, and also to document any side effects that may occur. Facilitating the improvement of chemotherapy treatments for ovarian cancer Ovarian cancer is the fourth commonest cancer overall, and the commonest gynaecological cancer among women in the UK with almost 6,000 new cases occurring annually. For the majority of women, standard treatment consists of surgery followed by chemotherapy. There are always some patients Clinical trials of new chemotherapy agents The Translational and Early Drug Development team based within the CTU at the NCCT is comprised of a clinical trials office 11 Translational Cancer Research within the Clinical Trials Unit in future ovarian cancer research. Understanding how the genetic make-up of the ovarian cancer cell will allow us to predict which patients will do best on certain treatments, and hopefully, tailor treatments to each patient so that the most effective chemotherapy treatment regime can be administered. Pfizer Research Innovation Award The Pfizer Research Innovation Award is given annually for innovative science to a researcher in Europe. It was awarded for my role in developing new drugs for the treatment of cancer. These include carboplatin, for which I derived a dosing formula which is used extensively, in particular for ovarian and lung cancer, and raltitrexed, a drug of the “antifolate” class used to treat colorectal cancer. The work on raltitrexed also stimulated work on pemetrexed, another antifolate which is the only drug licensed for the treatment of mesothelioma – cancer of the mesothelium which covers internal body organs, and asbestos-related lung tumour. A patient on a ward within the Northern Centre for Cancer Treatment at Newcastle General Hospital. Wright JG, Boddy AV, Highley M, Fenwick J, McGill A, Calvert AH. “Estimation of glomerular filtration rate in cancer patients.” British Journal of Cancer (2001) 84: 452 – 459. Hughes A, Calvert P, Azzabi A, Plummer ER, Johnson R, Rusthoven J, Griffin M, Fishwick K, Boddy AV, Verrill M, Calvert AH. “Phase I clinical and pharmacokinetic study of pemetrexed and carboplatin in patients with malignant pleural mesothelioma.” Journal of Clinical Oncology (2002) 20: 3533-3544. Niyikiza C, Baker SD, Seitz DE, Walling JM, Nelson K, Rusthoven JJ, Stabler SP, Paoletti P, Calvert AH, Allen RH. ”Homocysteine and Methylmalonic Acid: Markers to Predict and Avoid Toxicity from Pemetrexed Therapy.” Molecular Cancer Therapeutics (2002) 1: 545-552. Plummer ER, Middleton M, Wilson R, Jones C, Evans J, Robson L, Steinfeldt H, Kaufman R, Reich S, Calvert AH. ”First in human phase I trial of the PARP inhibitor AG-014699 with temozolomide (TMZ) in patients with advanced solid tumors.” Proceedings of the American Society for Clinical Oncology (2005) 24: abstract number 3065. who do and some who do not benefit from chemotherapy treatment. This is partly due to differences in the genetic make-up of the cancer cells which alter the way they respond to chemotherapy, either by making the cells more resistant or more susceptible to certain treatments. To facilitate research into the genetics of ovarian cancer, I have been awarded funding for the establishment of a tissue bank containing samples collected during the Medical Research Council (MRC) ICON3 clinical trial. The ICON3 trial was a large, multinational ovarian cancer clinical trial, comparing one chemotherapy treatment (carboplatin + paclitaxel) with two standard types of chemotherapy treatment (carboplatin alone or cyclophosphamide + doxorubicin + cisplatin). The establishment of the ICON3 Tissue Bank will bring together a large number of samples and make them available to be used 12 Dr Steve Clifford Brain tumours are the leading cause of death from cancer in children. Our research programme focuses on medulloblastoma, the most common malignant paediatric brain tumour, and is aimed at understanding the molecular mechanisms that drive its development, progression and clinical behaviour. We are applying this knowledge in translational research strategies; (i) to identify molecular markers of clinical behaviour, and (ii) to develop novel drugs and therapeutic strategies, for the improved treatment of children with medulloblastoma. NICR Team: Rachel Daniel, Janet Lindsey, Yuan Lu, Meryl Lusher, Agata Rozanska, Sarra Ryan, Kieran O’Toole, Hisham Megahed and in close collaboration with Nicola Curtin, Alan Boddy, Josef Vormoor, Chris Redfern and Debbie Tweddle. Introduction Medulloblastoma arises in the cerebellum and is the most common malignant paediatric brain tumour, accounting for almost 10% of all childhood cancer deaths. About 40% of children with medulloblastoma will die of their disease. This variable disease course is difficult to predict using established clinical and histopathological disease features, and current therapies are associated with significant long-term adverse effects in surviving patients, particularly intellectual and neuroendocrine impairment. A translational research programme has been established in medulloblastoma, involving research scientists, pathologists and oncologists. This programme is aimed at understanding the genetic and biological mechanisms that underlie medulloblastoma development, and exploiting these findings for therapeutic benefit. Such advances could lead to the identification of (i) more accurate molecular markers of disease course, for use in improved patient management and risk stratification strategies (i.e. targeted adjuvant therapies - intensive therapy for high-risk cases and reduced side-effects for responsive cases), and (ii) molecular targets for the development of novel drugs. Figure 1: Amplification of the MYC oncogene on chromosome 8 (A1, B1) and isochromosome (17q) (A2, B2) in medulloblastoma cells, detected by comparative genomic hybridisation (A; CGH) and fluorescence in situ hybridisation (B; FISH) methods. Identification and mapping of critical genetic events in medulloblastoma The identification of critical genetic lesions in medulloblastoma forms a major focus of our investigations. We are employing genome-wide and chromosome-specific high-density mapping strategies (including array-CGH, SNP arrays and fluorescence in situ hybridisation) and expression microarray analysis to identify key tumour suppressor genes (TSGs) and oncogenes in medulloblastoma (Figure 1). Using these strategies, we have made significant insights into mechanisms of chromosomal 13 Paediatric Neuro-Oncology Research: Genomics to Improved Therapies loss in medulloblastoma, and have identified critical genes and loci on chromosomes 8, 10 and 16, which are frequently disrupted in medulloblastoma pathogenesis. Epigenetic events in medulloblastoma Epigenetic events describe heritable alterations in gene expression that occur in the absence of DNA sequence changes, and have recently emerged as a major alternative mechanism of gene disruption in cancer. Over the last five years, we have demonstrated a significant involvement for epigenetic events in medulloblastoma. Our studies have identified a series of candidate tumour suppressor genes (e.g. RASSF1A, INK4C, MCJ, CASP8 and HIC1,) which are epigenetically inactivated by promoter hypermethylation in a significant proportion of cases, and represent some of the most frequently observed molecular events in this disease. Study Group (UKCCSG) and International Society of Paediatric Oncology (SIOP) clinical trials. Through these studies, we are developing novel indices which combine clinical, molecular and pathological markers for the positive discrimination of high-risk (e.g. chromosome 17p loss, MYC oncogene amplification, anaplastic pathology) and low-risk (e.g. beta-catenin status) disease, with a view to their validation in further trials prior to routine clinical application. Novel therapeutics for paediatric solid tumours We are developing novel molecularly-targeted therapeutic agents for the treatment of paediatric solid tumours, by collaborations within the NICR. Current studies are focussed on assessing the pre-clinical efficacy of specific inhibitors of critical DNA damage signalling molecules (eg. PARP-1, ATM and DNAPK) as potentiators of chemotherapeutic agents, in models of medulloblastoma and neuroblastoma. Our group forms part of the UKCCSG Division of Therapeutics and the pan-European Innovative Therapies for Children with Cancer (ITCC) consortium, which co-ordinate the pre-clinical evaluation of novel therapeutics in paediatric cancers throughout the UK and Europe, and their advancement into early clinical trials. Ellison DW, Onilude OE, Lindsey JC, Lusher ME, Weston CL, Taylor RE, Pearson AD, Clifford SC. “Beta-catenin status predicts a favorable outcome in childhood medulloblastoma.” Journal of Clinical Oncology (2005) 23: 7951-7957. Gilbertson RJ, Clifford SC. “PDGFRB is overexpressed in metastatic medulloblastoma.” Nature Genetics (2003) 35: 197-198. Lusher ME, Lindsey JC, Latif F, Pearson ADJ, Ellison DW, Clifford SC. “Biallelic epigenetic inactivation of the RASSF1A tumour suppressor gene in childhood medulloblastoma.” Cancer Research (2002) 62: 5906-5911. Uziel T, Zindy F, Xie S, Lee Y, Forget A, Magdaleno S, Rehg JE, Calabrese C, Solecki D, Eberhart CG, Sherr SE, Plimmer S, Clifford SC, Hatten ME, McKinnon PJ, Gilbertson RJ, Curran T, Sherr CJ and Roussel MF. “The tumor suppressors Ink4c and p53 collaborate independently with Patched to suppress medulloblastoma formation”. Genes and Development (2005) 19: 2657-2667. Pathways to medulloblastoma development Critical developmental cell signalling pathways are constitutively activated by genetic mechanisms in Medulloblastoma development, including the Wingless (Wnt/Wg) and Sonic Hedgehog (SHH) pathways, which each define a significant sub-set (~20%) of cases. We are performing detailed analyses of the genetic and epigenetic mechanisms which contribute to the activation of these pathways. These studies have revealed that each pathway can be disrupted by mutations of different genes encoding alternative pathway members. In addition, we have reported roles for the activation of additional cell signalling pathways (e.g. the PDGFRB-RAS/RAF/MAPK signalling cassette) in medulloblastoma. Translational research: Molecular pathology and clinical trials The identification of robust markers of clinical disease behaviour in medulloblastoma is paramount. In an extensive translational research programme, we are combining the assessment of genetic defects we have identified, alongside the refinement of histopathological indices, in uniformlytreated cohorts of patients entered into UK Children’s Cancer 14 Prof Nicola Curtin As part of the Drug Development Group, a multidisciplinary team of biologists and chemists, my group evaluates novel anticancer drugs and the optimisation of their therapeutic use. The main focus is on inhibitors of DNA damage signalling and repair proteins: PARP, DNA-PK and ATM. We also evaluate CDK inhibitors to control proliferation and nucleoside transport inhibitors to enhance antifolate activity. NICR Team: Huw Thomas, Lan-Zhen Wang, Mike Batey, Suzanne Kyle, Rachel Daniel, Evan Mulligan, Agata Rozanska, Jody Mitchell, Tomasz Zaremba, and in close collaboration with Steve Clifford, Barbara Durkacz, Roger Griffin, Ian Hardcastle, Herbie Newell and Ruth Plummer. by the ataxia telangiectasia mutated (ATM) protein kinase to both cell cycle checkpoints, halting proliferation, and to the DNA repair machinery. ATM kinase signals to DNA-PK, a trimeric enzyme consisting of its DNA-binding components (Ku) and the catalytic subunit (DNA-PKcs), which is integral to the repair of DNA double strand breaks by the non-homologous end-joining (NHEJ) pathway (Figure 1). Introduction The main focus of research undertaken by my group [in the Pre-clinical Pharmacology group] is the evaluation, and optimisation of compounds [provided by the Medicinal Chemistry group] developed either within the NICR or by external collaborators. Current projects are focussed around inhibitors of DNA damage signalling and repair proteins: poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase (DNA-PK) and ataxia telengiectasia mutated (ATM) kinase. We are also investigating pyrimidopyrimidine inhibitors of nucleoside transport and inhibitors of cyclin-dependent kinases. For Medicinal Chemistry see entries by Roger Griffin and Ian Hardcastle. DNA SSB DNA replication DNA DSB PARP XRCC1 Polβ Lig III Repair G1 arrest ATM/R γH2AX BRCA1 MRE11 Rad50 NBS1 G2 arrest FA core complex FANC D2 NHEJ repair Ku 70/80 BRCA2 HR repair Rad 51 DNA-PKcs RPA Rad 52/4 ERCC1 XRCC3 Inhibitors of DNA damage signalling and repair Many existing cancer therapies, both radiotherapy and chemotherapy, cause DNA damage. Signalling of this damage to DNA repair enzymes, and other enzymes that arrest cellular proliferation to allow time for repair, represents a potential mechanism of resistance to anticancer therapy. The nuclear enzymes, poly(ADP-ribose) polymerase-1 and -2 (PARP-1 and PARP-2) bind principally to DNA single strand breaks (SSB), stimulating PARP catalytic activity signalling the recruitment of base excision repair (BER) proteins to the DNA break and effecting repair. DNA double strand breaks (DSB) are signalled XRCC4 Ligase IV Figure 1: DNA single strand breaks (SSB) are repaired by PARP. ATM signals DNA double strand breaks (DSB) to cell cycle checkpoints and repair by DNA-PK dependent NHEJ or HR. Poly(ADP-ribose) polymerase inhibitors The compelling evidence for the role of PARP in the cellular response to genotoxic stress was the stimulus to develop 15 Drug Development – Pre-clinical Pharmacology inhibitors as therapeutic agents to potentiate DNA-damaging anticancer therapies. We have developed highly potent PARP inhibitors (Ki <5 nM) using structure activity relationships (SAR) and crystal structure analysis in collaboration with Agouron Pharmaceuticals, later Pfizer GRD. These PARP inhibitors significantly enhance the in vitro cytotoxicity of DNA damaging agents that are repaired by BER: monofunctional alkylating agents e,g. temozolomide, topoisomerase I poisons and ionising radiation used in the treatment of cancer. PARP inhibitors increase the antitumour activity of these three classes of anticancer treatments in vivo, in some models resulting in complete tumour regression. On the basis of these extremely promising preclinical data, clinical trials with a PARP inhibitor, in combination with temozolomide, commenced in June 2003 under the auspices of Cancer Research UK. Interestingly, cells defective in DNA DSB repair by the homologous recombination (HR) repair pathway are killed by concentrations of PARP inhibitors that are non-toxic to normal cells. This is of particular relevance in cancer because loss of BRCA1 and BRCA2, which are components of HR, is associated with hereditary (and some sporadic cases) of breast, ovarian, pancreatic and prostate cancer. This suggests that PARP inhibition may represent a truly tumour-specific, non-toxic, therapeutic manoeuvre for the treatment of such cancers. Our evidence also suggests PARP-1 competes with DNA-PK for double-strand ends and that inhibition of one enzyme blocks the activity of the other. cytotoxicity and antitumour activity. Following extensive preclinical evaluation, lead compounds have been identified for clinical evaluation. The PARP, ATM and DNA-PK inhibitors are currently under investigation in models of paediatric solid tumours– see section by Dr Steve Clifford. Inhibitors of nucleoside transport Dipyridamole, a cardiovascular agent, inhibits nucleoside transport and can therefore prevent salvage of exogenous nucleosides and rescue from antifolate cytotoxicity. We have synthesised a number of analogues of dipyridamole that prevent nucleoside uptake into cells and enhance the cytotoxicity of the antifolate, pemetrexed. These analogues have improved pharmacokinetics compared to dipyridamole and inhibit nucleoside uptake into tumours. We are currently evaluating their efficacy in combination with antifolates in tumour models. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, Kyle S, Meuth M, Curtin NJ, Helleday T. “Specific killing of BRCA2deficient tumours with inhibitors of poly(ADP-ribose)polymerase.” Nature (2005) 434: 913-917. Calabrese CR, Almassy R, Barton S, Batey MA, Calvert AH, CananKoch S, Durkacz BW, Hostomsky Z, Kumpf RA, Kyle S, Li J, Maegley K, Newell DR, North M, Notarianni E, Stratford IJ, Skalitzky D, Thomas HD , Wang L-Z, Webber SE, Williams KJ, Curtin NJ. “Preclinical evaluation of a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor, AG14361, with significant anticancer chemoand radio-sensitization activity.” Journal of the National Cancer Institute (2004) 96: 56-67. Hickson I, Zhao Y, Richardson CJ, Green SJ, Martin MNB, Orr AI, Reaper PM, Jackson SP, Curtin NJ, Smith GCM. “Identification of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM.” Cancer Research (2004) 64: 9152-9159. Veuger SJ, Curtin NJ, Richardson CJ, Smith GC, Durkacz BW. “Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADPribose) polymerase-1.” Cancer Research (2003) 63: 6008-15. DNA-PK and ATM inhibitors DNA double strand breaks (DSBs) are the most cytotoxic lesions induced by ionising radiation (IR) and topoisomerase II poisons used in cancer therapy. ATM and DNA-PK activity are required for efficient signalling to cell cycle checkpoints and repair of DNA DSBs. Novel potent ATP competitive inhibitors specific for ATM and DNA-PK have been developed in collaboration with KuDOS Pharmaceuticals. DNA-PK inhibitors increase the persistence of DNA DSB induced by topoisomerase II poisons and ionising radiation (IR) and both ATM and DNA-PK inhibitors increase topoisomerase II poison and IR -induced 16 Dr Barbara Durkacz Radio- and chemo-therapy acts primarily by damaging the DNA in tumour cells. Resistance to such treatment is mediated by the activity of DNA repair and signalling pathways. We are exploring the use of novel inhibitors of these pathways to sensitise patient-derived leukaemia cells and breast cancer cell lines. NICR Team: Elaine Willmore, Stephany Veuger, Clark Crawford, Martha Watson and in close collaboration with Nicola Curtin and Mike Tilby. to treat breast cancer and chronic lymphocytic leukaemia (CLL). In addition, we are investigating the underlying molecular mechanisms by which the inhibitors act. We have established assays to measure the activities of specific DNA repair pathways, e.g. DNA-PK enzyme activity, and the formation of H2AX foci as a measure of DNA double strand breaks (DSBs). We are also involved in a translational research project, demonstrating that both DNA-PK and ATM inhibitors sensitise both leukaemic cell lines and CLL patient-derived lymphocytes to chemotherapeutic drugs commonly used to Introduction The majority of currently used chemotherapy drugs act by damaging DNA. Tumour cells respond to DNA damage by the activation of DNA repair enzymes, including poly(ADPribose) polymerase (PARP-1), DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia-mutated (ATM). These enzymes not only mediate DNA repair processes that enable cells to survive the damage, but also activate cell cycle checkpoints and transcription factors such as NF-kB, which switches on the transcription of genes encoding proteins that prevent apoptosis (cell death). Significantly, NF-kB is aberrantly activated in many solid tumours and leukaemias. A 1.25 1.00 0.75 0.50 0.25 0.00 DNA-PK Inhibitor + Inhibitor - Inhibitor 1.00 ATM Inhibitor + Inhibitor - Inhibitor Viability Viability 0.75 0.50 0.25 0.00 Development of novel DNA repair inhibitors PARP-1, DNA-PK and ATM inhibitors have been developed within the Institute for clinical use. We have been closely involved in the target identification and preclinical evaluation stages of the novel inhibitors. Our hypothesis is that these inhibitors will sensitise tumour cells to chemotherapeutic drugs and ionising radiation, both by inhibiting specific DNA repair pathways, and by preventing the activation of NF-kB. B 0.00 0.10 0.20 0.50 1.00 0.00 0.10 0.20 0.50 1.00 Fludarabine μM Untreated + Fludarabine Fludarabine μM + Fludarabine + DNA-PK Inhibitor Untreated + Fludarabine + Fludarabine + DNA-PK Inhibitor Preclinical evaluation of inhibitors Our current research is aimed at assessing the ability of these novel inhibitors to increase the efficacy of drugs used Figure 1: A: CLL lymphocytes treated with fludarabine show increased chemosensitivity presence of DNA-PK or ATM inhibitors. B: The DNA-PK inhibitor increases fludarabine-induced γH2AX foci (stained red). 17 The Roles of DNA Damage-Activated Repair & Signalling Enzymes in Chemotherapeutic Response In addition, we are researching the effects of the inhibitors when combined with ionising radiation in breast cancer cell lines. NF-kB is very commonly aberrantly activated in breast cancer, and radiotherapy is important in the treatment of this disease. We have shown that ionising radiation treatment activates NF-kB in breast cancer (even when NF-kB levels are already high). The DNA repair inhibitors not only sensitised breast cancer cells to ionising radiation, but also prevented NF-kB activation. Significantly, by using paired cell lines proficient or deficient in NF-kB activity, we are able to demonstrate that the inhibitors only sensitised cells to ionising radiation in the cell line with functional NF-kB (Figure 2). This has led us to hypothesise that the inhibitors mediate cell death following DNA damage primarily by inhibition of NF-kB activation, and will have important ramifications in the design of clinical protocols using these inhibitors. 