research reports

University of Sheffield

2005/6

Director's Introduction
Professor B. W. Hancock

Cancer research in Sheffield has well-established programmes in both scientific and clinical research. The main scientific research programme is based in the YCR Institute for Cancer Studies, which occupies 800 sq. metres of refurbished space on the top floor of the Medical School. As well as research space for seven investigators, the Institute provides core facilities for cancer researchers throughout Sheffield. These facilities include high throughput DNA sample processing, deconvolutional microscopy, and a radiation source.

The Academic Unit of Clinical Oncology is located in the Cancer Research Centre (CRC), a purpose-built clinical trials facility at the Weston Park Hospital. The CRC provides facilities for the assessment and treatment of patients, office space for staff with fully networked computer systems, and specialist facilities including laboratory space and bone densitometry. The North Trent Cancer Research Network is co-ordinated from the CRC.

While these two research components are physically separate, excellent interactions between them have already been established, with numerous collaborations based in cancer genetics, genetic epidemiology and new therapies. Other important cancer research teams are based in the Medical School with specialised facilities within the Division of Genomic Medicine (Academic Units of Pathology, Infection & Immunity and Respiratory Medicine), the Division of Clinical Sciences (S)(Academic Units of Ophthalmology & Orthoptics, Palliative Medicine, Surgical Oncology [the Microcirculation Research Group] and Urology) and in the University Departments of Biomedical Science and Molecular Biology and Biotechnology. There are also researchers now established in the School of Clinical Dentistry (Department of Oral Pathology).

Director: Professor G.W. Duff
Deputy Director (Clinical): Professor B.W. Hancock

SECTION OF ONCOLOGY & PATHOLOGY
Section Head: Professor C.E. Lewis

YCR Institute for Cancer Studies
Head: Professor M. Meuth

The effect of single nucleotide polymorphisms on clinical outcome and survival in the FOCUS clinical trial of metastatic colorectal cancer
Dr. A. Cox and Profs. P. Quirke and M. Seymour, (University of Leeds)

The aim of this project, which was funded in December 2005, is to identify genetic variants that are associated with outcome and survival from metastatic colorectal cancer. The mainstay of first line chemotherapy for many years has been 5-fluorouracil, but the FOCUS clinical trial has helped determine the best way to use newer agents such as oxaliplatin and irinotecan. We have previously shown that inherited variation in the genes involved in the cellular response to DNA damage can affect survival. DNA samples are available from around 1200 patients enrolled in FOCUS, and since our YCR funding was awarded, we have obtained approval from the Local Research Ethics Committee to use these samples for this project. We aim to study a large number of single nucleotide polymorphisms in 8 genes involved in the DNA damage response, including XRCC2, ATM, XRCC3, RAD51, BRCA2, MRE11, RAD50 and NBS1. We are currently installing the SNPlexTM high-throughput genotyping system (Applied Biosystems) in our laboratory, and genotyping for this project using this system will commence shortly.

Here, we provide a completely novel concept for treating recombination defective cancers, such as inherited breast and ovarian cancers as well as many other cancers, using inhibitors of Poly(ADP-ribose) polymerase (PARP). Genetic rearrangements are a common cause for disruption of tumour suppressor genes that will eventually lead to cancer. PARP is an enzyme that controls recombination and may have an important role for development of cancer. Here, we study the mechanism how PARP controls recombination. Here we have seen that PARP does not play a direct role in recombination repair. However, it controls the levels of recombination within the cell and how damage is dealt with at replication forks. We have shown that PARP inhibitors specifically kill cells deficient in homologous recombination. This is important for cancer treatment, since several cancers develop due to a defect in this pathway, e.g., inherited form of breast cancer. In this work we have shown that we can specifically kill BRCA2 deficient tumours in xenograft mice. This finding provides a new concept for cancer treatments and provides hope for a cure of inherited breast cancer. During 2005, we published this in Nature, and the phase I clinical trials are soon to end and phase II trials will commence. With funding from this project we also published numerous other papers in the most prestigious journals, i.e., Nature Cell Biology, Human Molecular Genetics, Molecular and Cellular Biology, Nucleic Acids Research and Cell Cycle.
(Link to abstract of publication in Nature)

