research reports

University of York

2003/4


YCR Cancer Research Unit
Director: Professor N. J. Maitland

Gene therapy for prostate cancer
Prof. N.J. Maitland, Dr. L. Stanbridge, Ms. L. Hudson, Ms. R. Nugent, Ms. K. Tiemann, Mr. R. Birnie, Mr V. Dussupt
Collaborators: Dr. P. Berthon, Urogene SA, Evry, France
Prof. C. Bangma, University of Rotterdam, Netherlands
Prof. P. Mannoni, Dr. C. Bagnis, University of Marseille, France
Prof. T. Totterman, Dr. M. Essand, University of Uppsala, Sweden
Dr. C. Wrighton, ML Research, University of Keele
Dr. L. Seymour, Dr. M. Stevenson, University of Oxford
Prof. S. Bettuzzi, Mr. M. Scaltriti, University of Parma, Italy

This is a joint program funded by YCR and a FP5 award from the European Union, which is coordinated from York. The principal aims are to retarget viruses to preferentially infect human prostate cancer cells, and to direct expression of these genes by employing control sequences from some of the many genes expressed.

(I) GENERATION OF VIRAL VECTORS CONTAINING PROSTATE-SPECIFIC EXPRESSION CASSETTES
As a result of the EU program a set of transcriptional control sequences from genes whose expression is elevated in prostate cancer have been isolated and inserted into both adenoviral and baculoviral vectors. In most cases these promoters are driving either EGP or a hybrid EGFP-CAT indicator gene to enable visualisation and quantification of the expression targeting, in prostate cancer cell cultures, ex vivo models and human prostate tumour xenografts in nude mice. As prostate targeting has been confirmed in the various models, these indicators will now be replaced by therapeutic genes such as a new optimised HSV thymidine kinase gene for GDEPT therapies.

To broaden the specificity of the promoters, most of which are positively regulated by androgens, we have engineered a dual expression cassette, which allows the simultaneous expression of both the indicator/therapeutic gene, and a truncated, constitutively active androgen receptor protein.

(II) MODIFICATION OF THE ATTACHMENT PROPERTIES OF THE VIRAL VECTORS TO PREFERENTIALLY ATTACH TO PROSTATE TUMOURS
Baculovirus vectors attach with high efficiency, but low specificity to most human cell types, probably via heparan sulphate (HS) residues on the cell surface. This attachment may be partly responsible for the preference of many viruses to infect the liver after intravenous injection. We have adopted two strategies to retarget this attachment. We have inserted growth factor peptide coding sequences into surface -exposed sites in the gp64 membrane protein, which overlap with neutralising antibody binding sites. The peptides were selected to bind to receptors normally over-expressed on the surface of prostate cancer cells, e.g. Gonadotrophin releasing hormone receptor. The insertion of the peptide has also reduced non-specific binding, while promoting receptor-mediated infection of human cells. The retargeted viruses infect and grown in insect cells with normal efficiency, and can be purified to high titres, for in vivo testing.

Since destruction of HS residues abrogates baculoviral binding to human cells, we have also set out to genetically delete a putative HS biding motif in the gp64 molecule. As the effects of this modification on baculovirus infectivity are unknown, we have engineered this and other modifications into a soluble form of gp64, from which the membrane anchor has been deleted.

The immune system is a formidable barrier to the effectiveness of gene therapy vectors. To overcome this we are developing both virus-specific complement inhibitors (in collaboration with Prof T Totterman, Uppsala), and stealthing technology, using reactive polymers to cloak the gp64 molecule, (in collaboration with Dr L Seymour, Oxford).

Structure and functional studies of human papillomavirus E2 proteins
DR. J.E. BURNS, MS. E. HERNANDEZ-RAMON, MS. C. BURN, MS. H. WALKER, PROF. N.J. MAITLAND
Collaborators: Dr. A. Antson, Ms. O. Moroz, Dr. I. Bronstein, Prof. K. Wilson, Prof. G. Dodson, York Structural Biology Laboratory, University of York
DR. I. MORGAN, INSTITUTE OF COMPARATIVE MEDICINE, UNIVERSITY OF GLASGOW
DR. S. ALLEN, DR. W. ZHANG, SCHOOL OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NOTTINGHAM

We have been studying the E2 protein from two HPV types : HPV 16 (high risk: found in over 50% of cervical cancers), and HPV 2a (a low risk type which causes common warts but is also found in some oral carcinomas).

The E2 proteins of HPVs are multifunctional proteins that share a highly conserved tripartite structure and modulate viral replication and transcription through binding as dimers to specific sequences in the viral upstream regulatory region and interaction with the viral replication protein, E1, and several cellular proteins.