8 Relative luciferase activity 50000 40000 30000 20000 10000 0 Relative luciferase activity A1 IR IR + AG14361 A2 12500 10000 7500 5000 2500 0 0 IR IR + AG14361 0 5 10 20 50 5 10 20 50 Dose (Gy) Dose (Gy) B1 Survival: %Control 100 B2 100 10 Survival: %Control IR IR + AG14361 10 IR IR + AG14361 6 7 8 1 0 1 2 3 4 5 6 7 1 0 1 2 3 4 5 Gray Gray Figure 2: The effects of a PARP-1 inhibitor on DNA damage-activation of NFκB. A: The PARP-1 inhibitor AG14361 prevents induction of NF-κB dependent luciferase activity in breast cancer cell lines following ionising radiation. A1, MDA-MB-231 cells; A2, T47D cells. B: The PARP-1 inhibitor potentiates IR-induced cytotoxicity in B1, p65 +/+ (NF-kB proficient), but not B2, p65-/- (NF-kB deficient) cell lines. Willmore E, de Caux S, Sunter NJ, Tilby MJ, Jackson GH, Austin CA, Durkacz BW. “A novel DNA-dependent protein kinase inhibitor, NU7026, potentiates the cytotoxicity of topoisomerase II poisons used in the treatment of leukemia.” Blood (2004) 103: 4659-4665. Veuger SJ, Curtin NJ, Smith GC, Durkacz BW. “Effects of novel inhibitors of poly(ADP-ribose) polymerase-1 and the DNAdependent protein kinase on enzyme activities and DNA repair.” Oncogene (2004) 23: 7322-7329. Veuger SJ, Curtin NJ, Richardson CJ, Smith GC, Durkacz BW. “Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1.” Cancer Research (2003) 63: 6008-6015. Cowell I, Durkacz BW, Tilby M. “Sensitization of breast carcinoma cells to ionizing radiation by small molecule inhibitors of DNAdependent protein kinase and ataxia telangiectsia mutated.” Biochemical Pharmacology (2005) 71: 13-20. treat leukaemias (topoisomerase II poisons and nucleoside analogues such as fludarabine). Importantly, lymphocytes from patients with chemo-resistant disease were also sensitised by the inhibitors. Furthermore, using H2AX foci we have demonstrated that the DNA-PK inhibitor increased the level of fludarabine-induced DSBs, indicating that the inhibitor prevented DSB repair (Figure 1). Current research is investigating the effects of the inhibitors in poor prognosis patients known to have mutations in p53 or ATM. We have recently set up collaborations with Dr Pettitt and Dr Sherrington (Royal Liverpool University Hospital), and Dr Stankovic (University of Birmingham) to facilitate this research. 18 Dr Richard Edmondson The biology of ovarian cancer remains poorly understood. There is strong epidemiological evidence to link hormones with the development of ovarian cancer but the mechanisms underlying this are not understood. Moreover, the use of antihormonal treatment in ovarian cancer has not been successful. We therefore wish to investigate the effects and role of hormones on both ovarian cancer cells and also human ovarian surface epithelial cells, the tissue of origin of ovarian cancer. NICR Team: Ali Kucukmetin, Ann Fisher, Sarah Wilkinson and in close collaboration with Hing Leung, Craig Robson and Hilary Calvert. The role and effects of the androgen receptor co regulator, Tip 60, in determining hormone sensitivity of epithelial ovarian cancer Steroid hormone coactivators could play a role in the presence or the development of hormone resistance status of ovarian cancer. Coactivators interact directly with the activation domain of a nuclear receptor, leading to enhancement of receptor activation function. They also interact with components of the basal transcription machinery. Tip60 coactivates androgen receptors (AR) and potentiates the transcriptional activity of androgens. Some experiments show that Tip60 can also enhance transactivation through the oestrogen receptor and progesterone receptor in a liganddependent manner; thus identifying Tip60 as a nuclear hormone receptor coactivator. Upregulation of Tip60 mRNA and protein expression have been demonstrated in prostate cancer combined with a shift in Tip60’s cellular distribution from cytoplasmic to nuclear localisation as the disease progresses towards hormone resistance. We have demonstrated the presence of Tip60 at both the mRNA and the protein levels in ovarian cancer samples. Furthermore, using a tissue microarray of ovarian cancer samples that we have constructed we have examined Tip60 expression in relation to stage, grade and survival and demonstrated that Tip 60 expression correlates inversely with grade of tumour but not with overall survival. Our aims are firstly, investigate the effects of androgen administration and depletion on the level of Tip60 expression and distribution in ovarian cancer cells; secondly, to investigate Introduction Ovarian cancer remains a major health problem with over 6,000 cases and 4,500 deaths each year in the UK. There is strong epidemiological evidence to link hormones including androgens, oestrogens and gonadotrophin releasing hormone with the development of ovarian cancer but the exact role of these hormones is not fully understood. Moreover the use of antihormone therapy in ovarian cancer has been disappointing with poor results in phase II studies. We have therefore developed an interest in understanding the role of hormones in both ovarian cancer cells and also human ovarian surface epithelial cells, the cells of origin of ovarian cancer. We have previously established primary cultures of human ovarian surface epithelial (OSE) cells from patients undergoing surgery for benign disease. We have used these cells to demonstrate that OSE cells can respond to both androgens and gonadotrophins. OSE cells have a very short lifespan in the laboratory and we have therefore created immortalised cell lines from four of these cultures using a temperature sensitive SV40 large T antigen and hTERT. Latterly we have constructed a tissue microarray of 167 ovarian cancer cases with complete clinical data allowing us to correlate protein expression with clinical outcome. We are currently undertaking three projects investigating the role of hormones in ovarian cancer: 19 Understanding the Role and Effects of Hormones in Ovarian Cancer This project aims to investigate the control and effects of the growth factor pathway comprising hepatocyte growth factor and its receptor met with particular reference to the hormones contained within the underlying follicle. An investigation of the role of gonadotrophin releasing hormone II in ovarian cancer Recent work has identified a second human isoform of gonadotrophin releasing hormone known as GnRH II. Evidence has suggested that GnRH II may act to inhibit the growth of ovarian cancer cells in culture. We have investigated the expression of GnRH II in ovarian cancer samples using real time RT-PCR and immunohistochemistry and found a relative down regulation of expression of GnRH II compared to normal ovarian surface epithelium. This project aims to investigate the significance of this finding by firstly, examining the expression of the type 1 and type 2 GnRH receptors and to correlate this expression with GnRH II expression and clinical outcome; secondly, examine the mechanisms of GnRH II downregulation in ovarian cancer. An ovarian surface epithelial cell, the cell of origin of ovarian cancer, demonstrating cytokeratin 19 expression the effects of androgens on normal and malignant ovarian cells in the presence and absence of Tip60. An investigation of HGF/met expression and activity in the human ovarian surface epithelium and human epithelial ovarian cancer There is an association between the lifetime number of ovulations and the subsequent risk of developing ovarian cancer. There is also evidence to suggest that cell proliferation of the OSE occurs before ovulation rather than in response to damage. It is probable that this proliferation is mediated through an indirect mechanism; cell proliferation only occurs in the OSE cells directly overlying the developing follicle suggesting that local paracrine effects rather than systemic signalling control this process. The follicle itself contains supraphysiological concentrations of hormones including high levels of oestrogen. It is possible therefore that cell proliferation occurs in response to these local hormones contained in the follicle or other paracrine factors released from the granulosa cells which line the follicle and underlie the OSE. Edmondson RJ, Monaghan JM, Davies BR. “Gonadotrophins mediate DNA synthesis and protection from spontaneous cell death in human ovarian surface epithelium.” International Journal of Gynaecological Cancer (2005) 16: 171-177. Davies BR, Steele IA, Edmondson RJ, Zwolinski SA, Saretzki G, von Zglinicki T, O’Hare MJ. “Immortalisation of human ovarian surface epithelium with telomerase and temperature-senstitive SV40 large T antigen.” Experimental Cell Research (2003) 288: 390-402. Edmondson RJ, Monaghan JM, Davies BR. “The human ovarian surface epithelium is an androgen responsive tissue.” British Journal of Cancer (2002) 86: 879-885. Edmondson RJ, Monaghan JM. “The epidemiology of ovarian cancer.” International Journal of Gynaecological Cancer (2001) 11: 423-429. 20 Prof Roger Griffin As director of the Medicinal Chemistry section of the NICR, I am involved in the management of the drug development projects, with particular emphasis on the design and synthesis of novel anticancer agents. My direct areas of research centre on the development of small-molecule inhibitors of kinases, including CDK, DNA-PK and ATM, and the medicinal chemistry of pyrimidopyrimidine inhibitors of nucleobasenucleotide transport. NICR Team: Celine Cano-Soumillac, Celine Roche, Eric Valeur, Karen Haggerty, Sonsoles Rodriguez-Aristegui, Francesco Marchetti, Anna Watson, Christopher Wong, and in close collaboration with Hilary Calvert, Herbie Newell, Ian Hardcastle, Ross Maxwell, Ruth Plummer and Nicola Curtin. of DNA DSB repair, and ATM kinase responds to ionizing radiation-induced DSBs by signalling to downstream response factors involved in cell cycle regulation and DNA repair. DNA-PK and ATM kinase both recognise and initiate repair of DNA strand breaks produced by ionising radiation and certain cancer chemotherapeutic agents. Selective small-molecule inhibitors of DNA-PK and ATM may, therefore, have a potential therapeutic role as radio- and chemo-sensitizers in the treatment of cancer. Our studies are conducted in collaboration with KuDOS Pharmaceuticals. Introduction The design and synthesis of new molecules for biological testing is performed by the Medicinal Chemistry group. We are investigating drug-targets involved in either a) the drive, progression and spread of cancer, or b) resistance to cancer therapies, such as radiation and cytotoxic chemotherapy. Current targets include enzymes, e.g. kinases (DNA-PK, CDK) and acetyl transferases (e.g. Tip60), transporters (e.g. the nucleoside transporter) or protein-protein interactions (e.g. MDM2-p53). Where possible, structure-based drug design approaches are used, based on the X-ray crystal structure or NMR structure of the target protein. High-throughput synthesis methods are used frequently to optimise lead structures. For Pre-clinical Pharmacology see entries by Nicola Curtin and Herbie Newell. I) DNA-PK Inhibitors Research has centred on the development of potent and specific small molecule inhibitors of DNA-PK based on the lead inhibitor LY294002. A systematic structural modification of the core chromenone pharmacophore has enabled the elucidation of structure-activity relationships (SARs) for DNA-PK inhibition by this compound class, resulting in the identification of several interesting lead inhibitors based on benzochromen-4- Design of inhibitors of DNA damage-activated kinases The phosphatidylinositol (PI) 3-kinase related kinases (PIKKs) are a family of serine/threonine kinases. Two prominent members of the PIKK family, DNA-dependent protein kinase (DNA-PK) and ATM (ataxia telangiectasia mutated) kinase, play key roles in the cellular response to DNA damage, through DNA double-strand break detection and repair. DNA-PK is a key component of the non-homologous end joining (NHEJ) process S O N O O NU7441 NU7441 IC50 = 12 nM IC50 = 12 nM Figure 1: Crystal structure of the DNA-PK inhibitor NU7441 - For further information see Griffin et al. 2005. 21 Drug Development – Medicinal Chemistry Benzylamino confers activity comparable to piperidino Electron-donating groups enhance activity X Additional hydroxylation or chain lengthening does not enhance activity R alk Hydroxyl or alkoxy substituent favoured O N O N N Me Alkylation increases potency may restore AGP binding OH N N N N OH Confers potency and AGP binding in piperidino series tertiary amine associated with AGP binding? O for N replacement generally reduces activity Figure 2: Putative structure-activity relationships for pyrimidopyrimidine nucleoside transport inhibitors - For further information see Curtin et al. 2004 and pharmacokinetic properties of the drug. In particular, the avid binding of DP to the acute phase protein α1-acid glycoprotein (AGP) is thought to contribute to the disappointing in vivo activity observed. This problem was addressed by modifying the substituent pattern around the core pyrimidopyrimidine template of DP, with a view to determining structure-activity relationships for both nucleoside transport inhibition and AGP binding (Figure 2). These studies have resulted in the identification of a series of 4,8dibenzylaminopyrimidopyrimidines, which are at least as potent as DIP as nucleoside transport inhibitors, but that retain activity in the presence of supraphysiological concentrations of AGP. The development of water-soluble derivatives of the most promising analogues is underway, as a prelude to conducting more detailed biological studies. Griffin RJ, Henderson A, Curtin NJ, Echalier A, Endicott JA, Hardcastle IR, Newell DR, Noble MEM, Wang L-Z, Golding BT. “Searching for Cyclin-Dependent Kinase Inhibitors Using a New Variant of the Cope Elimination.” Journal of the American Chemical Society (2006) 128: 6012-6013. Griffin RJ, Fontana G, Golding BT, Guiard S, Hardcastle IR, Leahy JJJ, Martin N, Richardson C, Rigoreau L, Stockley ML, Smith GCM. “Selective benzopyranone and pyrimido[2,1-a]isoquinolin-4-one inhibitors of DNA-dependent protein kinase: synthesis, structureactivity studies, and radiosensitization of a human tumor cell line in vitro.” Journal of Medicinal Chemistry (2005) 48: 569-585. Davies TG, Bentley J, Arris CE, Boyle FT, Curtin NJ, Endicott JA, Gibson AE, Golding BT, Griffin RJ, Hardcastle IR, Jewsbury P, Johnson LN, Mesguiche V, Newell DR, Noble MEM, Tucker JA, Wang LZ, Whitfield HJ. “Structure-based design of a potent purine-based cyclin-dependent kinase inhibitor.” Nature Structural Biology (2002) 9: 745-749. Curtin NJ, Barlow HC, Bowman KJ, Calvert AH, Davison R, Golding BT, Huang B, Loughlin PJ, Newell DR, Smith PG, Griffin RJ. “Resistance-modifying agents. 11. pyrimido[5,4-d]pyrimidine modulators of antitumor drug activity; synthesis and structureactivity relationships for nucleoside transport inhibition and binding to α1-acid glycoprotein (AGP). Journal of Medicinal Chemistry (2004) 47: 4905-4922. one and pyrimidoisoquinolin-4-one templates. Guided by these initial results, a focused compound library approach was employed to probe the DNA-PK ATP-binding domain. These efforts were rewarded by the identification of the potent and highly selective ATP-competitive DNA-PK inhibitor NU7441 (IC50 = 12 nM) (Figure 1). Studies are underway to optimise the physicochemical and biological properties of this inhibitor class, as a prelude to selecting a clinical candidate. II) ATM Kinase Inhibitors Studies in this area originated from the observation that several simple pyran-4-one derivatives, developed as candidate DNA-PK inhibitors, exhibited weak activity as ATM kinase inhibitors. Utilising a solution phase multiple-parallel synthesis approach, a library of pyranone derivatives was prepared and evaluated for ATM kinase-inhibitory activity, resulting in the identification of KU-0055933 as a remarkably potent and selective ATM kinase inhibitor (IC50 = 10 nM). Lead optimisation studies have been completed for this inhibitor class, and a clinical candidate will shortly be selected. Synthesis of inhibitors of nucleoside transport The therapeutic potential of dipyridamole (DP) as an agent to potentiate the cytotoxicity of antimetabolite antitumour agents, has been overshadowed by the poor physicochemical 22 Prof Andy Hall Relapsed acute lymphoblastic leukaemia (ALL) is a major cause of death from malignancy in childhood. We aim to improve the outcome of these patients through the identification, validation and exploitation of novel targets for therapy, based on an improved knowledge of the biology of relapsed disease. NICR Team: Niki Brown, Marian Case, Sally Coulthard, Jose Luis Rodrigues de Brito, Sotiris Georgantopoulos, Linda Hogarth, Elizabeth Matheson, Lynne Minto, Lindsey Nicholson, Sarina Sulong and in close collaboration with Julie Irving and Josef Vormoor. Introduction Acute Lymphoblastic Leukaemia (ALL) is the most common form of malignancy in children. In this disease there is uncontrolled proliferation of lymphoid precursors in the bone marrow. Fortunately the introduction of intensive, multi-agent chemotherapy has produced dramatic improvement in the outcome of children who develop ALL, such that more than 80% with standard-risk disease now achieve long-term remission. However, relapsed ALL remains the most common cause of death from malignancy in childhood and there has been little improvement in outcome in this group of patients over the past 10 years. With the introduction of more intensive consolidation and continuation therapy the number of patients who can be salvaged using re-induction with standard therapies has declined. This suggests that relapsed ALL should be regarded as a different, less chemocurable form of ALL and treated with novel agents. We have been studying the biology of relapsed ALL with the aim of identifying novel targets for therapy. Studies performed as part of our ongoing programme have concentrated on candidate genes, selected on existing knowledge of their role in the determination of drug sensitivity in cell line models. Overall, this approach has identified relevant mechanisms in cell lines but generally these have not found to exist in patient cells, reinforcing the need to study primary samples wherever possible. We have produced evidence to suggest that the reason for this is that many cell lines used for in vitro studies are mismatch repair deficient, with a propensity to develop mutations not seen in vivo. However, most cases of childhood ALL are mismatch repair competent. For example, many reports have suggested that glucocorticoid resistance is frequently due to mutations in the glucocorticoid receptor. We confirmed that this is the case in the CCRF-CEM cell line, shown by our group to be mismatch repair deficient, but could find only one example in 50 cases of relapsed ALL. Screens for mutations of topoisomerase II alpha, the reduced folate carrier and MDR1 have failed to produce evidence of abnormalities in patient cells and we have found no clear correlation between asparaginase synthase expression and response in clinical samples, in contrast with established cell line models. The failure to find mutations or altered expression using the Figure 1: Fluorescence in situ hybridisation analysis of p16. Green probes show chromosome 9 centromeres. Red probes show p16 locus 23 The Biology of Relapsed Childhood Leukaemia candidate gene approach has led us to adopt a genome-wide approach to the study of relapsed ALL. We have done this at the DNA level, using Affymetrix single nucleotide polymorphism (SNP) mapping arrays, both 10k and 50k formats. SNPs have been identified throughout the genome and can be used as mapping tools to detect loss of heterozygocity (LOH) and alterations in gene copy number. Samples have been studied at presentation, after 8 days of drug exposure in vivo and at the time of overt relapse. SNP mapping has revealed areas of amplification and deletion not seen by cytogenetics. Notably, acquired isodisomy, in which there is LOH but no reduction in copy number occurred in more than 20% of cases at presentation indicating that this is a more frequent mechanism for allelic imbalance in childhood ALL than previously suspected. This was found to affect both whole chromosomes and chromosomal segments. Studies of paired samples have shown progressive changes, indicating that this approach may be used to identify key genes influencing chemoresisitance. Figure 2: Allelic imbalance affecting chromosome 9. A: A case with 9p deletion, B: A case with acquired isodisomy affecting 9p. The development of new therapies in relapsed ALL The lack of abnormalities in specific drug-metabolising enzymes or in the targets of drug action in clinical samples may be due in part to the fact that chemotherapy is used in complex combinations rather than as single agents. This concept is supported by evidence from early ALL trials where glucocorticoid receptor expression could be linked to clinical outcome. This relationship was lost with the introduction of more intensive therapy. An analogous situation is observed in chronic myleloid leukaemia treated with single agent imatinib where resistance due to mutations in the kinase domain is commonly observed. Our preliminary results using SNP mapping array analysis indicates that alterations in the control of the cell cycle may be important in the determination of chemosensitivity in vivo and may be more important in determining chemosensitivity than direct effects on drug metabolism or drug targets. Mapping array data is being combined with the analysis of mRNA levels in leukaemic cells to identify new drug targets which can be exploited in the treatment of relapsed ALL. Areas of gene amplification will be explored for the presence of genes encoding enzymes, including kinases, which can be inhibited with low molecular weight inhibitors. In addition areas of acquired isodisomy will be screened for the presence of mutations in key genes controlling cell proliferation of death as this is a recognised mechanism for malignant transformation. Irving JA, Bloodworth L, Bown NP, Case MC, Hogarth LA, Hall AG. “Loss of heterozygosity in childhood acute lymphoblastic leukemia detected by genome-wide microarray single nucleotide polymorphism analysis.” Cancer Research (2005) 65: 3053-3058. Irving JA, O'Brien S, Lennard AL, Minto L, Lin F, Hall AG. “Use of denaturing HPLC for detection of mutations in the BCR-ABL kinase domain in patients resistant to imatinib.” Clinical Chemistry (2004) 50: 1233-1237. Matheson EC and Hall AG. “Assessment of mismatch repair function in leukaemic cell lines and blasts from children with acute lymphoblastic leukaemia.” Carcinogenesis (2003) 24: 31-38. Leslie M, Case MC, Hall AG, Coulthard SA. “Expression levels of asparagine synthetase in blasts from children and adults with acute lymphoblastic leukaemia.” British Journal of Haematology (2006) 132: 740-742. 