Cells respond to DNA damage by triggering cell cycle arrest, DNA repair, or death. DNA damage response pathways are frequently disrupted during tumour development leading to genetic instability, loss of cell cycle checkpoints, defects in the induction of apoptosis, and altered responses to widely used chemotherapeutic agents. The objectives of our work are to elucidate the interactions of pathways controlling the response to DNA replication fork stress, to determine how these pathways are altered in tumour cells, and to determine how they may be exploited to improve therapy. One of our major lines of work is aimed at understanding the role of one of the primary regulators of the DNA damage response (a protein called ATM) in the cellular response to disruption of DNA replication. We have discovered that a subset of human colon cancer cell lines are defective in ATM activation following replication fork disruption and identified a mutant allele of another regulator (called MRE11) that suppresses ATM activation, ATM-mediated signaling through MRE11, and the induction of homologous recombination repair (HRR). Our results have important implications for the regulatory pathways and suggest a novel and therapy specific for tumour cells defective in this response. A second line of work is aimed at understanding how cells make the decision whether to repair DNA and restart DNA replication or die following disruption of replication. We have shown that the efficacy of a clinically relevant agent that disrupts DNA replication (camptothecin) can be enhanced by manipulation of S-phase signaling pathways. DNA replication inhibitors that do not normally induce cell death can be converted into potent inducers of apoptosis by depletion of the Chk1 checkpoint kinase. Thus combinations of agents that disrupt S-phase checkpoints with those that inhibit DNA replication may be particularly effective in the treatment of some types of tumors.

The papillomaviruses are the causative agents of warts but are also associated with certain cancers, principally carcinoma of the cervix. Two independent approaches are being employed towards combating viral infection and disease. The first is immunological or vaccine based, aimed at stimulating the bodys own immune response to the virus. The second is chemotherapeutic and involves an understanding of important viral proteins as specific targets for potential anti-viral drugs. We are undertaking a comprehensive structural and functional analysis of two viral proteins, known simply as E1 and E2, that coordinate and control viral DNA replication. We have previously determined the molecular structure of part of the E2 protein that interacts with E1 and controls viral replication. Our structure revealed the existence of a novel interaction between two E2 proteins that we now know can regulate its ability to associate with its partner E1. We have also made great in-roads into understanding how E1 functions in viral replication, and are on target to determine how the protein can process the viral DNA during replication.

Development of a macrophage-based system to target therapeutic viruses to prostate cancer
Professors Claire Lewis, Norman Maitland, Freddie Hamdy & Nicola Brown

This project is designed to use human macrophages as gene therapy vehicles to target the synthesis and delivery of therapeutic human adenoviruses to hypoxic regions of prostate tumours. The main advantages of this cell-based delivery system are the bypassing of the liver, the principal target for intravenously administered viral vectors, and the delivery of large quantities of virus into primary and/or metastatic tumours after intravenous injection. To achieve this, an adenovirus type 5 E1A/B gene cassette was inserted into a strongly hypoxia-regulated expression plasmid, and co-introduced into human macrophages with an E1A/B-deleted adenovirus. We then showed that these co-transfected macrophages migrate into hypoxic areas of tumour spheroids in vitro, where the hypoxia-induced expression of E1A/B results in production of the co-infected therapeutic virus. Within each viral particle, a reporter gene (GFP) has been placed under the control of promoter from a gene over-expressed in prostate (PSA). This ensures that when the virus replicates and is released by the host macrophage to infect surrounding cells, the expression of GFP is restricted to prostate cells. The triple targeting approach should remove the need for high-dose virus inoculations used at present in gene therapy protocols using adenoviruses and restrict therapeutic gene expression to the tumour site.

Novel use of hypoxia response promoters in Salmonella to target genes to hypoxic areas
Professors Claire Lewis & Jeff Green, Drs Judy Harmey & Rachel Ryan

Hypoxic areas are a hallmark feature of tumours but are difficult to access (and thus treat) by conventional therapies due to the poor vascular supply. This has prompted the development of alternative approaches to target therapeutic genes to destroy these otherwise inaccessible areas. We have developed a novel strategy for targeting gene therapy specifically to these sites using a non-pathogenic strain of the bacterium, Salmonella typhimurium, in which expression of gene (eg. the reporter gene, lacZ) is restricted to hypoxic areas by placing it under the control of an engineered hypoxia-responsive bacterial promoter. In the first year of the project we have constructed stable expression plasmids with various reporter and therapeutic genes under the regulation of hypoxia-responsive bacterial promoter, FFP*. The therapeutic genes include ones encoding the two anti-angiogenics, alphastatin and endostatin (to inhibit the development of new blood vessels in tumours) and a pore-forming cytotoxin (to allow selective killing of tumour cells). We are currently assessing the hypoxic inducibility of these transgenes in breast tumour monolayers and spheroids in vitro, and will shortly be investigating their delivery and therapeutic effects in murine tumours in vivo. Together, these studies will demonstrate the potential of this new strategy for delivering gene therapy to hypoxic areas of solid tumours.