24 Dr Ian Hardcastle The Drug Development group is a multidisciplinary team of biologists, pharmacologists and chemists. We develop novel drug treatments for cancer, based on an understanding of the key differences between normal and cancer cells. We are investigating drug-targets involved in either a) the development, progression and spread of cancer, or b) resistance to cancer therapies, such as radiation and cytotoxic chemotherapy. NICR Team: Celine Cano-Soumillac, Celine Roche, Eric Valeur, Karen Haggerty, Sonsoles Rodriguez-Aristegui, Francesco Marchetti, Anna Watson, Christopher Wong and in close collaboration with Roger Griffin, Herbie Newell, Ruth Plummer, Nicola Curtin, Craig Robson and Hilary Calvert. work in the NICR has validated Tip60 as a novel therapeutic target for prostate cancer. The principal aim of this project is to develop potent, selective, small-molecule inhibitors of Tip60 for the treatment of prostate cancer. In collaboration with OSI Pharmaceuticals, we have undertaken a high throughput screening programme for the identification of Tip60 inhibitors. Initial screening resulted in the identification of a number of novel series of structural leads with promising activity against Tip60. We are currently confirming the activity of the screening hits and expanding our understanding of their structure-activity relationships in this series of compounds. Introduction The design and synthesis of new molecules for biological testing is performed by the Medicinal Chemistry group. We are investigating drug-targets involved in either a) the drive, progression and spread of cancer, or b) resistance to cancer therapies, such as radiation and cytotoxic chemotherapy. Current targets include enzymes, e.g. kinases (DNA-PK, CDK) and acetyl 17 transferases (e.g. Tip60), transporters (e.g. the nucleoside transporter) or protein-protein interactions (e.g. MDM2-p53). Where possible, structure-based drug design approaches are used, based on the X-ray crystal structure or NMR structure of the target protein. High-throughput synthesis methods are used frequently to optimise lead structures. For Pre-clinical Pharmacology see entries by Nicola Curtin and Herbie Newell. A B MeO HO OMe Cl O N O Inhibitors of Tip60 acetyl transferase Prostate cancer accounts for 12% of all cancer deaths in the UK. The majority of patients initially respond to hormone-based therapies. However, patients invariably relapse with hormonally insensitive disease, which is resistant to most cytotoxic therapy, and so represents a major unmet clinical need. Tip60 histone acetyltransferase (HAT) has recently been identified as a coactivator of the androgen receptor (AR), a process implicated in hormone insensitive disease. Recent NU8231 IC50 = 5 mM Figure 1: A: X-ray structure of MDM2 with p53 bound showing key amino acid residues (purple); B: Chemical structure of NU8231, an isoindolinone inhibitor of the MDM2-p53 interaction – For further information see Hardcastle et al. 2005. 25 Drug Development – Medicinal Chemistry MDM2 p53* transcription translation MDM2 p53 p53 p53 MDM2 proteasome Figure 2: Activation of p53 by DNA damage leads to transcription of MDM2, p53 inactivation by binding to MDM2, and destruction by the proteasome - For further information see Hardcastle et al. 2005. with high expression of MDM2 protein and suppression of functional p53, promoting transformation and uncontrolled tumour growth. The rates of MDM2 amplification have been reported to be as high as 30% in soft tissue sarcomas. We have identified inhibitors of the MDM2-p53 interaction based on an isoindolinone scaffold. In collaboration with De Novo Pharmaceuticals, we employed computational screening methods to optimise the isoindolinones, resulting in the identification of inhibitors with improved potency, exemplified by NU8231 (IC50 = 5 μM) (Figure 2). Further studies showed that NU8231 had activity in intact cells consistent with the disruption and/or prevention of MDM2-p53 binding. Further SAR studies, guided by NMR structural studies with selected isoindolinones, have resulted in the identification of inhibitors with improved potency. Inhibitors of the MDM2-p53 interaction The p53 tumour suppressor protein plays a central role in protecting the genome from replicating in a damaged form. Cellular stresses, such as hypoxia and DNA damage, activate, and increase levels of p53. A number of genes that govern progression through the cell cycle, the initiation of DNA repair, and programmed cell death are regulated by p53. The activity of p53 is tightly regulated by the MDM2 protein, the gene for which is itself positively regulated by direct p53 transcriptional activation. MDM2 binds directly to p53, blocking its transcriptional activity and ubiquitylating the resulting complex, which is exported from the nucleus and destroyed by the proteasome. In normal cells the balance between active p53 and inactive MDM2bound p53 is maintained by this negative autoregulatory feedback loop (Figure 1). The X-ray crystal structure of MDM2 bound to a p53 peptide corresponding to the transactivation loop, reveals a hydrophobic pocket on the surface of MDM2, into which three key residues of p53 bind. Approximately 7% of human tumours overall show evidence of amplification of the MDM2 gene, which is associated Hardcastle IR, Cockcroft X, Desage El-Murr M, Leahy JJJ, Stockley M, Golding BT, Rigoreau L, Richardson C, Smith GCM, Griffin RJ. “Discovery of potent chromen-4-one inhibitors of the DNAdependent protein kinase (DNA-PK) using a small-molecule library approach.” Journal of Medinical Chemistry (2005) 48: 7829-7846. Hardcastle IR, Ahmed SU, Atkins H, Farnie G, Golding BT, Griffin RJ, Guyenne S, Hutton C, Källblad P, Kemp SJ, Kitching MS, Newell DR, Norbedo S, Northen JS, Reid RJ, Saravanan K, Willems HMG, Lunec J. “Isoindolinone based inhibitors of the MDM2-p53 protein-protein interaction” Bioorganic and Medicinal Chemistry Letters (2005) 15: 1515-1520. Hardcastle IR, Arris CE, Bentley J, Boyle FT, Chen Y, Curtin NJ, Endicott JA, Gibson AE, Golding BT, Griffin RT, Jewsbury P, Menyerol J, Mesguiche V, Newell DR, Noble MEM, Pratt DJ, Wang L-Z, Whitfield HJ. “N2-Substituted-O6-cyclohexylmethylguanine Derivatives: Potent Inhibitors of Cyclin-Dependent Kinases 1 and 2.” Journal of Medicinal Chemistry (2004) 47: 3710-3722. Davies TG, Bentley J, Arris CE, Boyle FT, Calvert AH, Curtin NJ, Endicott JA, Gibson AE, Golding BT, Griffin RJ, Hardcastle IR, Jewsbury P, Johnson LN, Mesguiche V, Newell DR, Noble MEM, Tucker JA, Wang L, Whitfield HJ. ‘Structure-based development and cellular pharmacology of a potent purine-based inhibitor of cyclin dependent kinases 1 and 2’ Nature Structural Biology (2002) 9: 745-749. 26 Mr Rakesh Heer Prostate cancer is a major clinical issue with abnormal stem cell growth considered to be a key underlying mechanism. Our research interest is to characterise the molecular pathways involved in the regulation of adult prostate stem cells through various degrees of maturation as this could provide an insight into how these processes may contribute to diseases of the prostate and outline new therapeutic targets. NICR Team: In close collaboration with Hing Leung and Craig Robson. the role of the ECM and its interaction with growth factors in the context of differentiation in the prostate. Introduction Adult stem cells are self-renewing cells that can differentiate and proliferate to maintain homeostasis of a particular tissue or organ. In the prostate, abnormal regulation of epithelial stem differentiation is thought to contribute towards both benign and malignant conditions in the human prostate. The prostate epithelium comprises of an immature basal layer and a differentiated luminal layer with specialised secretory functions. The basal layer contains the stem cell compartment as it can give rise to the mature luminal cells. The basal layer resides on the collagen rich basement membrane and this interaction is thought to be key in maintaining this population. The stem cell enriched basal layer can be isolated by its high expression of α2β1 integrin - a cell surface adhesion molecule that specifically regulates adhesion onto type-1 collagen. Furthermore, we have previously demonstrated that integrin α2β1 high population can be sub divided into stem (α2β1hi CD133+) cells and, its immediate immature progeny, the transient amplifying population (TAP) (α2β1hi CD133-). The molecular mechanism(s) controlling the commitment and regulation of these cells through various compartments during differentiation remains unclear and forms the focus of our studies. ECM in stem cell regulation Integrins are important cell surface adhesion molecules that bind cells to the ECM and are involved in maintaining stem cells in their niches in a number of tissues. In keratinocytes, binding of β1 integrins to the extracellular matrix inhibits terminal differentiation and maintains the epidermal stem cell compartment. This finding is of particular relevance to the prostate, as epithelial stem and (TAP) cells, are rich in α2β1 integrin expression. 100 % of cell expressing marker 80 60 stem cells 40 20 0 PAP CK18 AR α2β1 treated with KGF KGF + SB 202190 Markers of differentiation Prostate development Prostatic in utero development is dependent on the interplay between the epithelium, mesenchyme and extracellular matrix (ECM). Members of the fibroblast growth factor (FGF) family have been implicated in the control of prostate epithelial development. Of particular interest is the role of keratinocyte growth factor (KGF/FGF7) which is involved in the androgen regulation of murine prostate organogenesis. Little is known of Figure 1: KGF regulated differentiation in α2β1hi basal cells from human primary culture. Bar chart summarising the effect of KGF (and p38-MAPK inhibitor SB202190) on α2β1hi cell expression of differentiation specific markers (measured by FACS) New work from our laboratory demonstrates β1 integrin function is required for the maintenance of the basal prostatic The role of β1 integrin in regulating adult prostate epithelial differentiation 27 Human Prostate Epithelial Differentiation epithelial cells. Suppression of β1 integrin function by either methylcellulose suspension or, more specifically, β1 neutralising antibody induces differentiation, with associated expression of differentiation-specific markers - prostate acid phosphatase (PAP) and cytokeratin 18 (CK18). High AR expressing stromal cells 1 Testosterone The role of KGF in regulating adult prostate epithelial differentiation We show that treatment with KGF potently induces epithelial differentiation with concomitant suppression of α2β1 integrin expression as well as the induction of androgen receptor expression (AR). Specifically, p38-MAPK appears to be involved and the presence of SB202190, a p38 inhibitor, significantly blocks KGF-induced differentiation (figure 1). Furthermore, the expression of the high affinity receptor tyrosine kinase to KGF (FGFR2) is predominantly detectable in the α2β1hi CD133- TAP cells when compared to the stem cells (α2β1hi CD133+) - which would therefore be relatively unresponsive to the differentiating effect of KGF. Taken together, using a human primary culture model, we have demonstrated key roles for interaction between KGF and integrin-mediated function in the regulation of prostate epithelial differentiation. Our data supports a model where the α2β1 integrin maintains the prostatic epithelial basal cell compartment, with its expression negatively regulated by stroma-derived KGF (androgen regulated) acting through p38-MAPK (Figure 2). These findings add to the growing importance of β1 integrin in epithelial cell differentiation across a number of tissues. Other FGFs 2 3 FGFR2 II/IIIc STROMAL compartment KGF FGFR2 II/IIIb Figure 2: Model of KGF and androgen regulation of prostate development and differentiation. (1) Testosterone acts on the AR +ve stromal cell which leads to the production of KGF. (2) KGF (and FGF10) then acts in a paracrine fashion to stimulate the epithelial stem (and TAP) cell differentiation. (3) Testosterone itself has a direct role in maintaining the differentiated luminal cell. Proposed work In collaboration with Dr Gabriele Saretzki (Institute of Human Ageing, Newcastle University) we aim to generate an immortalised stem cell line by transfection of normal selected cells with hTERT, the catalytic subunit of human telomerase. The use of hTERT has proven to be a powerful tool for generating cell lines with an extended lifespan, but with very little additional changes. Characterisation of the established cell line will be performed to examine their in vitro and in vivo ability to regenerate prostate like gland as well as to differentiate under the appropriate conditions, such as stimulation by KGF or treatment with blocking antibody against a2b1 integrins. Using this in vitro model KGF treated and control cells, in addition to differentiation specific selections (stem vs TAP vs differentiated) from the generated cell line will be studied using the Affymetrix gene array (Human Genome UI22A system). This will outline additional mechanisms of stem cell regulation and potential targets for differentiation inducing treatments. Heer R, Collins AT, Robson CN, Shenton BK, Leung HY. “KGF suppresses a2b1 integrin function and promotes differentiation of the transient amplifying population in human prostatic epithelium.” Journal of Cell Science (2006) 119: 1416-1424. McKie AB, Douglas DA, Olijslagers S, Graham J, Omar MM, Heer R, Gnanapragasam VJ, Robson CN, Leung HY. “Epigenetic inactivation of the human sprouty2 (hSPRY2) homologue in prostate cancer.” Oncogene (2005) 24: 2166-2174. Heer R, Douglas D, Mathers ME, Robson CN, Leung HY. “Fibroblast growth factor 17 is over-expressed in human prostate cancer.” Journal of Pathology (2004) 204: 578-586. FGF 10 EPITHELIAL compartment Differentiation Stem cell Luminal cell 28 Dr Julie Irving Childhood acute lymphoblastic leukaemia (ALL) is the most common childhood malignancy and despite cure rates approaching 80%, remains the most common cause of death from malignancy in children. Our research aims to optimise chemotherapy so that survival rates are further increased and cure is attained with minimal toxicity. NICR Team: Linda Hogarth, Sally Coulthard, Elizabeth Matheson, Lynne Minto, Marian Case, Lindsay Nicholson, Jose Luis Rodrigues de Brito, Sarina Sulong, and in close collaboration with Andy Hall, Herbie Newell, Josef Vormoor, Chris Redfern and Hing Leung. Introduction ALL is a clonal disorder affecting the lymphoid lineage and is the most common malignancy in children, with between 400 and 450 new cases presenting each year in the UK. While the introduction and escalation of combination chemotherapy has led to an improvement in survival rates to over 80%, the rate of improvement now appears to have slowed and there are more deaths from relapsed ALL than from any other childhood malignancy. Although certain molecular features such as the presence of the Philadelphia chromosome or 11q23 abnormalities are associated with a poor prognosis, most deaths occur in patients who present with medium or low-risk disease. There is therefore an urgent need to improve our understanding of the mechanisms which underlie chemoresistance in patients without established poor-risk molecular abnormalities and to identify new targets for drugs used to treat children at relapse. In contrast, there are concerns that some children have very ‘low risk’ disease and may be cured with a less intensive chemotherapeutic regime than standard. proteins in haematological malignancies, either singly (e.g. Imatinib in chronic myeloid leukaemia) or in combination with standard chemotherapeutic drugs to potentiate the apoptotic response (e.g. PS-341 in multiple myeloma). This targeted therapeutic approach with novel small molecule inhibitors may be superior to more traditional chemotherapeutic agents both in terms of efficacy and toxicity. Candidate genes implicated in chemo resistance are sought in leukaemic blasts evading chemotherapeutic insult in vivo. Using four-colour fluorescent activated cell sorting (Figure 1), residual blasts are identified, isolated and mRNA expression The identification and validation of target genes which may allow chemotherapeutic intervention. New therapeutic opportunities are emerging using small inhibitory molecules which specifically target deregulated Figure 1: The identification and isolation of leukaemic blasts from bone marrow aspirates which have evaded 15 days of chemotherapeutic insult in vivo, by four colour fluorescent activated cell sorting. (Cells shown are gated on a CD19+ /CD34+ region). 29 Optimisation of Chemotherapy in Childhood Acute Lymphoblastic Leukaemia Wildtype G12V G13D G12D G12D Positive control than for the PCR-based method. Thus it is may prove a valuable, alternative/supplementary methodology in future clinical trials and our group is one of 6 laboratories in the UK involved in a feasibility study to address this issue. Related Projects The theme of targeted therapies for haematological malignancies is also addressed in a project in which FGFR3 has been validated as a target for therapeutic intervention in multiple myeloma patients bearing the t(4;14) translocation. This translocation is found in approximately 15% of multiple myeloma patients and results in the overexpression of the FGFR3, a receptor tyrosine kinase which appears critical to tumour cell survival. In a collaboration with Astex Therapeutics Limited (Cambridge, UK), a company which specialises in fragment-based drug design, novel small molecule inhibitors are being developed against FGFR1, 2 , 3 and 4 which should have a role in the treatment of multiple myeloma and other malignancies including prostate cancer – see entry by Herbie Newell. Irving JAE, Minto L, Hall AG. “Loss of heterozygosity and somatic mutations in the glucocorticoid receptor gene are rarely found at relapse in paediatric acute lymphoblastic leukaemia but can occur in a subpopulation early on in the disease course.” Cancer Research (2005) 65: 9712-9718. Irving JAE, Bloodworth L, Hogarth LA, Hall AG. “Loss of heterozygosity in childhood acute lymphoblastic leukaemia detected by genome-wide microarray single nucleotide polymorphism analysis.” Cancer Research (2005) 65: 3053-3053. Velangi MR, Matheson EC, Morgan GJ, Jackson GH, Taylor PRA, Hall AG, Irving JAE. “DNA mismatch repair pathway defects in the pathogenesis and evolution of myeloma.” Carcinogenesis (2004) 25: 1795-1803. Irving JAE, O’Brien SG, Lennard AL, Lin F, Hall AG. “The use of denaturing high-performance liquid chromatography for the detection of mutations in the BCR-ABL oncogene in patients resistant to Imatinib.” Clinical Chemistry (2004) 50: 1233-1237. Figure 2: Mutational screening of NRAS exon 1 by DHPLC identifies the G12V and G13D in acute lymphoblastic leukaemia samples at relapse. performed using Affymetrix micro arrays. Preliminary data implicate pathways involving MEK/ERK and NF-κB in chemo resistance for which potential small molecule inhibitors exist. These include PARP inhibitors, MEK/ERK inhibitors and fenretinide. In addition, a mutational screening programme of genes shown to activate these pathways using the sensitive technique of denaturing high-performance liquid chromatography (DHPLC), (Figure 2) has revealed a significant number of somatic mutations in samples both at diagnosis and relapse, which further validates this approach. Detection of minimal residual disease by flow cytometry While most children attain clinical remission which is assessed by morphological examination of bone marrow smears, submicroscopical disease levels, defined as minimal residual disease (MRD) is apparent in some. The principal aim of the current UK childhood ALL clinical trial is to test whether MRDbased stratification can optimise therapy so that maximal cure rate is obtained with minimal toxicity. In this trial, levels of MRD are measured using a molecular, DNA-based real-time quantitative PCR (RQ-PCR) method which is sensitive and robust but labour intensive and expensive. Recent progress in an alternative methodology, namely 4-colour flow cytometry, has shown that it is relatively cheap and quick and can achieve a sensitivity considered to have clinical significance, i.e. 1 in 104. Importantly, the clinical applicability of Flow-MRD is higher 30 Prof Hing Leung Our focus is to perform translational studies aimed at advancing our knowledge of the pathobiology in prostate carcinogenesis, discovering new targets for treatment and developing novel therapies for clinical prostate cancer. We are particularly interested in understanding how abnormal signalling critically contributes to prostate cancer. Our group has been developing two complementary strategic themes in prostate cancer research, namely androgen receptor and growth factor receptor signalling. NICR Team Emma Clark, Susan Cook, Luke Gaughan, Julia Graham, Ian Logan, Arthur McKie, Hesta McNeill, Mark Tones, Stuart McCracken, Sarah Wilkinson and in close collaboration with Richard Edmondson, Craig Robson, Rakesh Heer, Julie Irving and clinical staff based at the Freeman Hospital. MEK5/ERK5 pathway We recently reported our original findings linking the MEK5/ERK5 pathway to prostate cancer, with evidence for upregulation of MEK5 and ERK5 expression in clinical tumours. Such abnormal expression and function was found to be closely associated with aggressive disease with unfavourable survival outcome. Using an in vitro prostate cancer cell model, MEK5/ERK5 over-expression drives migration, invasion and the expression of proteases. Collaboration with Professor Philip Cohen (University of Dundee) has facilitated the progress of this project significantly. Introduction Prostate cancer is a major international public health issue: its incidence has been increasing and is continuing to rise in many countries. Our efforts continue to aim at examining how abnormal expression and function of androgen receptor with its associated co-factors and growth factor receptor tyrosine kinases along with the downstream intracellular signalling partners drive prostate carcinogenesis. Androgen receptor (AR) We are excited with our recent findings of novel interacting proteins for the androgen receptors. Detailed biochemical and functional analysis have confirmed the role of these novel genes in androgen receptor function, including mdm2, Tip60, PIRH2 and HIP1 -see section by Craig Robson. Institute for Institute for Human Human Genetics Genetics Medicinal Medicinal Chemistry Chemistry Northern Northern Institute for Institute for Cancer Research Cancer Research ProMPT ProMPT Institute Institute for for Human Human Ageing Ageing FP6 PRIMA FP6 PRIMA Prostate Cancer Research Proteomics (Slabas , Proteomics Durham) ProtecT ProtecT Fibroblast Growth Factor (FGF) system Aberrant expression of Fibroblast Growth Factors and their high affinity receptor tyrosine kinases have been implicated in clinical prostate cancer. Work from our group as well as others have revealed over-expression of multiple members of FGFs and their receptors (FGFRs). Proof of principle experiments using an adenoviral mediated gene transfer of a functional dominant soluble FGFR1 construct confirm the functional significance of FGF mediated signalling in prostate cancer cells. Clinical Urology Medical Medical & & Clinical Clinical Oncology Oncology Oncology (Slabas, Durham) CANCURE CANCURE Cross-Faculty Infrastructure Research The Prostate Research Group has an extensive collaborative network, and is involved in ProMPT, FP6 Prima and ProtecT clinical trials, and the European CANCURE translational prostate cancer research project. 31 Prostate Cancer Research Drug Development Exploiting the important findings from the above three distinct pathways, we have successfully collaborated with the Drug Development Group in the NICR resulting in exciting programmes: Tip60 (OSI), FGFR (Astex Therapeutics Limited) and signal transduction targets (Cancer Research Technology). Suppression of Physiologic Inhibitory Mechanisms: Sprouty and SEF In the past 18 months or so, we have initiated a programme of work to test the hypothesis whether abnormalities resulting in the loss of function for physiologic inhibitory molecules such as sprouty and SEF (Similar in Expression to Fibroblast Growth Factor) are involved in prostate carcinogenesis. Our highly significant findings, for the first time, implicated both Sprouty and SEF in prostate carcinogenesis. Our ongoing work supports a critical role for Sprouty and SEF, implicating them as potential roles as tumour/metastases suppressor genes. Differentiation of the Prostate Epithelium Building on our existing collaboration with Dr Anne Collins and Prof Norman Maitland (University of York) and Dr Gabi Saretzki (Institute for Human Ageing), we have recently developed a highly innovative programme of work studying factors regulating prostate epithelial differentiation. With the appropriate consent, clinical materials are applied and the role of growth factors and androgens in the determination and commitment of prostate epithelium to undergo differentiation is uncovered - see section by Rakesh Heer. Open retropubic radical prostatectomy represents one curative option for treating early prostate cancer; other options include external bean conformal radiotherapy and brachytherapy. Better treatment modalities are however urgently needed for relapsed and hormone refractory prostate cancer. Gaughan L, Logan IR, Neal DE, Robson CN. “Regulation of Androgen Receptor and Histone Deacetylase 1 by Mdm2-Mediated Ubiquitylation.” Nucleic Acids Research (2005) 33: 13-26. Mehta PB, Jenkins BL, McCarthy L, Thilak L, Robson CN, Neal DE, Leung HY. “MEK5 over-expression is associated with metastatic prostate cancer, and stimulates proliferation, MMP-9 expression and invasion.” Oncogene (2003) 22: 1381-1389. McKie AB, Douglas DA, Olijislagers S, Graham J, Heer R, Robson CN, Leung HY. “Epigenetic Inactivation of the Human Sprouty2 (hSPRY2) Homologue in Prostate Cancer.” Oncogene (2005) 24: 2166-2174. Heer R, Collins AT, Robson CN, Shenton BK, Leung HY. “Keratinocyte Growth Factor (KGF) Suppresses a2b1 Integrin Function and Promotes Differentiation of the Transient Amplifying Population in Human Prostatic Epithelium.” Journal of Cell Science (2006) 119: 1416-1424. Training of clinical and basic researchers of the future We place particular emphasis on training and nurturing of future researchers, and are highly successful in recruiting and training of gifted young clinical and basic researchers, as indicated by a number of prestigious national awards from MRC, CR UK and Royal College training fellowships as well as a CR UK Clinician Scientist award. 32 Dr John Lunec We investigate the molecular biology of human cancers, including ovarian and breast carcinoma, sarcoma, mesothelioma and neuroblastoma. Our aim is to establish determinants of clinical response to optimise existing and new therapies undergoing clinical trials, and to identify novel drug development targets. We have particular interests in the MDM2/p53/p14ARF and associated pathways, the development of MDM2-p53 antagonists, MYCN in neuroblastoma and antifolates. NICR Team: Christine Challen, Jane Margetts, Joyce Nutt, Sandy Beare, Amy Smith, Claire Hutton, Xiaohong Lu, Jane Carr, Emma Bell, Lindi Chen, Pei-Ju Sung and close collaboration with Hilary Calvert, Ruth Plummer, Ian Hardcastle, Debbie Tweddle, Chris Redfern, Richard Edmondson, Andy Hall, Herbie Newell, Mark Verrill and Josef Vormoor. of the p53 gene or overexpression of MDM2, which is a negative regulator of p53, tumours tend to be more aggressive and respond less well to treatment. We have investigated the MDM2/p53/p14ARF pathway in a range of tumour types and have initiated a large scale study of ovarian cancers from the MRC ICON3 trial which compared the efficacy of carboplatin and taxol. We have also recently reported that although p53 mutations are rare in neuroblastoma at diagnosis, cell lines derived from relapsed tumours show frequent inactivation of the MDM2/p53/p14ARF pathway. Introduction Developments in our understanding of the cellular and molecular processes that are responsible for the initiation and progression of cancers have opened up possibilities for the improvement of diagnosis and treatment, including the rational design of therapeutics targeted against specific molecular pathway faults. We now know that the development of cancer requires a multistep accumulation of genetic alterations. Although many of the genes involved have been identified, we are only just beginning to use this information to design new treatments aimed at specific molecular targets. One of the major hurdles to overcome is the molecular heterogeneity of cancer. Not all tumours are the same. Even within a given organ-specific cancer type the pattern of genetic alterations varies and this must be defined in order to tailor treatments to the individual. MDM2-p53 antagonists Tumours which do not have mutant p53, frequently have the The MDM2/p53/p14ARF pathway Many cancers have genetic alterations which involve or converge on the MDM2/p53/p14ARF cellular stress response pathway. The p53 protein is not only frequently involved in the development of cancer, but it is also an important mediator of the response to radiotherapy and chemotherapy. When the p53 pathway is functionally inactivated, either by direct mutation Figure 1: MDM2-p53 antagonists as non-genotoxic activators of p53. Western blot of MDM2 and p21 induction by isoindolinones (left) and molecular model of an isoindolinone binding to the hydrophobic pocket of MDM2 (right). 33 Molecular Oncology & Developmental Therapeutics activity of p53 suppressed by overexpression of MDM2. This can arise from amplification, in which there are multiple copies of the MDM2 gene, or from loss or downregulation of the gene encoding p14ARF, which is a negative regulator of MDM2. We are developing small molecule inhibitors of the MDM2-p53 interaction as a potential non-genotoxic approach to the reactivation of p53 in such circumstances (Figure 1). MYCN and the p53 pathway in neuroblastoma The MYCN oncogene is frequently amplified in neuroblastoma, for which it is a well established indicator of poor prognosis, used to identify patients who need more intensive therapy. It encodes a transcription factor involved in the regulation of tumour growth, differentiation status and response to chemotherapy. One of our major findings has been that the delayed entry of cells into the DNA synthetic phase of the cell cycle, which is normally mediated by the p53 pathway in response to DNA damage, is reduced in MYCN amplified neuroblastoma cells. We have found this to be associated with a reduced induction of the p21 cyclin dependent kinase inhibitor protein. This has implications for response to treatment and tumour progression, which we are exploring by manipulating the expression of MYCN. In addition, we have developed a reporter gene assay to screen compound libraries for potential inhibitors of MYCN dependent transcription. mesothelioma. Clinical trials of pemetrexed are also underway for breast and ovarian cancer. We are conducting translational studies in conjunction with these and other trials to investigate potential molecular pathological determinants of response to antifolates, including folate receptor, FPGS and thymidylate synthase expression. One of the folate receptors, FR-α has gained considerable interest as a potential determinant of response to pemetrexed and as a target for therapeutic intervention in cancer by virtue of its limited expression in normal tissues, where it is largely restricted to luminal surfaces, not directly accessible to the bloodstream, and the ability of the recycling receptor to bind and internalize certain antifolate compounds and folate conjugates. In spite of its importance, monoclonal antibodies to FR-α suitable for immunohostochemical analysis of formalin fixed and paraffin embedded biopsy samples, or that can be used for Western blot analysis of FR-α expression, have not been available. We have recently addressed this by successfully developing an FR-α antibody on a BioNet studentship project in partnership with Novocastra Laboratories Limited. Carr J, Bell E, Pearson AD, Kees UR, Beris H, Lunec J, Tweddle DA. “Increased frequency of aberrations in the p53/MDM2/p14(ARF) pathway in neuroblastoma cell lines established at relapse.” Cancer Research (2006) 66: 2138-2145. Hardcastle IR, Ahmed SU, Atkins H, Calvert AH, Curtin NJ, Farnie G, Golding BT, Griffin RJ, Guyenne S, Hutton C, Kallblad P, Kemp SJ, Kitching MS, Newell DR, Norbedo S, Northen JS, Reid RJ, Saravanan K, Willems HM, Lunec J. “Isoindolinone-based inhibitors of the MDM2-p53 protein-protein interaction.” Bioorganic and Medicinal Chemistry Letters (2005) 15: 1515-1520. Liang H, Atkins H, Abdel-Fattah R, Jones SN, Lunec J. “Genomic organisation of the human MDM2 oncogene and relationship to its alternatively spliced mRNAs.” Gene (2004) 338: 217-223. Tweddle DA, Malcolm AJ, Cole M, Pearson AD, Lunec J. “p53 cellular localization and function in neuroblastoma: Evidence for defective G(1) arrest despite WAF1 induction in MYCN-amplified cells.” American Journal of Pathology (2001) 158: 2067-2077. Determinants of response to antifolates Folates are essential vitamins required for the de novo synthesis of DNA precursors and hence cellular growth and division. They are taken up into cells via specific cell surface receptors and covalently modified by the enzyme folylpolyglutamate synthetase (FPGS). Polyglutamated forms are retained in cells and their affinity as substrates for subsequent steps in folate linked metabolic pathways is markedly increased. The development of folate analogue inhibitors targeting these pathways is an active area of anticancer drug development in which Newcastle has a strong track record. New antifolates for cancer are currently under development, and in particular the multi-targeted antifolate pemetrexed has been shown to prolong patient survival in 34 Dr Ross Maxwell Magnetic resonance imaging/spectroscopy and positron emission tomography techniques will be developed for studies in preclinical tumour models and early phase clinical trials. These methods will be co-ordinated with the anti-cancer drug development programme from an early stage to allow non-invasive evaluation of the presence of biological targets and for pharmacokinetic and pharmacodynamic measurements. NICR Team: In close collaboration with Herbie Newell and Roger Griffin. pharmacodynamic effects in hypothesis-driven Phase I clinical trials of anti-vascular (e.g. 5,6-dimethylxanthenone-4-acetic acid, combretastatin-A4-phosphate, and ZD6126) or antiangiogenic (e.g. VEGF inhibitors) agents. In the case of drugs primarily acting on vascular targets, the clinical availability of MR contrast agents (especially the gadolinium chelate, Gd-DTPA) together with relatively rapid imaging techniques, means that MRI has provided specific measurements relevant to their proposed mechanism of action. However, for many other drugs classes, appropriate MR biomarkers have yet to be developed and/or validated. A key objective of this programme is to generate non-invasive MR measurements providing specific information about biological targets and their responses to classes of drugs of Introduction Functional imaging allows the non-invasive investigation of disease processes and drug action in patients. Modern molecular pathology has provided a vastly improved understanding of disease mechanisms, and contemporary drug development approaches are yielding a new generation of treatments that target the underlying pathology of cancer. In developing these “molecular medicines” it is necessary to demonstrate that the drug is distributed to the required sites in the patient and interacting with its target in the intended manner. Functional imaging techniques can allow such information to be obtained and facilitates the use of these drugs in individualised medicine. For Drug Development see entries by Nicola Curtin, Roger Griffin, Ian Hardcastle and Herbie Newell. Two key techniques in functional imaging are magnetic resonance imaging and spectroscopy (MRI/S) and positron emission tomography (PET) (Figure 1). Newcastle University, in collaboration with the Newcastle upon Tyne Hospitals NHS Trust, are developing a comprehensive (clinical research and laboratory) functional imaging centre on the Newcastle General Hospital campus. a) Dynamic Contrast Enhanced MRI b) 18F-fluorodeoxyglucose PET Current role of MRI/S in anti-cancer drug development MRI and, to a lesser extent, MRS are beginning to have important roles in anticancer drug development and evaluation. In particular, dynamic contrast-enhanced MRI (DCE-MRI) is proving to be increasingly valuable for assessing Figure 1: Dynamic Contrast Enhanced MRI (a) and 18F-fluorodexoyglucose PET (b) scans from a P22 tumour showing heterogeneous blood flow and glycolytic uptake and metabolism. 35 Oncology Functional Imaging Research interest to the NICR and clinical researchers in Newcastle. In order to have “functional biomarkers” deployed in time for use in early stage clinical trials, they will need to be developed and validated as an integral part of the pre-clinical drug development process. Both pre-clinical and clinical magnetic resonance studies will be co-ordinated with the chemical synthesis of drug (and probe) molecules and in vitro evaluation. Manchester) and Dr Mike Carroll (School of Natural Life Sciences). The proposed PET oncology research programme will involve the design, synthesis and evaluation of novel PET probes for drugs designed to exploit targets being studied at the NICR. Functional ligands for imaging in cancer This programme is funded by an EPSRC Lifescience Interface Platform Grant in collaboration with Prof Andy Blamire (School of Clinical and Laboratory Sciences), Dr Mike Carroll (School of Natural Sciences), Prof Pat Price (Wolfson Molecular Imaging Centre, University of Manchester) and Prof David Parker (University of Durham). Three complimentary approaches will be pursued, namely, the development of: i) novel MR probes to measure the tumour microenvironment and response to therapy; ii) new PET probes to define the pharmacokinetics and pharmacodynamics of novel antitumour drugs; iii) innovative combined PET/MR probes to evaluate the potential of combined modality imaging. Galbraith SM, Maxwell RJ, Lodge MA, Tozer GM, Wilson J, Taylor NJ, Stirling JJ, Sena L, Padhani AR, Rustin GJ. “Combretastatin A4 phosphate has tumor antivascular activity in rat and man as demonstrated by dynamic magnetic resonance imaging.” Journal of Clinical Oncology (2003) 21: 2831-2842. Nielsen FU, Daugaard P, Bentzen L, Stodkilde-Jorgensen H, Overgaard J, Horsman MR, Maxwell RJ. ”Effect of changing tumor oxygenation on glycolytic metabolism in a murine C3H mammary carcinoma assessed by in vivo nuclear magnetic resonance spectroscopy.” Cancer Research (2001) 61: 5318-5325. Maxwell RJ, Frenkiel TA, Newell DR, Bauer C, Griffiths JR. “19F nuclear magnetic resonance imaging of drug distribution in vivo: the disposition of an antifolate anticancer drug in mice.” Magnetic Resonance in Medicine (1991) 17: 189-196. Maxwell RJ, Martinez-Perez I, Cerdan S, Cabanas ME, Arus C, Moreno A, Capdevila A, Ferrer E, Bartomeus F, Aparicio A, Conesa G, Roda JM, Carceller F, Pascual JM, Howells SL, Mazucco R, Griffiths JR. “Pattern recognition analysis of 1H NMR spectra from perchloric acid extracts of human brain tumor biopsies.” Magnetic Resonance in Medicine (1998) 39: 869-877. Newcastle Magnetic Resonance Centre The Newcastle Magnetic Resonance Centre was formally opened on 6th March 2006 by Prof. Colin Blakemore. Located in a new building on the Newcastle General Hospital site, the Centre has a 3 Tesla Philips MR system with full multi-nuclear imaging and spectroscopy capability. The facility provides nearly 1,000 square metres of laboratory space including offices, seminar room, space for 2 MRI systems (1 installed in January 2006, 1 planned) and full facilities for clinical magnetic resonance research. Pre-clinical magnetic resonance facility A 7 Tesla MR Imaging and Spectroscopy system has been funded through Cancer Research UK and SRIF3. Installation is expected early in 2007. The facility will be located in a new Institute for Ageing and Health building at Newcastle General Hospital. This system will enable the development of new MR techniques, probes and contrast agents in pre-clinical models. It will also allow in vivo evaluation of candidate drugs at the pre-clinical stage. Positron emission tomography With the announcement from the Department of Health for the provision of positron emission tomography (PET) facilities across the UK, plans are now being developed for the establishment of a PET Centre adjacent to the MR Centre. The complementary use of PET will ensure that multiple functional imaging approaches will be available. The proposed PET research will involve close collaboration with the Professors Pat Price and Terry Jones (Wolfson Molecular Imaging Centre, 36 Dr Felicity EB May Trefoil proteins are small signalling molecules secreted by a significant number of human adenocarcinomas. We are studying the structure and function of different molecular forms of trefoil proteins found in vivo. Our research aims to identify the effects that trefoil proteins have on cancer cells and to understand the mechanisms by which trefoil proteins exert their actions in tumours. NICR Team: Neil Jennings, Paul Wright, Rehan Saif, Nahed Hawsawi, Claire Worrall, Gail de Blaquière and in close collaboration with Bruce Westley and Mark Verrill. Characterisation of these interactions will further understanding of trefoil protein biology and allow the pathological consequences to be targeted. Thirdly, the expression and role of trefoil proteins in disease; especially in Introduction The human trefoil factor family contains three members, TFF1, TFF2 and TFF3 (May and Thim, 2005). Trefoil factors are small secreted proteins co-expressed with mucins by epithelial cells that line mucus membranes. Expressed at highest levels in the gastrointestinal tract, individual trefoil factors are secreted by different cells: TFF1 by the superficial gastric cells whereas TFF2 is largely restricted to cells of the basal gastric glands and Brunner’s glands of the duodenum. TFF3 is expressed in goblet cells of the small and large bowel. They are present at lower levels in other tissues including salivary gland, lung, pancreas, breast and cornea. Trefoil factors are expressed, sometimes at high levels and sometimes apparently ectopically, in many human adenocarcinomas. The normal role of trefoil proteins is in mucosal protection. They are involved in the packaging, secretion and rheological properties of mucins. They are implicated in mucosal repair following damage caused by various insults including abrasion, acidic pH and ingestion of noxious substances including alcohol. Repair includes a rapid movement of epithelial cells over damaged areas. Our group is involved in three aspects of trefoil protein biology. Firstly, determination of the molecular forms of human trefoil proteins found in normal and malignant cells. Analysis of the structures and diverse functions of these different molecular forms. Secondly, the biological consequences of trefoil protein expression result from inter-molecular interactions. Figure 1: Structures of molecular forms of mammalian trefoil proteins. Ribbon diagrams are shown of the known structures of the trefoil proteins. The trefoil domains are shown in blue. The regions of the protein outside the trefoil domain are shown in grey. The three loops of the trefoil domain are numbered. A schema of the TFF1:TFIZ1 heterodimer is shown. 