SECTION OF FUNCTIONAL GENOMICS
Head: Professor S.K. Dower

Academic Unit of Infection & Immunity
Head: Dr J.R. Sayers

Novel, highly immunogenic ganglioside-based cancer vaccines
Dr A.W. Heath

Gangliosides are small sugary molecules present on a range of human cells, but present in higher numbers on many tumour cells. Ganglioside based vaccines have potential application for therapy of a variety of tumours , but tend to be very poorly immunogenic, and hence methods are required to induce stronger immune responses to these so-called therapeutic vaccines. We have received funding from YCR for a pilot study to address this issue.

We have shown in other work that conjugates of vaccine antigens with antibodies able to bind an immune cell surface molecule called CD40 (CD40mAb), are very highly immunogenic. The pilot study is aimed at producing conjugates of CD40 mAb and ganglioside antigens, with the end result hopefully being that the resultant conjugates are much more immunogenic. Ganglioside-based vaccines have potential applications for therapy of a variety of tumours. Chemical conjugates produced between the gangliosides and the antibody must retain ganglioside antigen recognisable to the immune system, and also CD40 binding activity. We have now produced conjugates of gangliosides with CD40 antibodies, and are assessing immunogenicity.

Antibody idiotypes present on the surface of certain tumours of the immune system, such as B cells lymphomas, are highly tumour specific antigens. Vaccines derived from tumour idiotypes have had some success in clinical trials, but their success has been limited by the poor immunogenicity of the vaccine. We have shown that CD40 antibodies are very potent adjuvants, enhancing immune responses against a variety of antigens attached to them. This project involves the production of a vaccine with tumour idiotype and CD40 binding antibody within the same construct. This approach should very strongly enhance the immune response against the tumour idiotype, and ultimately the efficacy of idiotype vaccines.

DIVISION OF CLINICAL SCIENCES (S)
Director: Professor F.C. Hamdy

Academic Unit of Urology
Head: Professor F.C. Hamdy

The role of osteoprotegerin on prostate cancer survival
Dr C. Eaton, Professor F.C. Hamdy and Dr I. Holen (Academic Unit of Clinical Oncology, Division of Genomic Medicine)

Our previous in vitro studies using breast, prostate and thyroid cancer cell lines have shown that osteoprotegerin (OPG) produced by tumour cells protects these cells from induction of apoptosis by a member of the TNF family, TRAIL. The initial part of this study demonstrated that reduction of OPG expression in prostate cancer cells was induced by stable integration of a construct expressing siRNA directed against mature OPG and that these clones appeared more sensitive to TRAIL in vitro. The central aim of the next phase of the project was to extend these in vitro observations into a xenograft model and test the hypothesis that OPG protects prostate cancer cells from TRAIL induced apoptosis in vivo as well as in vitro. We have now engineered clones of prostate cancer cells that express green fluorescent protein allowing them to be tracked by in vivo monitoring. To ensure reliable growth in vivo these cells have been serially passaged in nude mice, initially subcutaneously and latterly in intraosseous sites and subsequently re-engineered to overexpress OPG at high levels. Strains have now been isolated and expanded that expressgreater than10 times the levels of OPG expressed by parent cells or empty vector controls in vitro under the same conditions. The continued expression of OPG and the sensitivity of these cells to induction of apoptosis TRAIL in vivo is currently being evaluated as intraoseous xenografts. These cells are also being engineered for knock down of induced OPG expression using RNAi technologies and again re-evaluated in vivo/in vitro. Studies to test whether OPG secretion is androgen regulated in prostate cancer have so far indicated that this is not the case.

Academic Unit of Surgical Oncology
Head: Professor M.W.R. Reed

The relationship between vascular endothelial growth factor and tissue factor in breast cancer
Dr CA Staton, Professor MWR Reed & Professor NJ Brown

Many haemostatic proteins modulate tumour angiogenesis resulting in either stimulation or inhibition, thus understanding the relationships between these processes has implications for cancer therapy. Tissue Factor (TF), a key haemostatic molecule, up-regulates vascular endothelial growth factor (VEGF), a potent pro-angiogenic factor. In colorectal cancer a close correlation exists between TF, VEGF and microvessel density (MVD), a measure of angiogenesis, but this has not been evaluated in breast cancer. Thus far, our studies have identified the presence of TF on the cell surface of the fast growing and highly metastatic breast cancer cell line, MDA-MB-436, but the absence of TF on the cell surface of two slower growing and less metastatic cell lines, T47D and MCF-7 cells, using flow cytometry. Western blotting analysis has confirmed this data. In contrast VEGF is expressed in all three cell lines, with the highly metastatic MDA-MB-436 cells expressing the most (Western and FACs). These data suggest that TF and VEGF may both influence the growth of tumours. RT-PCR studies are currently underway to establish TF/VEGF mRNA levels present in these cell types, thereby confirming that MDA-MB-436 cells are the relevant cell type for siRNA inhibition of TF and VEGF. Concurrently immunohistochemistry studies are underway to confirm the relative roles of TF and VEGF in human pre-invasive and invasive breast cancer.