37 Structure & Function of Trefoil Proteins in Normal & Malignant Cells cancers of the gastrointestinal tract and breast. Expression of trefoil proteins may predict response to therapy or invasive potential. Structure and function of trefoil proteins Trefoil proteins contain a 42-43 amino acid conserved domain that includes six cysteine residues. TFF1 and TFF3 contain a single trefoil domain whereas TFF2 contains two domains. We collaborate with structural biologists to determine the molecular structures of trefoil proteins. The trefoil domain is characterised by three loops stacked parallel to each other and by three disulphide bonds (Figure 1). TFF1 and TFF3, form homo-dimers via a seventh cysteine residue at the carboxyterminus and have the potential to form intra-molecular disulphide bonds. The TFF3 dimer forms a more compact structure than the TFF1 dimer in which the two trefoil domains are connected by a flexible region at variable distances from each other and in many different orientations (Muskett et al., 2003). The shorter carboxy-termini and an additional 310-helix lock the TFF3 dimer into a constrained structure. Although TFF2 is also a compact structure, the dispositions of its monomer units are very different. The TFF1 dimer is more potent than the monomer in a variety of in vitro biological assays and in animal models. The majority of TFF1 in normal human gastric mucosa is present as a heterodimer. This has profound implications for TFF1 function. We have purified the TFF1 heterodimer and identified the protein partner by amino-terminal sequencing and MALDI TOF mass spectrometry as a novel protein called TFIZ1 for trefoil factor interactions(z) 1 (Westley et al., 2005). TFIZ1 is a secreted protein of 18.3 kDa that contains five cysteine residues one of which forms an inter-molecular disulphide bond with TFF1 (Figure 1). class I carcinogen that colonises the gastric mucosa. In collaboration with Dr Marguerite Clyne and Prof Brendan Drumm (University College Dublin), we have shown that H. pylori interact avidly with the dimeric form of TFF1, and that this interaction enables it to bind gastric mucin (Clyne et al., 2004). This suggests that TFF1 acts as a receptor for H. pylori and may explain its tropism for gastric tissue and colocalization with MUC5AC. Expression and role of trefoil proteins in cancer Gastric cancer has a propensity to metastasize and kills 640,000 people worldwide each year. The main site of expression of TFF1 in normal tissues is the stomach. Work with TFF1-null mice shows that TFF1 is a tumour suppressor. TFF1 expression is lost early during carcinogenesis in a significant proportion of human gastric tumours. Conversely, TFF1 is frequently overexpressed or expressed ectopically by carcinoma cells. It stimulates migration and invasion of tumour cells and is thought to be involved in tumour dissemination. It is likely that trefoil proteins exert different effects depending on when they are expressed during the conversion of a normal cell into a cancer cell. Inter-molecular interactions may also determine if trefoil protein expression is beneficial or detrimental. Thim L, May FEB. “Structure of mammalian trefoil factors and functional insights.” Cellular and. Molecular Life Sciences (2005) 62: 2956-2973. Muskett FW, May FEB, Westley BR, Feeney J. Solution structure of the disulphide-linked dimer of human intestinal trefoil factor (TFF3): the intermolecular orientation and interactions are markedly different from those of other dimeric trefoil proteins. Biochemistry (2003) 42: 15139-15147. Westley BR, Griffin SM, May FEB. “Interaction between TFF1, a gastric tumor suppressor trefoil protein, and TFIZ1, a Brichos domain-containing protein with homology to SP-C.” Biochemistry (2005) 44: 7967-7975. Clyne M, Dillon P, Daly S, O'Kennedy R, May FEB, Westley BR, Drumm B. “Helicobacter pylori interacts with the human single domain trefoil protein TFF1.” Proceedings of the National Academy of Sciences, USA (2004) 101: 7409-7414. Trefoil protein interactions TFF1 is co-expressed with the secreted mucin MUC5AC in superficial cells of the gastric mucosa. We have shown that TFF1 co-sediments with mucin glycoproteins on caesium chloride gradients and that TFF1 interacts with MUC5AC but not MUC6 in vivo. The TFF1 dimer is the predominant molecular form bound to MUC5AC. The bacterium Helicobacter pylori is a 38 Prof Herbie Newell In the CR UK Drug Development Programme at the NICR, I work with Nicola Curtin, Roger Griffin, Ian Hardcastle and other NICR colleagues to develop novel small molecule anticancer agents, and with Ross Maxwell to apply functional imaging to cancer drug development. Targets for therapeutic exploitation are either linked to the molecular pathology of cancer or implicated in drug resistance. NICR Team: Amy Peasland, Mike Batey, Luke Harrison, Suzanne Kyle, Huw Thomas, Lan-Zhen Wang, and in close collaboration with Mike Tilby, Hilary Calvert, Julie Irving, Ruth Plummer, Josef Vormoor, Ross Maxwell, Nicola Curtin, Ian Hardcastle and Roger Griffin. Introduction The main focus of our research group is the evaluation, and optimisation of compounds developed by either the medicinal chemists of the NICR, or external collaborators. Current projects are focussed around inhibitors of DNA damage signalling and repair proteins: poly(ADP-ribose) polymerase (PARP), DNAdependent protein kinase (DNA-PK) and ataxia telengiectasia mutated (ATM) kinase. We are also investigating pyrimidopyrimidine inhibitors of nucleoside transport and inhibitors of cyclin-dependent kinases and the TIP60 acetyltransferase. Structure-based design has been used to elaborate NU2058, and pyrimidine isosteres, exploring in particular the ribosebinding and specificity pockets. These studies have resulted in CDK2-selective inhibitors, which are over 1,000-fold more potent than NU2058, for example NU6102 (Figure 1). The compounds produce cell cycle phase arrest and inhibition of cellular protein phosphorylation consistent with CDK inhibition at growth inhibitory concentrations. Lead optimisation is underway to generate a candidate for clinical evaluation. A particular property of NU2058 is the ability to potentate the antitumour activity of platinum complexes. Work in collaboration with Professor Eileen Dolan (University of Chicago) is probing the mechanism underlying the potentiation of cisplatin by NU2058, with a view to exploiting the phenomenon in clinical trials. Cyclin-dependent kinase inhibitors Cyclin-dependent kinases (CDKs) are key regulators of cell cycle progression whose function is frequently deregulated in human tumours, for example by increased expression of activating cyclins (e.g. cyclin D), decreased expression of inhibitory peptides (e.g. p16 and p21), or substrate loss (e.g. retinoblastoma gene deletion). In a collaboration with Professors Jane Endicott and Martin Noble (Oxford University), structure-based drug design is being used to develop small molecule CDK inhibitors. The initial “hit” O6cyclohexylmethylguanine (NU2058), was shown to bind to CDK2 at the ATP site of the enzyme with the purine ring in a different orientation to that of the inhibitors olomoucine and Rroscovitive. Furthermore, the cellular pharmacology of NU2058 was shown to differ from that of olomoucine and flavopiridol. Figure 1: Structure-based design of potent CDK inhibitors - The purine inhibitor NU6102 bound in the active site of CDK2. 39 Drug Development – Pre-clinical Pharmacology Additional targets for exploitation The molecular genetic and mechanistic studies undertaken by a number of the groups in the NICR have identified a broad range of novel targets with potential for exploitation for drug development. The close clinical-laboratory interface in the NICR facilitates the analysis of patient samples with linked clinical data, a resource that is central to effective target identification and validation. Targets have been identified in paediatric and adult, haematological and solid, tumours. Molecular genetic and pharmacological approaches are used to further validate targets in cell-based systems, and the development of novel pharmacological probes is a particular strength of the NICR. The most recent target to be identified, the FGF receptor, is being exploited in a collaboration with Astex Therapeutics Limited (Cambridge, UK). Using fragment-based drug design, colleagues at Astex Therapeutics Limited are developing inhibitors which studies in the NICR have shown should have activity in the treatment of multiple myeloma and prostate cancer. Figure 2: PET Scanning as a pharmacodynamic tool in patients - Parametric images of the liver showing the change in 11C-thymidine tracer delivery (Upper Images) and retention (Lower Images) before (Left Images) and following (Right Images) administration of the thymidylate synthase inhibitor nolatrexed. The white areas represent the areas of highest tracer delivery or retention, the dark areas represent necrosis within liver metastases. Functional imaging For the rational development of anticancer drugs it is essential that both the pharmacokinetics (PK) and pharmacodynamics (PD) of the agents are defined in pre-clinical models in order to develop PK and PD endpoints for mechanistic early clinical trials. In addition to conventional PK and PD studies based on samples obtained by invasive procedures, such as blood sampling and tumour biopsy, non-invasive methods such as magnetic resonance imaging (MRI) and positron emission tomography (PET) are also increasingly important. Such methods permit functional imaging and for developments and research in this area. A particular application of PET in drug development is the use of 11 C-thymidine uptake and retention to demonstrate thymidylate synthase inhibition following the administration of thymidylate synthase inhibitors (Figure 2). In a collaboration with Professor Pat Price (Wolfson Molecular Imaging Centre, Manchester) the feasibility of 11C-thymidine PET scanning was demonstrated and a strong collaboration has been established to develop additional novel functional imaging probes for cancer. Davies TG, Bentley J, Arris CE, Boyle FT, Curtin NJ, Endicott JA, Gibson AE, Golding BT, Griffin RJ, Hardcastle IR, Jewsbury P, Johnson LN, Mesguiche V, Newell DR, Noble MEM, Tucker JA, Wang L-Z, Whitfield HJ. “Structurebased design of a potent purine-based cyclin-dependent kinase inhibitor.” Nature Structural Biology (2002) 9: 745-749. Wells P, Aboagye E, Gunn RN, Osman S, Boddy AV, Taylor GA, Rafi I, Hughes AN, Calvert AH, Price PM, Newell DR. “2-[C-11]thymidine positron emission tomography as an indicator of thymidylate synthase inhibition in patients treated with AG337.” Journal of the National Cancer Institute. (2003) 95: 675-682. Newell DR. “How to develop a successful cancer drug - molecules to medicines or targets to treatments?” European Journal of Cancer (2005) 41: 676-82. 1. Pennati M, Campbell AJ, Curto M, Binda M, Cheng Y, Wang LZ, Curtin N, Golding BT, Griffin RJ, Hardcastle IR, Henderson A, Zaffaroni N, Newell DR. “Potentiation of paclitaxel-induced apoptosis by the novel cyclindependent kinase inhibitor NU6140: a possible role for survivin downregulation.” Molecular Cancer Therapeutics (2005) 4: 1328-37. 40 Dr Ruth Plummer Phase I studies are the first clinical steps in the development of novel anti-cancer agents and are designed to establish a safe dose and examine the potential toxicity of new drugs. Work in the Clinical Trials Unit (CTU) has a strong translational component and samples are brought back to the NICR from the clinic for further study of cancer cell biology, drug interactions and drug/target effects. NICR Team Evan Mulligan, Tomasz Zaremba and in close collaboration with Hilary Calvert, Herbie Newell , Roger Griffin, Nicola Curtin, Alan Boddy, John Lunec, Josef Vormoor, Mark Verrill, Joyce Nutt and clinical staff based at the NCCT. Phase I studies Phase I studies involve taking novel agents “first-in-human” and are carried out in patients with incurable cancer. These studies are designed to examine the safety and toxicity of new compounds, and to find a safe dose for on-going development. Patients with a range of tumour types are referred to the unit from a wide catchment area, all aspects of the studies are discussed at length with them by medical and dedicated nursing staff to ensure that they understand the process before entering any trial. As well as evaluating dose by assessing side effects many blood samples are taken for pharmacokinetic (drug level) and pharmacodynamic (drug effect on the body) tests. These samples may return to the Institute for analysis or be sent to a range of collaborating laboratories in the UK and overseas. Introduction For many patients with solid tumours, chemotherapy can be effective but is rarely curative when the disease has spread beyond surgical control. The mission statement of the Institute embodies the need to develop for better and less toxic treatments, and to this end the drug development programme aims to identify targets, design drugs and eventually introduce these into treatment regimens. The Early Clinical Trials Unit is a University unit within the Northern Centre for Cancer Treatment, a large regional cancer centre (Figure 1). On the trials unit, patients are recruited into both phase I and II studies looking at novel agents, those developed within NICR, by Cancer Research UK and the pharmaceutical industry. It is the end point of the pre-clinical drug development process, and the start of the careful clinical evaluation of drugs that happens before they are submitted for regulatory approval. The majority of my research is based around these early studies. In addition, I treat patients with sarcomas, both bone and soft tissue, and metastatic melanoma both with standard chemotherapy regimens but also on phase II studies, evaluating the activity of new agents in these disease types. All trials on the unit have a strong translational component, where blood or tumour samples are requested from patients, to be returned to the laboratory for further analysis and research into tumour biology. Figure 1: A patient being prepared for receiving chemotherapy treatment at the Northern Centre for Cancer Treatment at Newcastle General Hospital. (Photograph courtesy of Cancer Research UK Photography Department) 41 Early Clinical Trials, Melanoma and Sarcoma Sarcoma PARP activity as % of pre-treatment 25 20 15 10 5 0 4 mg/m 2 12 mg/m 2 18 mg/m 2 Figure 2: Results from immunoblotting assay showing that AG014699 inhibits its target enzyme in metastatic melanoma tumour biopsies at 4 hours after a single intravenous dose. Sarcomas are tumours of bone and soft tissue, which occur predominantly in the younger age groups. Primary treatment for these diseases is generally surgery, although there is an increasing role for chemotherapy both with established agents and with new therapies. Newcastle is a large sarcoma centre, having supra-regional funding for the treatment of this disease. In addition to treating a significant number of patients we have a portfolio of sarcoma studies allowing access to new agents for local patients. There is a strong sarcoma clinical team which facilitates the collection of research samples and recruitment of patients into both paediatric and adult clinical trials. We also have an expanding programme of translational research looking at the biology of this disease and trying to identify new targets for drug development – completing the circle of translational research, bench to bedside and back again. One particular area of interest in the Institute over recent years has been the development of DNA repair enzyme inhibitors and I have been closely involved in the late preclinical and early clinical trials of the first of these agents to enter the clinic, the poly(ADP-ribose)polymerase inhibitor, AG014699. This agent was designed to inhibit the target enzyme, being given with chemotherapy to prevent the tumour cell repairing DNA damage caused by the treatment. A pharmacodynamic assay carried out to GCLP standards within the Institute demonstrated that this drug does hit its target effectively and recruitment to a Phase II study in melanoma has recently completed recruitment. Plummer ER, Middleton MR, Jones C, Olsen A, Hickson I, McHugh P, Margison G, McGown G, Thorncroft M, Watson AJ, Boddy AV, Calvert AH, Harris AL, Newell DR, Curtin NJ. “Temozolomide pharmacodynamics in patients with metastatic melanoma: DNA damage and activity of repair enzymes ATase and PARP-1.” Clinical Cancer Research (2005) 11: 3402-3409. Plummer R, Rees C, Hughes A, Beale P, Highley M, Trigo J, Gokul S, Judson I, Calvert H, Jackman A, Mitchell F, Smith R, Douglass P. “Clinical phase I trial of ZD9331, a water-soluble, nonpolyglutamatable, thymidylate synthase inhibitor.” Clinical Cancer Research (2003) 9: 1313-1322. Plummer R, Ghielmini M, Calvert P, Voi M, Renard J, Calvert H, Sessa C. “Phase I and pharmacokinetic study of the new taxane analogue BMS-184476 given weekly in patients with advanced malignancies.” Clinical Cancer Research (2002) 8: 2788-2797. Boddy AV, Plummer ER, Todd R, Sludden J, Griffin M, Robson L, Cassidy J, Bissett D, Bernareggi A, Verrill MW, Calvert AH. “A Phase I and pharmacokinetic study of paclitaxel poliglumex (XYOTAX), investigating both every 3-weekly and every 2-weekly schedules.” Clinical Cancer Research (2005) 11: 7834-7840. Melanoma Malignant melanoma is a relatively rare skin cancer with an increasing incidence. When metastatic this disease is incurable with a poor prognosis and there is an urgent need for the development of more effective therapies. In addition to the continuing investigation of the properties of AG014699 as a potential agent useful in melanoma (Figure 2), we participate in a number of national and international early studies in this disease and are starting to establish other research interests in this area. 42 Dr Christopher Redfern Vitamin A is the precursor to a natural signalling molecule that controls cell behaviour. Compounds related to vitamin A, referred to as retinoids, are being studied to increase our understanding of the aetiology and characteristics of cancer cells and to develop novel therapeutic strategies. Our approach integrates a wide range of conventional molecular and cellular techniques with the development of new methods to study cell behaviour. NICR Team Helen Imrie, Jane Armstrong, Frida Ponthan, Danielle Lindley, Lindsey Nicholson, Boj Goranov and in close collaboration with Julie Irving, Josef Vormoor, Gareth Veal, Steve Clifford, Debbie Tweddle and John Lunec. also include glucocorticoids (in collaboration with Dr. Jola Weaver and Dr. Akheel Syed, School of Clinical Medical Sciences), and on the identification of genes involved in neuroblastoma tumourigenesis in collaboration with Dr. Mike Jackson of the Institute for Human Genetics. Introduction Vitamin A (retinol) is derived from the diet and metabolised within cells to retinoic acid. Compounds derived from retinol or related by function are termed retinoids. Retinoic acid is a vital intracellular signalling molecule, and is transported to the nucleus where it regulates gene expression via nuclear receptors (retinoic acid receptors or RARs). Mammalian cells have well-developed systems for regulating the levels of retinoic acid. The conversion of retinol to retinoic acid is regulated by cellular retinol binding proteins and retinoid dehydrogenases. Similarly, the metabolism and transport of retinoic acid to nuclear receptors is regulated by cellular retinoic acid binding proteins and cytochrome P450 retinoic acid hydroxylases. Treating cells with retinoic acid in vitro can induce some cancer cells such as neuroblastoma to differentiate to a less malignant form, and affect other cell properties such as proliferation and programmed cell death (apoptosis). We are investigating the molecular mechanisms of retinoic acid action to enhance the clinical efficacy of retinoids and identify new ways to use retinoid-signalling pathways for cancer treatment. Neuroblastoma cells are a good model system, and since retinoic acid is currently used clinically to reduce the incidence of relapse in high-risk neuroblastoma patients, we collaborate with colleagues on projects to enhance the efficacy of retinoids and elucidate retinoid control pathways in neuroblastoma. My research interests Retinoid receptors and cellular responses Three types of RAR (RAR-α, -β and -γ), are expressed in cell-typespecific patterns and work as heterodimers with related receptors called RXRs (Figure 1). To understand the role of these receptors in neuroblastoma biology and facilitate the design of receptorspecific compounds, we are studying the effects of overexpressing and knocking out expression of each receptor. Recent All-trans retinoic acid RAR RXR AGGTGA NNNNN AGGTGA DR5 RARE Figure 1: RXR-RAR heterodimer associated with a ‘retinoic acid response element’ (RARE) within the promoter of a retinoic-acid-regulated gene: the binding of all-trans retinoic acid (ATRA) stimulates transcription and expression of the gene. 43 Cell and Molecular Biology of Retinoids results show that over-expression of RAR-γ markedly changes the way neuroblastoma cells respond to retinoic acid, inducing apoptosis rather than differentiation.Therefore, receptor-specific agonists or antagonists may have considerable value for neuroblastoma therapy, allowing tumour cells to be removed from the body rather than lying dormant as differentiated cells capable of regression to a malignant phenotype. Other collaborative projects include fenretinide as a therapy for Ewing’s Sarcoma (with Dr. Sue Burchill, University of Leeds), and the development of non-invasive bio-imaging methods for investigating the role of fenretinide and other retinoids for the therapy of paediatric cancers. New methods for high throughput cell biology Much of cell and molecular biology research relies on measuring changes in cell proliferation, motility, shape or death. Current methods to achieve this are still rudimentary and timeconsuming. If we could develop tiny sensors to measure changes in cell shape, motility, attachment and force-generating properties, this could have a marked effect on the speed of cancer research. We are actively collaborating with researchers in the Institute of Nanotechnology (Dr. John Hedley and team) to develop silicon-chip-based sensors which can used to investigate the physical properties of cells at a depth and scale which has not been possible before. These promise to be exciting developments for basic cell and molecular biology research. Corazzari M, Lovat PE, Oliverio S, Pearson AD, Piacentini M, Redfern CPF. “Growth and DNA damage-inducible transcription factor 153 mediates apoptosis in response to fenretinide but not synergy between fenretinide and chemotherapeutic drugs in neuroblastoma.” Molecular Pharmacology (2003) 64: 1370-1378. Hewson QD, Lovat PE, Corazzari M, Catterall JB, Redfern CPF. “The NF-kappaB pathway mediates fenretinide-induced apoptosis in SHSY5Y neuroblastoma cells.” Apoptosis (2005) 10: 493-498. Lovat PE, Oliverio S, Ranalli M, Corrazzari M, Rodolfo C, Bernassola F, Aughton K, Maccarrone M, Campbell Hewson QD, Pearson ADJ, Piacentini M, Redfern CPF. “GADD153 and 12-Lipoxygenase mediates fenretinide-induced apoptosis of neuroblastoma.” Cancer Research (2002) 62: 5158-5167. Lovat PE, Di Sano F, Corazzari M, Fazi B, Donnorso RP, Pearson AD, Hall AG, Redfern CPF, Piacentini M. “Gangliosides link the acidic sphingomyelinase-mediated induction of ceramide to 12lipoxygenase-dependent apoptosis of neuroblastoma in response to fenretinide.” Journal of the National Cancer Institute (2004) 96: 1288-1299. Retinoic acid and other signalling pathways Research in collaboration with Drs. Tim Cheek and Anna Brown (Institute of Cell and Molecular Biosciences) has shown that retinoic acid-differentiated neuroblastoma cells differ in their calcium-signalling properties, and suggest that distinct signalling pathways characterise a small population of tumour cells which might represent cancer stem cells. These signalling pathways might be used to target chemotherapy more effectively to overcome drug resistance and relapse. Retinoids may also interact with other nuclear receptor signalling pathways and other collaborative projects are in progress to elucidate the potential for combining retinoids with glucocorticoids for leukaemia therapy. Novel retinoids for chemotherapy Synthetic retinoids which induce apoptosis rather than differentiation have been identified. One of these is fenretinide, which induces apoptosis in neuroblastoma and Ewing's Sarcoma cells. Our research into the molecular mechanisms of fenretinide has shown that the induction of intracellular reactive oxygen species (ROS) is a critical event, and leads to NF-kappa Bdependent apoptosis via activation of the transcription factor GADD153. Cellular stress as a consequence of increased ROS results in the induction of chaperone proteins. These are produced by cells in an attempt to counteract stress - a homeostatic response. Knocking down the expression of chaperone proteins enhances cell killing by fenretinide, and may be a general strategy that could be used to enhance the efficacy of chemotherapeutic drugs. These ideas are being pursued as part of collaborative projects involving Prof. Mark Birch-Machin and Dr. Penny Lovat of the School of Clinical Laboratory Sciences. 44 Dr Helen Reeves Cancers develop as a result of either inherited or acquired genetic damage to key growth regulatory genes. Identifying and characterising the key genetic events associated with initiation and progression of gastrointestinal cancers is our primary aim. These events contribute to our understanding of critical regulatory pathways, but may also lead to improved cancer surveillance and therapy. NICR Team: Gary Beale, Dipanker Chattopadyhay, Arvind Rajagopal, Luca Miele, Rajiv Lojan and in close collaboration with the Liver Group and NCCT. Introduction The interests of our group include gastrointestinal (GI) cancers and the chronic diseases predisposing to their development. In particular, our studies focus on the roles of the tumour suppressor gene Kruppel-like factor 6 (KLF6) in these processes, as well as the application of advanced molecular and proteomic technologies aimed at identifying novel biomarkers and therapeutic targets. Dysfunction of KLF6 is clearly important in CRC. Wild type KLF6 translocates to the nucleus and is rapidly degraded. The mutant and dominant negative and splice forms have a longer half life and accumulate in the cytoplasm. Identifying the protein partners regulating localisation and degradation is a step toward identifying the means to manipulate KLF6 as a therapeutic target. We have created tagged vectors to express KLF6 isoforms in cells in order to identify their protein partners. In addition, we are currently using microarray technology and in vitro models to identify KLF6 downstream target genes. The Role KLF6 in the development and progression of GI cancers KLF6 is a ubiquitously expressed transcription factor with roles in the regulation of cell growth and differentiation. It is inactivated by a combination of loss of an allele and/or mutation in a number of cancers, including colorectal cancers (CRC). We have confirmed reduced expression of wild type KLF6 in 85% of CRC, often occurring in ‘normal’ as well as cancer tissues. Additionally, dominant negative alternative splice forms accumulate in up to 70%. In over half of CRCs these abnormalities are associated with either genetic or epigenetic changes at the KLF6 gene locus, supporting an emerging role for this gene as a key tumour suppressor gene inactivated in CRC (Figure 1). Ongoing studies include the characterisation of KLF6 inactivation in pre-neoplastic diseases such as polyps and ulcerative colitis, as well as in metastatic disease. This work is being performed in collaboration with Mr Olagunju Oginbiyi (Royal Free Hospital), as well as local collaborator Mr Derek Manas (Freeman Hospital, Newcastle). Figure 1: KLF6 is a growth suppressive transcription factor. It is inactivated at the DNA level by loss or mutation of the gene in colorectal cancers. KLF6 inactivation contributes to accelerated growth and malignant transformation. We believe that smaller isoforms of the gene accumulate (brown colour) in both early lesions such as polyps, as well as the cancers. These isoforms inactivate the full length protein and also promote cancer development and progression. 45 The Molecular Pathogenesis of Gastrointestinal Cancers – The Search for Key Biomarkers and Therapeutic Targets KLF6 studies are also ongoing in hepatocellular (HCC), pancreatic and ovarian cancers. In addition, in collaboration with Professor Anne Daly (School of Clinical and Laboratory Sciences) and Mr Richard Charnley (Freeman Hospital, Newcastle), we are characterising DNA repair and its disruption in pancreatic cancers. The search for biomarkers and therapeutic targets in hepatocellular cancers (HCC) HCC complicates chronic liver diseases and has risen dramatically over the last 5 years, in part because of an increase in obesity which predisposes to the development of ‘NASH’. NASH (non-alcoholic fatty liver disease), and ASH (alcoholic fatty liver disease) are the key causes of cirrhosis and HCC in the UK. HCC has a dismal prognosis. The only cures include surgical resection, which is usually not appropriate in those with cirrhosis, or liver transplantation, which only 1 or 2% are suitable for. Mr Manas has introduced a therapy called radiofrequency ablation (RFA), but only small tumours can be treated by RFA. Hence, there is a desperate need to both improve our means of detecting this cancer at earlier stages amenable to treatment, as well as to identify alternative treatment strategies. The majority of HCC patients in Newcastle (approximately 100 per year and rising) are cared for on the Freeman Hospital Liver Unit. In collaboration with Mr Manas, these patients are enrolled into an ethically approved prospective follow-up study. This study includes the upkeep of a clinical database, but also serial serum and tissue collection for molecular studies. Presently, we are performing serum proteomic studies in collaboration with Dr Joe Gray of the Newcastle University Proteomics Facility. Work to identify a NASH/ASH associated HCC diagnostic and prognostic biomarkers is ongoing. cirrhosis and are at risk of developing HCC. Being able to identify cirrhosis easily (i.e. without the need for liver biopsy which is costly and invasive) would be very helpful, in part because it would identify the population who would benefit from HCC surveillance. In collaboration with Professors Chris Day (School of Clinical Medical Sciences) and Alastair Burt (School of Clinical and Laboratory Sciences), ethically approved serum and liver biopsy samples are collected from patients with different stages of alcoholic and non-alcoholic liver diseases for research purposes. We have optimised the methodology for sensitively and quantitatively comparing the serum proteome in individuals with simple fatty liver, versus fatty liver with inflammation, versus fatty liver disease with inflammation and fibrosis. In addition, we are using microarray technology to investigate the gene expression profiles associated with these different disease groups. In combination, we hope to identify candidate genes for genetic susceptibility studies, as well as identify biomarkers of disease processes to aid the clinical management of these common diseases. Narla G, Heath KE, Reeves HL, Li D, Giono LE, Kimmelman AC, Glucksman MJ, Narla J, Eng FJ, Chan AM, Ferrari AC, Martignetti JA, Friedman SL. “KLF6, a candidate tumor suppressor gene mutated in prostate cancer.” Science (2001) 294: 2563-2566. Reeves HL, Narla G, Oginbiyi O, Haque A, Katz A, Benzeno S, Kramer-Tal S, Friedman S, Martignetti JA. “Kruppel-like factor 6 (KLF6) is a tumor-suppressor gene frequently inactivated in colorectal cancer.” Gastroenterology (2004) 4:1090-103. Tal-Kremer S, Reeves HL, Narla G, Friedman SL. ”KLF6 is a Tumour Suppressor Gene inactivated in Hepatocellular Cancers.” Hepatology (2004) 40: 1047-1052. Narla G, DiFeo A, Reeves HL, Schaid DJ, Hirshfield J, Hod E, Katz A, Isaacs B, Hebbring S, Komiya A, McDonnell SK, Wiley KE, Jacobsen SJ, Isaacs SD, Walsh PC, Zheng SL, Chang BL, Xu J, Thibideau SN, Friedman SL, Martignetti J. “A germline DNA polymorphism associated associated with increased prostate cancer risk enhances alternative splicing of the KLF6 tumor suppressor gene.” Cancer Research (2005) 65: 1213-1222. The search for candidate genes and biomarkers associated with progressive liver disease Abnormal liver function tests (LFTs) are extremely common in the UK population. Some people with abnormal LFTs have 46 Prof Craig Robson Prostate cancer accounts for the death of over 10,000 males in the UK per annum. The disease is characterised by a transition from androgen dependence, through androgen sensitivity and ultimately to androgen independence. Understanding the mechanisms that are responsible for this transition to androgen independence is a major goal of our research. NICR Team: Luke Gaughan, Ian Logan, Emma Clark, Steven Darby, Kelly Armstrong, Natasha Rigas, Susan Cook, Julia Graham, Hesta McNeill, and in close collaboration with Hing Leung, Richard Edmondson, Rakesh Heer and Ian Hardcastle. Several of these proteins have associated enzymatic activities that can modulate the transcriptional activity of androgen receptor target genes. Our studies have concentrated on the role of androgen receptor co-regulator proteins and the importance of post-translational modifications in androgen receptor function. Major projects include investigation of acetylation in androgen Introduction Prostate cancer is the most prevalent malignancy in Western men and contributes directly to approximately 10,000 deaths per year in the UK. The androgen receptor is implicated in prostate malignancy and remains the primary target for therapeutic intervention in prostate cancer patients. Initial treatment with androgen ablation, which inactivates the androgen receptor transcriptional response, is usually successful and causes regression of the prostate tumour. Most malignancies, however, become refractory and progress as a consequence of numerous molecular events, such as mutation or amplification of the androgen receptor gene, enabling cell growth in an androgenindependent manner, for which there is currently no effective treatment. Our research is directed towards unravelling the mechanisms by which prostate cancer cells progress from androgen dependence, through androgen sensitivity and finally to androgen independence. A better understanding of the mechanisms by which tumour cells acquire androgen independence may identify novel targets of treatment for hormone refractory prostate cancer. Research interests The research programme of the group is primarily focussed on hormone receptor signalling in prostate cancer. Using both yeast and mammalian model systems we have identified several proteins that interact with the human androgen receptor protein. Figure 1: Imaging of red and green fluorescently-tagged proteins in prostate cancer cells. Co-localisation of proteins is evident in the merged lower image as a yellow signal. 47 Molecular Analysis of Prostate Cancer receptor action and the roles of the histone acetyl transferase, TIP60 and histone deacetylases as important transcriptional modifiers for the androgen receptor. Other ongoing projects include the study of ubiquitin-dependent proteasome-mediated destruction of the androgen receptor and the role of the E3 ubiquitin ligases, Mdm2 and PIRH2 (a novel coregulator of the androgen receptor). Recently, this research has been extended to include the action of lysine- and arginine- methylation in androgen receptor function. Major collaborative projects We have exploited strong links between the laboratory and the clinic at local, national and international levels to develop a programme of translational cancer research. We are involved in several projects to exploit the changes in cellular signalling pathways that occur in prostate cancer. We have established drug development programmes that apply small molecule inhibitors to prevent tumour growth. Recent collaborations within the NICR are pursuing alternative targets for drug therapy by investigating (i) the role of NFkB and (ii) the therapeutic potential of novel cyclin dependent kinase inhibitors in prostate cancer. Furthermore, we are involved in the design of new small molecule inhibitors that can be applied to selectively target prostate cancer cells that no longer respond to current hormone therapy. In an alternative strategy, we use viral vectors that target prostate cancer cells and we are now involved in the first UK gene therapy trial for prostate cancer. Prostate cancer is a heterogeneous disease necessitating the development of methodologies that can be applied to small numbers of cancer cells. We have developed laser capture microdissection of patient biopsies and are applying gene microarray and proteomics to identify novel markers associated with disease progression. High throughput analysis of clinical material is facilitated by our establishing of tissue microarrays. We are part of a large UK prostate cancer research collaborative called ProMPT (Prostate cancer: Mechanisms of Progression and Treatment). Linking Newcastle with Cambridge, Sheffield, Bristol, York and Manchester Universities, the collaborative brings together groups with diverse areas of expertise, so new laboratory discoveries can be quickly turned into new treatments. Collaborative projects include studying the function of adaptor proteins in androgen receptor signalling in prostate cancer (Prof David Neal and Dr Ian Mills of Cambridge University) and investigating bone morphogenetic proteins in skeletal metastases using prostate cancer models of angiogenesis and metastases (Profs Freddie Hamdy and Nicky Brown of Sheffield University). A further collaboration exists with Prof Norman Maitland and Dr Anne Collins (York University) to identify and characterise prostate stem cells. I am a founding member of an MRC Cancer Cell Bioimaging and eScience Consortium with Drs Trevor Jackson (School of Clinical and Laboratory Sciences) and Peter Andras (School of Computing Sciences). Within this consortium, we are applying real-time fluorescent imaging of normal and mutated proteins in prostate cancer cells to unravel their importance in cancer progression. The group has established links to major European prostate cancer research groups. We are partners in a multi-centre, European Union, Framework VI programme to identify and characterise new targets for the disease. Recently we have been awarded a four year Marie Curie Early Stage Training Programme, CANCURE. I am the Programme Director and principal investigator for this partnership of 11 European Institutions for the training of 11 postgraduate fellows. Gaughan L, Logan IR, Neal DE, Robson CN. “Regulation of androgen receptor and histone deacetylase 1 by Mdm2-mediated ubiquitylation.” Nucleic Acids Research (2005) 33: 13-26. Halkidou K, Logan IR, Cook S, Neal DE, Robson CN. “Putative involvement of the histone acetyltransferase Tip60 in ribosomal gene transcription.” Nucleic Acids Research (2004) 32: 1654-1665. Logan I, Sapountzi V, Gaughan L, Neal DE, Robson CN. “Control of human PIRH2 protein stability: involvement of tip60 and the proteasome.” Journal of Biological Chemistry (2004) 279: 1169611704. Mills IG, Gaughan L, Robson CN, Ross T, McCracken S, Kelly J, Neal DE. “Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors.” Journal of Cell Biology (2005) 170: 191-200. 48 Dr Mike Tilby Many important anti-cancer drugs combine with the genome of tumour cells to cause lethal DNA modifications. Many aspects of how these drugs act remain obscure and our work aims to better understand the effects of DNA-reactive drugs in cells, how cells respond to the resulting DNA damage and how to increase the sensitivity of tumour cells to these drugs. NICR Team: Hazel McCartney, Emma Meczes, Luke Harrison, Ian Jarvis, Tyrone John, and in close collaboration with Alan Boddy, Herbie Newell and Barabara Durkacz. A A B B C C Introduction Anticancer drugs that kill cancer cells by reacting with genomic DNA have been in clinical use for over 50 years. They react with DNA to form so-called drug-DNA adducts which are either tolerated and repaired or lead to cell death. How a tumour responds to treatment with these types of drugs depends upon how much drug reaches its intracellular target (i.e. DNA), what type of chemical modifications are caused to the DNA, how well the tumour cells are able to remove the damage and to what extent they are able to tolerate the presence of persistent adducts. In both experimental and clinical tumours, important questions remain to be answered concerning, for example, how many and what type of DNA adducts are formed in tumours in patients, how active drug actually reaches the DNA, whether the damage is preferentially located in certain regions of the genome and what are the most important cellular responses to the presence of damage. These topics are relevant to understanding why the outcome of treatment differs between patients, why certain drugs are more effective against certain types of tumour, how to optimise treatment for individual patients and how to improve the efficacy of these drugs with additional drugs. Several aspects of our work benefit from an ongoing collaboration with Dr. D.G. Pearson in the Geochemistry Laboratory of the Earth Sciences Department (Durham University). They have extensive analytical instruments and expertise for quantification of very low levels of platinum. Since the only way that platinum levels in cells will increase is from the exposure to platinum-based drugs, measurement of platinum can be used to quantify drug-induced DNA modifications. D Figure 1: Drug adducts on DNA (A) are analysed by mass spectrometry after vaporisation in an argon plasma at 10,000 ºC (B), by immunoassay (C), and by chromatography (D). Drug-DNA adducts in patients Knowledge of the levels of DNA adducts achieved in tissues of patients during therapy reveals information that could help explain why certain patients respond better than others and what levels of drug-DNA interactions in experimental situations are clinically relevant. Measurement of such adducts is technically challenging because they are formed at only very low levels. We have developed sensitive methods for quantifying adducts in clinical biopsies (Figure 1). These have so far been applied to analysis of easily obtainable peripheral blood cells. Application of these 49 Intra-Cellular Pharmacology of DNA-Reactive Anticancer Drugs Figure 2: Locations of DNA damage caused by radiation in nuclei of cells is revealed by immunofluorescent staining. Addition of repair inhibitor prevents repair of the damage and decreases survival of these breast cancer cells. abilities of cells to remove or tolerate the resulting adducts. We are investigating the mechanism by which novel compounds developed in the NICR cause a marked increase in sensitivity of cells to cisplatin and melphalan. We are also involved in studying the ability of other compounds developed in the NICR (in collaboration with KuDOS Pharmaceuticals, to enhance sensitivity to therapy by inhibiting the ability of cells to repair DNA damage. This has involved analysis of sites within the nuclei where biochemical changes occur at places of DNA damage. These sites can be visualised under a microscope as small spots which are induced by DNA damaging treatment. Repair