Medical Imaging and Medical Physics

FDG dual-headed PET identifies those patients with non-small cell lung cancer who are suitable for surgery? (Ref S272)
W B Tindale, E Lorenz, M B Hanney, S Matthews, R Vaughan, S Mitchell, S Fleming, A Connolly, E J R van Beek.

Background
The accurate identification of patients who may benefit from surgical resection remains a significant challenge in the treatment of non-small cell lung cancer (NSCLC), and is critically dependent on reliable preoperative staging. Conventionally, preoperative staging is based on CT imaging, although Positron Emission Tomography (PET) has been shown to offer improved accuracy, particularly for assessing mediastinal lymph-node involvement.
Although set to increase, access to PET scanners in the UK is limited, and a possible alternative where such systems are unavailable is the use of modified dual-headed gamma cameras (GC-PET). Whilst the imaging characteristics of GC-PET are inferior to those of dedicated PET, the clinical significance of the reduced image quality is unclear. This study aims to evaluate the accuracy of GC-PET for staging NSCLC.

Study Methodology
98 patients with known or suspected NSCLC were recruited. All underwent standard CT imaging as part of their routine management, followed by GC-PET imaging. GC-PET scans were reported blind of CT findings, and results compared with histological findings following subsequent surgery or mediastinoscopy.

Results
GC PET was able to differentiate between resectable and unresectable mediastinal lymph-node involvement with an accuracy of 91% (sensitivity 80%, specificity 95%)
The accuracy of CT for differentiating resectable from non-resectable lymph node involvement was 72%, and its sensitivity and specificity 67% and 73% respectively.

Conclusion
Although dedicated PET is the imaging modality of choice, the results of this study indicate that GC-PET offers increased accuracy relative to CT for staging mediastinal lymph node involvement in NSCLC. Where dedicated PET is not available, the use of GC-PET may improve patient management.

DEPARTMENT OF BIOMEDICAL SCIENCE
Chairman: Professor P.W. Andrews
Head of Department: Professor C.G.W. Smythe

A screen of candidate regulatory genes that may play a role in the progression of testicular germ cell tumours
Professor P.W. Andrews

Teratocarcinomas are a significant subset of testicular germ cell tumours (TGCT), the most common cancers of young men. Recent research has highlighted several genes that may play a role in regulating self-renewal and preventing differentiation of human embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas. With this pilot study grant we used RNA interference (RNAi) to screen several human EC cell lines and showed that, indeed, the transcription factor Oct4 is generally required for maintenance of human EC cells. When levels of Oct4 were reduced, these cells ceased proliferation and differentiated. Thus, Oct4 could be a potential target in developing new therapies for TGCT. In addition we used RNAi to screen several other candidate genes that might play a role in maintenance of the EC phenotype. From this study we have found preliminary evidence that the a gene, RCP32, that encodes a subunit of RNA polymerase, and another gene, dppa4, that encodes a nuclear protein, are required for ES cell survival. This information provides the basis for further studies to explore the function of these genes in stem cell maintenance. The results of such studies could help provide insights into the regulation of stem cell self renewal and its role in carcinogenesis. In this pilot study we also further developed the RNAi methodology using an inducible system that will help in the further analyses of the role in EC cell biology of these specific factors.

Regulation of Pluripotency in Malignant Stem Cells
Professor P W Andrews

Teratocarcinomas are a subset of germ cell tumours, comprising a mixture of differentiated cell types and undifferentiated embryonal carcinoma (EC) cells. Undifferentiated EC cells constitute the malignant stem cell component of the tumour, whilst their differentiated derivatives display a limited proliferative capacity and are non-tumorigenic. Aberrations which promote EC self-renewal in preference to differentiation may play an important role in germ cell tumour progression. A further understanding of the pathways involved in stem cell self-renewal and differentiation is important in order to enhance our current knowledge of tumorigenesis and aid the development of novel therapies.

The cyclin-dependent kinase inhibitor (CDKI) protein p27KIP1is a negative regulator of cell cycle progression, whose expression is frequently lost in a number of tumour types. Differentiating cells often contain elevated p27KIP1protein levels, suggesting an additional role in the control of cellular differentiation. Work in our lab has previously identified a requirement for p27KIP1during the commitment and terminal differentiation of human EC cells. The aim of this present study is to further investigate the precise mechanisms by which p27KIP1mediates differentiation. Our current data suggests that p27KIP1acts via the various cyclin-CDK cell cycle mediators to induce a differentiated phenotype. In addition to causing cell cycle arrest, the treatment of EC cells with specific CDK inhibitor peptides directly induces differentiation, as illustrated by a loss of stem cell marker expression. We are currently using an inducible RNAi system to further characterise the role of p27KIP1in EC cell differentiation