processes normally cause a reduction in numbers of spots with time but the inhibitors block this decline (Figure 2). Distribution of adducts in different regions of the genome The DNA in every human cell has a total length of approximately 2 metres. The expressed genes only occupy a few percent of this length and are mainly clustered into small regions. We are investigating whether the formation and repair of DNA damage is uniform across the genome or focused on certain regions. Meczes EL, Azim-Araghi A, Ottley CJ, Pearson DG, Tilby MJ. “Specific adducts recognised by a monoclonal antibody against cisplatinmodified DNA.” Biochemical Pharmacology (2005) 70: 1717-1725. Cowell, IG, Durkacz, BW, Tilby, MJ. “Sensitization of breast carcinoma cells to ionizing radiation by small molecule inhibitors of DNA-dependent protein kinase and ataxia telangiectsia mutated.” Biochemical Pharmacology (2005) 71: 13-20. Gould KA, Nixon C, Tilby MJ. “p53 elevation in relation to levels and cytotoxicity of mono- and bifunctional melphalan-DNA adducts.” Molecular Pharmacology (2004) 66: 1301-1309. Veal GJ, Dias C, Price L, Parry A, Errington J, Hale J, Pearson ADJ, Boddy AV, Newell DR, Tilby MJ. “Influence of Cellular Factors and Pharmacokinetics on the Formation of Platinum-DNA Adducts in Leukocytes of Children Receiving Cisplatin Therapy.” Clinical Cancer Research (2001) 7: 2205-2212. methods has shown that for platinum-based drugs, the levels of adducts formed in white blood cells varies between patients and is not simply determined by the amount of drug present in the blood. In collaboration with surgeons we are now analysing DNA adducts in samples of solid tumour removed from patients after drug administration to investigate the relationship between adduct formation in blood cells and in tumours. Nature of adducts formed in cells Our recent studies have revealed evidence that a new, previously unrecognised, class of DNA adducts are formed by cisplatin in cells. These adducts comprise a significant proportion of all the adducts formed (about 30%). This finding resulted from the application of sensitive mass spectrometry techniques to identify drug-derived products in chromatographic analyses of DNA purified from drugtreated cells. Ongoing work aims to determine the nature of these adducts, their effects on cells and the extent of their formation in various cell types, including tissues from patients. Enhancing the effects of DNA damaging therapies It is possible to increase the effects of DNA damaging drugs by increasing the extent of drug-DNA interaction or by decreasing the 50 Dr Deborah Tweddle High risk neuroblastoma often initially responds to therapy, but later relapses with chemoresistant disease. We have shown that acquired abnormalities in the p53 pathway are important in neuroblastoma drug resistance and are currently investigating p53 abnormalities in paired diagnosis and relapsed neuroblastoma tumours. We are also investigating the role of the oncogene MYCN, amplified in around 50% high risk neuroblastoma, and its relationship with p53. NICR Team Jane Carr, Emma Bell, Lindi Chen, Hayley Moore, Helen Imrie, Rachel Daniel, Agata Rozanska, Angela Baker, Julian Board, and in close collaboration with John Lunec, Steve Clifford, Chris Redfern, Josef Vormoor and Gareth Veal. including p53 mutations, MDM2 amplification and p14ARF methylation. In our series 9/16 (53%) of cell lines established at relapse had abnormalities in the p53 pathway, but none established at diagnosis. We and others have shown that many of the cell lines with p53 pathway abnormalities are more resistant to chemo- and radio-therapy. Introduction Neuroblastoma is the commonest extracranial solid tumour with 120 cases diagnosed annually in the UK. Despite advances in other childhood cancers, high risk neuroblastoma (around 50% of all neuroblastoma) is curable in less than 30% of cases despite intensive multimodal therapy. High risk neuroblastoma, comprising MYCN amplified localised and infant neuroblastoma and stage 4 neuroblastoma over 1 year of age, often initially responds to therapy but later relapses with chemoresistant disease. New treatments and a better understanding of drug resistance mechanisms are needed to improve survival rates. p53 pathway abnormalities in relapsed neuroblastoma We are currently investigating the frequency of p53 pathway abnormalities in paired neuroblastomas from diagnosis and DNA damage + p14ARF MYCN _ MDM2 + + p21WAF1/CIP1 _ p53 p53 + p53 and drug resistance in neuroblastoma cell lines Mechanisms of drug resistance in neuroblastoma are incompletely understood. p53 is the most commonly mutated tumour suppressor gene in human cancer but is rarely mutated in neuroblastomas at diagnosis. A simplified diagram of the p53 pathway is shown in Figure 1. We have been investigating p53 pathway abnormalities in cell lines established from neuroblastomas at different stages of therapy. We identified an inactivating p53 mutation in a cell line established from a neuroblastoma at relapse, which was not present in the cell line established at diagnosis, and have now tested a further 23 neuroblastoma cell lines for p53 pathway abnormalities _ G1 phase Rb S phase Rb-P + Apoptosis Cell cycle arrest Figure 1: p53 activation upregulates genes causing cell cycle arrest and apoptosis. Loss of p53 function can occur through p53 mutation, MDM2 amplification and p14ARF methylation and deletion. MYCN may induce p14ARF. 51 The Role of MYCN and p53 in Neuroblastoma relapse. From 14 paired tumours analysed so far we have identified 2 relapsed tumours with p53 mutations, 2 relapsed tumours with p14ARF deletion and 1 diagnostic and relapsed tumour with MDM2 amplification. We are now increasing our sample size by including neuroblastomas from the United Kingdom Children’s Cancer Study Group and by collaborating with colleagues in the U.S. and Germany. If p53 pathway abnormalities occur frequently in relapsed neuroblastoma this may lead us to alter our current therapies to include agents which do not depend on functional p53 to exert their cytotoxicity. New therapies for neuroblastoma Current neuroblastoma therapy regimes include DNA damaging agents such as temozolamide and topotecan which are potentiated by DNA repair inhibitors, including PARP1 inhibitors, in a p53 independent manner. We are investigating the effect of these inhibitors in preclinical models of neuroblastoma and medulloblastoma. Novel therapies for neuroblastoma are currently being investigated in preclinical models and include the p53 independent synthetic retinoid fenretinide, and its synergism with conventional chemotherapy and proteosome inhibitors alone and in combination with fenretinide. MYCN, and its role in the p53 response to DNA damage Previously we reported that 3 MYCN amplified p53 wild-type neuroblastoma cell lines failed to undergo a G1 arrest after irradiation-induced DNA damage despite induction of p53regulated genes including the p21WAF1 cyclin-dependent kinase inhibitor. The hypothesis that this was causally linked to MYCN expression has been tested by studying a larger panel of cell lines, manipulating MYCN expression in a regulatable system, and knocking down MYCN in MYCN-amplified cell lines using RNA interference. The results suggest that MYCN expression is not responsible for the failure of MYCN amplified cell lines to G1 arrest after DNA damage. However, knocking down MYCN did increase the G1 population in exponentially growing cells, and we are currently investigating which genes might be involved in this process using expression microarrays, and also p53 and MYCN expression in tumours from MYCN transgenic mice. Newcastle UKCCSG reference laboratory for neuroblastoma molecular investigations The intensity of neuroblastoma therapies in current clinical trials is dependent on the presence or absence of MYCN amplification, which must be carried out by robust, reproducible techniques in a national reference laboratory. Newcastle is the UK reference laboratory and MYCN testing is undertaken in collaboration with Mrs Angela Baker and Mr Julian Board under the supervision of Dr Nick Bown (Cytogenetics Department, International Centre For Life). Carr J, Bell E, Kees UR, Pearson ADJ, Beris H, Lunec J, Tweddle DA. “Increased frequency of aberrations in the p53/MDM2/p14ARF pathway in neuroblastoma cell lines established at relapse.” Cancer Research (2006) 66: 2138-45. Tweddle DA, Malcolm AJ, Bown N, Pearson ADJ, Lunec J. “Evidence for the development of p53 mutations after cytotoxic therapy in a neuroblastoma cell line.” Cancer Research (2001) 61: 8-13. Tweddle DA, Malcolm AJ, Cole M, Pearson ADJ, Lunec J. “p53 cellular localisation and function in neuroblastoma: Evidence for defective G1 arrest despite WAF1 induction in MYCN amplified cells.” American Journal of Pathology (2001) 158: 2067-2077. Cohn SL, Tweddle DA. “MYCN Amplification Remains Prognostically Strong Twenty Years Following its ‘Clinical Debut’” European Journal of Cancer (2004) 40: 2639-2642. p53 localisation in differentiating neuroblastoma Our previous immunohistochemical studies have demonstrated nuclear p53 in undifferentiated neuroblastoma and both nuclear and cytoplasmic p53 in differentiating neuroblastoma tumours. We are investigating this further in cell lines undergoing retinoic acid induced differentiation. Altered subcellular localisation of p53 has been reported by others to be an alternative mechanism of p53 inactivation in neuroblastoma. 52 Dr Gareth Veal In addition to the development of new therapeutics, selected on the basis of the molecular and cellular pathology of the target tumour, it is important to explore more effective ways of using current drugs for the treatment of children with cancer. Our aims are to increase the understanding of the factors that determine drug efficacy in paediatric oncology and use this information to optimise clinical utility. NICR Team Jane Armstrong, Girish Chinnaswamy, Julie Errington, Sophie Rowbotham and in close collaboration with Alan Boddy, Chris Redfern and Mike Tilby. Clinical pharmacology studies with actinomycin D The antitumour antibiotic actinomycin D is used routinely to treat certain forms of cancer in both adults and children and is a key component in the successful treatment of Wilms’ tumor, with more than 80% of patients treated now being cured of the disease. However, its use is associated with the incidence of lifethreatening veno-occlusive disease and a clearly defined relationship between dose intensity and the development of hepatotoxicity in this patient group has been indicated. We have developed an analytical assay which allows the quantification of actinomycin D in clinical samples and have recently completed a pilot study investigating actinomycin D pharmacokinetics and the extent of inter-patient variation in drug exposure. Data indicates that dosing of actinomycin D based on surface-area is not optimal, either in younger patients, in whom there may be an increased risk of toxicity, or in older patients Introduction The current practice of basing drug doses on body size in paediatric oncology is associated with a large degree of interpatient variation in clinical outcome following standard doses of chemotherapy. This may be optimised for many chemotherapeutics by identifying more rational approaches to drug dosing, based on patient characteristics such as pharmacogenetic factors and drug metabolizing activity. It is anticipated that the use of this information together with therapeutic drug monitoring and adaptive dosing to achieve targeted systemic drug exposures in individual patients will lead to more consistent clinical outcomes (Figure 1). However, for the vast majority of anticancer drugs used in paediatric oncology, limited clinical pharmacology data is available to dose patients based on any meaningful scientific rationale and there is much work that can be done to optimise treatment. In order to advance this area of research, our work focuses on both clinical pharmacology studies and translational laboratory-based research involving key drugs used for the treatment of children with cancer. We are currently running a number of clinical studies associated with a wide range of approaches to cancer treatment. Studies are carried out at a national level, in collaboration with the United Kingdom Children’s Cancer Study Group (UKCCSG), involving patients at centres throughout the UK and have successfully recruited several hundred patients over the past 5 years. Drug exposure (AUC) Toxicity Therapeutic Window Efficacy Standard Therapy Alternative/modified Therapy Figure 1: Schematic representation of the relationship between drug exposure, clinical toxicity and response. Pharmacokinetic or pharmacogenetic variation may result in increased drug toxicity or decreased drug efficacy in individual patients. 53 Translational & Clinical Pharmacology Studies in Paediatric Oncology where doses are capped. This has led to a more detailed investigation of possible relationships between actinomycin D dosing, pharmacokinetics and toxicity in infants with Wilms’ tumour, a patient subpopulation with an increased risk of venoocclusive disease. Translational studies with 13-cisRA in neuroblastoma Despite the successful use of 13-cis-retinoic acid (13-cisRA) for the treatment of high-risk neuroblastoma, it is widely accepted that its clinical utility could be further improved. This may be achieved either through the development of new synthetic retinoids, by the co-administration of agents to inhibit drug metabolism, or by improving the dosing regimens of currently used retinoids. Results from a preliminary clinical pharmacology study indicate that the method of drug administration is a major determinant of systemic drug exposure, with lower 13-cisRA plasma levels observed in younger patients who are unable to swallow the capsules and who require the drug to be extracted and mixed with food prior to administration (Figure 2). These data also indicate a significant relationship between the extent of drug metabolism during treatment and incidence of disease relapse. This has led to the initiation of a larger study to investigate the pharmacokinetics of 13-cisRA in conjunction with pharmacogenetic data concerning the expression of key cytochrome P450 enzymes thought to play a role in retinoid metabolism. In vitro studies investigating ways of optimising the use of retinoids by inhibiting drug metabolism and isomerisation with R116010, an inhibitor of the cytochrome P450 enzyme CYP26, have produced promising results and have now progressed to in vivo studies. These data, along with results from an ongoing project investigating mechanisms of synergy and retinoid interactions in neuroblastoma therapy drug combinations, will potentially impact on the future clinical use of these agents. In addition to these two important areas of research, ongoing clinical and laboratory studies are being carried out involving the drugs carboplatin, etoposide, vincristine, melphalan and busulphan for the treatment of childhood malignancies such as rhabdomyosarcoma, Wilms’ tumour, neuroblastoma, brain tumours and lymphoma. Figure 2: Peak plasma concentrations of 13-cisRA in children who swallowed 13-cisRA capsules versus those patients for whom the contents were extracted prior to administration. Optimisation of cancer treatment with the use of pharmacologically guided drug therapy, to determine the drugs and drug doses most likely to produce a therapeutic effect, in conjunction with pharmacogenetic data concerning enzymes involved in drug metabolism or resistance, represents a very realistic and achievable goal. Veal GJ, Cole M, Errington J, Parry A, Hale J, et al. “Pharmacokinetics of dactinomycin in a pediatric patient population – a United Kingdom Children’s Cancer Study Group study.” Clinical Cancer Research (2005) 11: 5893-5899. Armstrong JL, Ruiz M, Boddy AV, Redfern CPF, Pearson ADJ, Veal GJ. Increasing the intracellular availability of all-trans-retinoic acid in neuroblastoma cells. British Journal of Cancer (2005) 92: 696-704. Cooper B, Veal GJ, Radivoyevitch T, Tilby MJ, Meyerson HJ, et al. “A Phase I and pharmacodynamic study of fludarabine, carboplatin and topotecan in patients with relapsed, refractory, or high-risk acute leukemia.” Clinical Cancer Research (2004) 10: 6830-6839. Veal GJ, English MW, Grundy RG, Shakespeare C, Glaser A, et al. “Pharmacokinetically guided dosing of carboplatin in paediatric cancer patients with bilateral nephrectomy.” Cancer Chemotherapy and Pharmacology (2004) 54: 295-300. 54 Dr Mark Verrill Clinical Trials in breast cancer with a focus on translational endpoints. Main laboratory studies relate to pharmacogenomics in patients receiving Doxorubicin/ Cyclophosphamide adjuvant chemotherapy and Paclitaxel in the National Cancer Research Network (NCRN) Will Weekly Win trial for which I am the Chief Investigator. Clinical trials include the NCRN portfolio and a number of early studies of novel anticancer drugs. NICR Team: Sandra Beare, Johanne Lee, and in close collaboration with Alan Boddy, Hilary Calvert, John Lunec, Felicity May, Ruth Plummer, Bruce Westley and clinical staff based at the NCCT. Introduction Breast cancer is the commonest cancer in women with more that 40,000 new cases in the UK each year. Despite notable advances in treatment in recent years, many women still experience recurrences of their cancer after initial treatment given with curative intent and there is an unmet need for better treatments. We have a number of different research interests in breast cancer, bridging the gap between the laboratory and clinic. Northern Cancer Research Network clinical trials The National Cancer Research Network (NCRN) supports a number of clinical trials in breast cancer and the Newcastle Breast Unit have taken a major role in many of these with representation on the trial working groups and substantial recruitment. Achievements include >100 patients in the TACT (‘A randomised trial of standard anthracycline-based chemotherapy with fluorouracil, epirubicin and cyclophosphamide (FEC) or Epirubicin and CMF (Epi-CMF) vs FEC followed by sequential docetaxel as adjuvant treatment for women with early breast cancer’) and >50 patients in the tAnGo (‘A Phase III randomised trial of gemcitabine in paclitaxel-containing, epirubicin based adjuvant chemotherapy for women with early stage breast cancer’) adjuvant studies. I am also the Chief Investigator in the NCRN Will Weekly Win Figure 1 Patient receiving her first treatment with Epirubicin in the NCRN TACT breast cancer trial 55 Breast Cancer & Sarcoma Research trial (‘A randomised 2-arm, prospective, multi-centre, openlabel Phase III trial comparing the activity and safety of a weekly versus a 3 weekly Paclitaxel treatment schedule in patients with advanced or metastatic breast cancer’) and the leading recruiter with >60 patients. The TACT-2 (‘Trial of accelerated adjuvant chemotherapy with capecitabine in early breast cancer’) trial will open in 2006. We also contributed to the pivotal HERA study of adjuvant Herceptin. Sarcoma I also have an interest in sarcoma – a cancer of the connective tissues, being an active participant in the EORTC Soft Tissue and Bone Sarcoma Group and also contributes to its clinical trial portfolio. Trials include Euro-E.W.I.N.G. 99 (‘European Ewing Tumour Working Initiatives of National Groups: Ewing Tumour Studies 1999’), Euramos (‘A randomized trial of the European and American Osteosarcoma study Group to optimize treatment strategies for resectable osteosarcoma based on histological response to pre-operative chemotherapy’), a randomised trial of doxorubicin versus doxorubicin plus ifosfamide in advanced soft tissue sarcoma and trials of adjuvant imatinib and sunitinib in gastrointestinal stromal tumours (GIST). In the laboratory there is an ongoing tissue collection programme supporting investigation of the role gene mutations in the pathogenesis of sarcomas, in particular p14-ARF. Pharmacogenomics Cancer treatments, in general, use crude means to tailor treatment to patients based on height and weight. It is known that there are variations in the genes encoding drug handling and metabolism and these may lead to major differences in exposure to cytotoxic agents between individuals. Many of these differences are due to single nucleotide polymorphisms in genes. We have acquired samples from both the national NCRN Will Weekly Win trial and from more that 200 patients receiving a standard anthracycline containing adjuvant chemotherapy regimen in Newcastle in order to analyse the effect of single nucleotide polymorphisms (SNP’s) on drug toxicity and treatment outcome. Other phase II and III trials. The Clinical Trials Unit at Newcastle General Hospital is involved in a series of phase II and phase III trials including Novel tubulin binding agents (Abraxane, Tocosol, CT 2103, Epothilone B, Oral Vinorelbine, Vinflunine). Other current studies include a phase II trial of a novel kinesin spindle poison, a patient preference bisphosphonate study and an EORTC translational neoadjuvant Taxane study. Leonard RCF, Lind M, Twelves C, Coleman R, van Belle S, Wilson C, Ledermann J, Kennedy I, Barrett-Lee P, Perren T, Verrill M, Cameron D, Foster E, Yellowlees A, Crown J. “Conventional adjuvant chemotherapy versus single-cycle, autograft-supported, high-dose, late-intensification chemotherapy in high-risk breast cancer patients: A randomized trial.” Journal of the National Cancer Institute (2004) 96: 1076-1083. Serin D, Verrill M, Jones A, Delozier T, Coleman R, Kreuser E-D, Mross K, Longerey B, Brandely M. “Vinorelbine alternating oral and intravenous plus epirubicin in first line therapy of metastatic breast cancer: Results of a multicenter phase II study.” British Journal of Cancer (2005) 92: 1989-1996. Judson I, Leahy M, Whelan J, Lorigan P, Verrill M, Grimer R, Robinson M. “A guideline for the management of gastrointestinal stromal tumour (GIST).” Sarcoma, (2002) 6: 83-87. Morse R, Rodgers J, Verrill M, Kendell K. “Neuropsychological functioning following systemic treatment in women treated for breast cancer: A review”. European Journal of Cancer (2003) 39: 2288-2297. Cognitive function It is well known that cognitive function may be impaired in breast cancer survivors and that this may be a result of treatment, both with chemotherapy and hormones. This has been examined and we have reported deficits in cognitive function as a result of use of either treatment modality. 56 Prof Josef Vormoor By studying fundamental aspects of tumour cell biology (leukaemic stem cells, Ewing’s sarcoma metastasis) our research tries to advance the understanding of these childhood malignancies and their resistance to chemotherapy. This will allow us to take a translational research approach and develop new treatments for children and adolescents with cancer. NICR Team: In close collaboration with Alan Boddy, Steve Clifford, Andy Hall, Julie Irving, John Lunec, Herbie Newell, Ruth Plummer, Chris Redfern, Debbie Tweddle and Gareth Veal. Identification and characterisation of candidate malignant stem cell populations in childhood leukaemia There is increasing evidence that, as in normal haematopoiesis, the malignant growth of the leukaemic cell clone is maintained by only a small fraction of highly specialized stem cells. Unravelling the exact biology of these leukaemic stem cells as the disease-maintaining cells is essential for understanding the leukaemic process. Our research aim is to identify, isolate and characterise candidate leukaemic stem cells in the most common type of childhood leukaemia, i.e. acute lymphoblastic leukaemia (ALL). Using modern multi-parameter (4- and 6-colour) flow cytometry, candidate leukaemic stem cell populations are defined by patterns of cell surface markers (reflecting the earliest stages of lymphoid differentiation and leukaemia-specific expression profiles). These candidate leukaemic stem cell populations are purified by cell sorting for further analysis. Molecular testing for the presence of leukaemia-associated chromosomal abnormalities provides information on whether these cells belong to the leukaemic clone and thus indirect information on the cell of origin in which the leukaemia arises. In the context of work by many other groups, these experiments have provided the basis for a model of a stem cell hierarchy in childhood leukaemia: while good prognosis ALL/t(12;21) only involves the CD19+ B lymphoid compartment (Fig. 1: dark blue lightning bolt), certain types of high risk ALL/t(9;22) and t(4;11) appear to originate in a more primitive (CD19-) but already lymphoid-committed cell (light blue lightning bolt) and acute myeloid leukaemia (AML) arises in very primitive cells with a phenotype characteristic of pluripotent stem cells (CD34+CD38-) (Fig. 1: brown lightning bolt). The stem cell properties of the candidate leukaemic stem cell populations are now being tested by transplantation onto immune-deficient mice. With this experimental approach we will be able to identify the cells that maintain the disease in vivo and thus have the ability of causing relapse. The long term goal of this project is to investigate stem cellspecific mechanisms of chemotherapy resistance and to identify leukaemic stem cell-specific therapeutic targets by direct characterisation of the gene expression profile (transcriptome) of leukaemic stem cell populations in childhood ALL. Figure 1: Model of the stem cell hierarchy in childhood leukaemia. 57 Cellular Biology of Childhood ALL and Ewing’s Sarcoma and Phase I/II Studies on New Agents for Children with Cancer Mechanisms of organ-specific metastasis of Ewing’s sarcoma Ewing’s sarcoma is the second most common bone tumour in children and young people. The most important negative prognostic factor for patients with Ewing’s sarcoma predicting relapse and failure of therapy is metastatic spread. While most patients with localised disease can be cured, the chance for long term survival of patients with lung metastases drops down to approximately 30% and the survival of patients with primary bone metastases unfortunately is below 20%. To study the mechanisms of Ewing’s sarcoma metastasis our group has established a mouse model that reflects tumour spread in patients. Human Ewing’s sarcoma cells are injected intravenously into immune deficient mice and form lung, bone and bone marrow metastases at high frequency. Ewing’s sarcomas appear to use a similar mechanism to haematopoietic stem cells to home onto bone and bone marrow and the bone marrow micro-environment provides important survival signals that help, rendering human Ewing’s sarcoma cells resistant to chemotherapy (tumour cell niche). Ewing’s sarcoma cells adhere to bone marrow endothelium via binding to VCAM-1, egress into the bone marrow induced by the chemokine SDF-1 and receive important survival signals by adhesion to stroma cells and extracellular matrix components, e.g. fibronection (FN). By isolation of tumour clones with preferential metastasis to lungs and bone, and subsequent gene expression profiling of these clones, we are currently using the mouse model for the identification of genes involved in organ-specific metastasis. In collaboration with Dr. Franzius (Department of Nuclear Medicine, University of Munster, Germany) we have been able to visualise Ewing’s sarcoma metastases in living mice using FDG-PET (micro-PET). The Ewing’s sarcoma mouse model is, therefore, not only a tool to study experimental tumour metastasis but also allows pre-clinical development and testing of new PET tracers for functional imaging. Phase I/II studies on new agents for children with cancer From a clinician’s perspective there is a great need to develop new treatments for those of our patients who have so far not benefited from the progress made in paediatric oncology and who still have a very poor chance of long term survival. These include patients with relapsed leukaemia, patients with advanced stage sarcomas (including Ewing’s sarcoma) and neuroblastoma and certain patients with brain tumours. We are aiming at increasing the number of phase I/II studies in children with cancer. Our research in Newcastle spans all areas of drug development from basic biology (target identification), drug development, preclinical drug testing and pharmacology to phase I/II studies in children. The studies will be organised within the national framework provided by the UKCCSG Division of Therapeutics Committee, and Newcastle is one of the phase I/II centres accredited by the European Consortium for Innovative Therapies for Children with Cancer (ITCC). Hotfilder M, Röttgers S, Rosemann A, Schrauder A, Schrappe M, Pieters R, Jürgens H, Harbott J, Vormoor J. “Leukemic stem cells in childhood high risk ALL/t(9;22) and t(4;11) are present in primitive lymphoid-restricted CD34+CD19- cells.” Cancer Research (2005) 65: 1442 – 1449. Hotfilder M, Röttgers S, Rosemann A, Jürgens H, Harbott J, Vormoor J. “Immature CD34+CD19- progenitor/stem cells in TEL/AML1positive acute lymphoblastic leukemia are genetically and functionally normal.” Blood (2002) 100: 640 – 646. Hotfilder M, Sondermann P, Senß A, van Valen F, Jürgens H, Vormoor J. “PI3K/Akt is involved in mediating survival signals that rescue Ewing tumour cells from fibroblast growth factor 2-induced cell death.” British Journal of Cancer (2005) 92: 705 – 710. Vormoor J, Baersch G, Decker S, Hotfilder M, Schäfer KL, Pelken L, Rübe Ch, van Valen F, Jürgens H, Dockhorn-Dworniczak B. “Establishment of an in vivo model for paediatric Ewing tumors by transplantation into NOD/scid mice.” Paediatric Research (2001) 49: 332 - 341. 58 Prof Bruce R Westley Oestrogens are extremely important in breast cancer progression and treatment. Our research aims to identify and understand the mechanisms by which oestrogens exert their effects on breast cancer cells and tumours. Our findings are being translated to the clinic in collaboration with Medical Oncologists. NICR Team: Paul Wright, Gail de Blaquière, Rehan Saif, Nahed Hawsawi, Claire Worrall and in close collaboration with Felicity May, Mark Verrill and Hilary Calvert. receptor. Genes that are involved in mediating the effects of oestrogen may prove worthwhile targets for breast cancer therapy. The IGF signal transduction pathway The insulin-like growth factors, IGF-1 and IGF-2, are synthesised principally in the liver but also in other peripheral tissues. They control normal growth and development. We demonstrated that oestrogens sensitise breast cancer cells to the proliferative effects of IGFs. We have shown that oestrogens increase the expression of Introduction Breast cancer kills approximately 15,000 women in the UK each year. Oestrogens are important in the progression of breast cancer and strategies to inhibit their activity are widely used in its treatment. These include the inhibition of the effects of oestrogens by anti-oestrogens such as Tamoxifen and the inhibition of endogenous oestrogen synthesis in the ovary and peripheral tissues by aromatase inhibitors such as arimidex. Oestrogens exert their effects by binding to an intracellular protein called the oestrogen receptor (Figure 1). The oestrogen receptor controls the expression of a repertoire of genes (Westley and May, 2006). The proteins encoded by these genes mediate oestrogen action and some are responsible for the progression of breast cancer. Our research programme is aimed at the identification of the oestrogen-regulated genes which are most important for controlling the progression of breast cancer. The identification of key oestrogen regulated genes in breast cancer may firstly elucidate the biological process of tumour progression. The identification of these genes could help to understand tumour progression in a wide variety of cancers including breast cancer and identify novel targets for therapeutic intervention. Secondly it may provide a panel of accurate markers of oestrogens responsiveness in breast cancer. Only a proportion of women with breast cancer benefit from hormone therapy. This would individualise treatment for women with breast cancer thereby restricting the use of hormone therapy to those who would be most likely to obtain benefit. Thirdly it may present novel therapeutic targets that are downstream of the oestrogen Figure 1: Effects of oestrogen in breast cancer cells. Oestrogen binds to a receptor protein (orange) and activates the transcription of a repertoire of genes. The genes we are studying encode: proteins involved in mediating the effects of the insulin like growth factors (IGFs) (pale blue), the trefoil proteins TFF1 and TFF3 (dark blue), and proteins that regulate exocytosis (pink). 59 Oestrogen Responsive Breast Cancer cancer cells. We have demonstrated that TFF1 stimulates the motility of breast cancer cells and that the TFF1 dimer is more potent than the TFF1 monomer (Prest et al., 2002) and have found that TFF1 is expressed at high levels in metastatic tumour cells and in primary breast tumours that progress rapidly and become symptomatic within the interval between screenings for breast cancer (Crosier et al., 2001). This is consistent with trefoil proteins being involved in tumour progression. We are investigating the possibility that breast cancer cells subvert the activity of trefoil proteins in the protection of normal cells to facilitate the invasion and metastasis of breast cancer cells. Proteins encoded by oestrogen-regulated genes Figure 2: Identification of oestrogen regulated genes in MCF-7, EFF3 and EFM19 breast cancer cells. The columns on the left show the number of genes regulated by oestrogen in one, two and three cell lines. The heat map on the right shows the nineteen consistently regulated genes. components of the insulin-like growth factor signal transduction pathway in breast cancer cells thereby increasing the responsiveness of breast cancer cells to IGFs. This may mediate the effects of oestrogens on breast cancer cells. Thus far, we have shown that oestrogens increase the expression of the type I IGF cell surface receptor and the immediate downstream signal transduction protein IRS-1 (Molloy et al., 2000). We are exploring the possibility that targeting the IGF signalling pathway may be effective in anti-oestrogen resistant breast cancer. We have used Affymetrix Hu U133 Plus 2.0 Genechips to identify oestrogen-regulated genes in breast cancer cells. A panel of nineteen genes were regulated consistently by oestrogen in a number of oestrogen-responsive breast cancer cell lines (Figure 2). Some of these genes had been identified previously as oestrogenregulated, but many are novel oestrogen-regulated genes. Genes that are regulated consistently by oestrogen in several cell lines are likely to have a central and fundamentally important role in the dependence of breast cancers on oestrogen. Many of the genes encode proteins that are either involved directly in intracellular vesicle formation or trafficking including exocytosis (7 genes), or are themselves secreted (7 genes). Surprisingly, only three of the consistently regulated genes encode proteins involved in the cell cycle. We are currently examining the function of these genes in breast cancer cells and in breast tumours. Westley BR, May FEB. “Identification of steroid hormone regulated genes in breast cancer.” Methods in Molecular Medicine (2006) 120: 363-388. Molloy CA, May FEB, Westley BR. “Insulin receptor substrate-1 expression is regulated by estrogen in the MCF-7 breast cancer cell line.” Journal of Biological Chemistry (2000) 275: 12565-12571. Prest SJ, May FEB, Westley BR. “The estrogen-regulated protein, TFF1, stimulates migration of human breast cancer cells.” FASEB Journal (2002) 16: 592-594. Crosier M, Scott D, Wilson RG, Griffiths CDM, May FEB, Westley BR. “High expression of trefoil protein TFF1 in interval breast cancers.” American Journal of. Pathology (2001) 159: 215-221. Trefoil proteins The human trefoil proteins, TFF1, TFF2 and TFF3 are expressed at highest levels in normal tissues in the mucosa of the gastrointestinal tract and at lower levels in other tissues including breast. Trefoil proteins are expressed in a variety of cancers including breast cancer where the expression of TFF1 and TFF3 is increased dramatically by oestrogens. The evidence that a normal role of trefoil proteins is to aid restitution of the gastrointestinal mucosa by stimulating epithelial cell migration led us to propose that the role of TFF1 and TFF3 in breast cancer cells could be to aid tumour cell dissemination by stimulating movement of breast 60 List of Sponsors Ali's Dream Association for International Cancer Research Astex Therapeutics Charlie's Challenge CLIC Sargent Dame Allan's School Department of Health AstraZeneca Donaldson J A Aventis Eli Lilly Biotechnology and Biological Sciences Research Council Bioscience Technologies Biotica Breast Cancer Campaign Brian Brennan Bequest British Biotech British Urological Foundation Cancer and Polio Research Fund Cancer Research UK Centre for Excellence for Life Sciences Erasmus MV European Brewers Association Research European Union Gateshead Health NHS Trust GlaxoSmithKline Institute Gustave Roussy JGWP Katie Trust KuDOS Pharmaceuticals Lawrence K 61 We would like to thank the following sponsors who have contributed more than £1,000 to the Northern Institute for Cancer Research, between 2004 – 2006: Leukaemia Research Fund Lionesses of Darlington Liver North Medical Research Council National Cancer Institute (US) Royal College of Surgeons Samantha Dickson Trust Sembcorp Utilities UK Sport Aiding Medical Research for Kids (SPARKS) Taxolog National Cancer Research Institute (UK) Tyneside Leukaemia Research Fund Neuroblastoma Society Newcastle Healthcare Trust North of England Childrens Cancer Research Fund Northern Cancer Care & Research Society OSI Pharmaceuticals Pattison JH & D Pfizer Ralph Shackman Trust United Kingdom Childrens Cancer Study Group (UKCCSG) University of York Vernalis Wallace G & S Wellcome Trust Wilkins B We would also like to thank other contributors who have donated funds in the past but who are not listed here. 62 Staff list Scientific Director Prof Andy Hall a.g.hall@ncl.ac.uk Dr Julie Irving j.a.e.irving@ncl.ac.uk Research Staff Senior Research Associates Ms Sandy Beare Dr Christine Challen Dr Sally Coulthard Dr Linda Hogarth Dr Janet Lindsey Dr Xiaohong Lu Dr Meryl Lusher Dr Arthur McKie Dr Joyce Nutt Mrs Julieann Sludden Mr Gordon Taylor Dr Elaine Willmore Prof Hing Leung h.y.leung@ncl.ac.uk Clinical Director Prof Hilary Calvert Contact Institute Secretary Dr John Lunec john.lunec@ncl.ac.uk Assistant Director Dr Christopher Redfern chris.redfern@ncl.ac.uk Dr Ross Maxwell ross.maxwell@ncl.ac.uk Institute Manager Mrs Jill Hogg jill.hogg@ncl.ac.uk Dr Felicity EB May f.e.b.may@ncl.ac.uk Prof Herbie Newell herbie.newell@ncl.ac.uk Academic Staff Prof Alan Boddy alan.boddy@ncl.ac.uk Dr Ruth Plummer e.r.plummer@ncl.ac.uk Dr Steven Clifford s.c.clifford@ncl.ac.uk Dr Helen Reeves h.l.reeves@ncl.ac.uk Research Associates Dr Jane Armstrong Ms Angela Baker Mr Michael Batey Dr Garry Beale Mr Julian Board Dr Celine Cano-Soumillac Ms Jane Carr Ms Marion Case Dr Emma Clarke Mr Mike Cole Dr Steven Darby Dr Gail De Blaquiere Dr Luke Gaughan Dr Helen Imrie Dr Ian Logan Mrs Elizabeth Matheson Dr Emma Meczes Mr Kieran O’Toole Prof Nicola Curtin n.j.curtin@ncl.ac.uk Prof Craig Robson c.n.robson@ncl.ac.uk Dr Barbara Durkacz b.w.durkacz@ncl.ac.uk Dr Mike Tilby m.j.tilby@ncl.ac.uk Dr Richard Edmondson richard.edmondson@ncl.ac.uk Dr Debbie Tweddle d.a.tweddle@ncl.ac.uk Prof David Ellison d.w.ellison@ncl.ac.uk Mr Nikhil Vasdev nikhil.vasdev@ncl.ac.uk Dr Vincent Gnanapragasm v.j.gnanapragasm@ncl.ac.uk Dr Gareth Veal g.j.veal@ncl.ac.uk Prof Roger Griffin r.j.griffin@ncl.ac.uk Dr Mark Verrill mark.verrill@ncl.ac.uk Dr Ian Hardcastle i.r.hardcastle@ncl.ac.uk Prof Josef Vormoor h.j.vormoor@ncl.ac.uk Mr Rakesh Heer rakesh.heer@ncl.ac.uk Prof Bruce R Westley b.r.westley@ncl.ac.uk 63 Dr Amy Peasland Dr Frida Pontham Dr Anastacia Rigas Dr Celine Roche Mr Huw Thomas Mr Eric Valeur Dr Stephany Veuger Mrs Lan-Zhen Wang Mr Simon Pridgeon Dr Klaus Rohe Dr Arvind Rajagopal Dr Rehan Saif Dr Mark Tones Dr Paul Wright Administrative Staff Institute Secretary Mrs Sandra Cartwright sandra.cartwright@ncl.ac.uk PA Mrs Lynn Driscoll l.k.driscoll@ncl.ac.uk Technical Staff Senior Research Technicians Mrs Susan Cook Ms Rachel Daniel Dr Karen Haggerty Dr Claire Hutton Mrs Suzanne Kyle Mrs Hazel McCartney Mrs Lynne Minto Secretary Ms Pauline Stephenson pauline.stephenson@ncl.ac.uk Junior Research Associates Ms Kelly Armstrong Mr Matthew Gorton Finance Secretary Ms Trudie Bryson t.a.bryson@ncl.ac.uk Research Fellow Dr Girish Chinnaswamy Computing Officer Mr Graham Bray g.p.bray@ncl.ac.uk Clinical Fellow Dr Ali Kucukmetin Research Technicians Ms Julie Errington Mrs Melanie Griffin Ms Hesta McNeill Mr Evan Mulligan Ms Agata Rozanska Ms Sarah Wilkinson Clinical Research Fellows Mr Dipanker Chattopadhay Dr Ann Fisher Dr James Hayden Dr Neil Jennings Dr Johanne Lee Dr Jane Margetts Dr Stuart McCracken Dr Luca Miele Building Administrator Dr Rebecca Kenyon r.m.kenyon@ncl.ac.uk Research Support Officer Dr Penney Gray p.a.gray@ncl.ac.uk Laboratory Technicians Mrs Norma Marsh Mrs Karen Perry CRT Business Manager Dr Phil Elstob pelstob@cancertechnology.com 64 How to find us… The Paul O’Gorman Building is well placed for local, national and international travel. Maps around the University campus show the location of the Paul O’Gorman Building and both pedestrian and disabled access routes. By Metro: The Tyne and Wear Metro is the most convenient way to get from Gateshead, Sunderland, Newcastle Airport, Tynemouth and also to many suburban areas to the centre of Newcastle. The nearest station to the University campus is Haymarket. By Rail: Newcastle Central Station is situated on the east coast mainline making it easily accessible to London Kings Cross, Edinburgh and Glasgow. On arrival, taxis are usually available immediately outside (though there can sometimes be a queue). Newcastle Central Station is a short 10 minute drive away. Alternatively, a Metro station is located in the Central Station. By Air: Newcastle International airport has direct flights to many UK and European cities and is only around a 20 minute drive away. On arrival, taxis are usually available at the taxi rank outside. Alternatively, there is a Metro station located in the airport terminal. CRAMLINGTON A1 A193 By Road: Follow the A1 towards Newcastle, or if using the A69, travel west bound until the junction with A1 where you should join the A1 northbound. Whether you are traveling from the north or south, you should leave the A1 at the junction with the A167/A696 and follow the signs for the 'City Centre' (A167). Take the exit marked ‘Universities, RVI, Eldon Square’ (B1318, City North) and turn right to go across the flyover. Take the third exit off the roundabout and then first left. The Paul O’Gorman Building is in front of you. There is a public car park located on B1318 (Claremont Road). WHITLEY BAY Newcastle International A1 A1058 A19 A1058 A168 NORTH SHIELDS GOSFORTH A193 A69 WALLSEND NEWCASTLE UPON TYNE A184 Tyne SOUTH SHIELDS A183 A1 GATESHEAD A167 A184 A184 A194(M) A19 A1231 SUNDERLAND Wear WASHINGTON A1(M) 65 To A1, A69 & Airport Cla rem on tR oad Newcastle upon Tyne Coast Road & Tynemouth A1058 A167(M) ers nt Hu ad Ro Ri ch on ds ar A167(M) P Royal Victoria Infirmary a Ro d Claremont Road St. James’ Park yS tre et Haymarket Newcastle United FC Inset map here Detailed section Paul O’Gorman Building rry be aw e Str Plac Per c A167(M) A189 A186 St. Jam es Bou levard Westg ate Road e Tyn ge d Bri Manors Bigg Market Central Station ing Sw idge Br A167(M) A189 e yn rT ve Ri To Gateshead International Stadium & Sunderland 66 Northern Institute for Cancer Research Paul O’Gorman Building Newcastle University Newcastle upon Tyne UK Tel: +44 (0) 191 246 4300 Fax: +44 (0) 191 246 4301 www.ncl.ac.uk/nicr Please direct any enquiries to: nicr-enquiries@ncl.ac.uk