Our crystal structure of the E2 N-terminus revealed a novel second dimerisation interface which we postulated might explain the formation of DNA loops seen with full-length but not C-terminal E2 proteins. We have now tested this hypothesis in HPV16 E2 by introducing structurally directed mutations into the N-terminal dimer interface. These mutants fail to transactivate but retain the ability to bind DNA containing E2 binding sites. Atomic force microscopy reveals that wild type E2 forms DNA loops but this function is greatly reduced in the mutant proteins. A similar set of mutations in E2 from HPV 2 shows that the mutant proteins remain able to stimulate replication although transactivation is severely reduced. Since HPV 2a is a highly replicative virus and these mutations have been cloned back into the intact virus genome in order to study their effects on the virus life cycle in organotypic raft cultures of human keratinocytes.

Alterations in the activation pathways of Protein Kinase B and Erk in the progression to metastasis of human prostate cancer
Dr. R.M. Sharrard, Ms. J. Spalton, Prof. N.J. Maitland
Collaborators: Dr. D. Alessi, University of Dundee
Prof. C. Cooper, Dr. J. Clark, Institute for Cancer Research, Sutton
Dr. H. Isaacs, University of York

Alterations to the PKB and erk activation signalling pathways that regulate cell proliferation and survival, are common in prostate cancer. However the interactions between GF receptors, src, PI3K, PKB, MEK, and erk are complex, as are the multiple ways in which these interactions may be altered in tumour progression. In non-tumour PNT2 cells, cross-talk between the EGF and IGF-I receptor allows both EGF and IGF-I to stimulate MEK/erk as well as PI3K/PKB activation. This is lost in PC3 and LNCaP metastatic cells, possibly via alterations in expression of GF receptors and their downstream adaptor proteins such as the IRS family in IGF signalling. While the non-tumour cells show positive cross-talk or co-dependence between the PKB and erk pathways, metastatic tumour cells show mutual inhibition between PKB and erk.

Inhibition of PI3K by LY294002 inhibits growth of metastatic cells through G1 arrest, while non-tumour and P4E6 cells continue to grow in the presence of the PI3K inhibitor. Short-term treatment with LY294002 has a relatively small retarding effect on cell cycling in PNT2 and in P4E6 (non-metastatic tumour) cells; in contrast, progression through S phase and G2/M is slowed in PC3 and dramatically retarded in LNCaP cells. The kinetics of PI3K/PKB inhibition by LY294002 suggeststhat, unlike LNCaP, PC3 cells express PIP3-inactivating phosphatases, which may substitute for PTEN function during S and G2/M, though not in the progression from G1 to S phase.
Specific combinations of protein kinase inhibitors directed against the PKB and erk pathways should offer a rational therapy to target prostate cancers and their metastases.

In vitro modelling of the human prostate
Dr. A. Collins, Dr. S. Lang, Mrs. K. Hyde, Ms. C. Brinham, Dr. S. Bryce, Prof. N.J. Maitland
Collaborators: Mr. N. Clarke, Department of Surgery, Christie Hospital NHS Trust and Department of Urology, Withington Hospital, Manchester
Mr. M. Stower, Dept. of Urology, York District Hospital, Wigginton Road, York
Prof. D. Edwards, Mr. A. Riddick, Dept of Biological Sciences, University of East Anglia
Prof. A. Turner, Dr. B. Usmani, Ms. L. Dawson, School of Biochemistry and Molecular Biology, University of Leeds
Prof. C. Cooper, Institute of Cancer Research, Sutton, Surrey
Prof. M. Stratton, Dr. R. Wooster, Sanger Centre, Cambridge

This project has several arms, and is partly funded by funds from the National Cancer Research Institute. We have previously demonstrated that organotypic reconstructions of normal human prostate are can be achieved by reconstitution in a defined matrix and medium, including prostate stromal cells. We have now demonstrated that undifferentiated cell lines from both tumours and normal origins, can be induced to undergo partial or complete differentiation in these systems. However, even using clonal cell populations, only a small proportion of the epithelial cells introduced into these systems ever form organoids. It is likely that these colony-forming cells represent stem cell like, or at least undifferentiated epithelial cells. We have therefore set out to isolate and characterise these populations of cells from human material.

(i) Identification of cancer stem cells in human prostate tumours
The goal of existing therapies for prostate cancer has been to eradicate the bulk of cells within a tumour. However, most patients go on to develop androgen-independent disease, which remains incurable by current treatment strategies. One hypothesis to explain resistance is that existing therapies fail to kill cancer stem cells effectively. The focus of this project is to identify and characterise the properties of the prostate cancer stem cell. Putative tumour stem cells have been isolated from primary and metastatic prostate tumours using the markers a2b1 integrin and CD133 markers for normal prostate epithelial stem cells. We have demonstrated, for the first time, that these putative tumour stem cells have the ability to self-renew and form all the cells present within the original tumour, both in vitro and in vivo; properties consistent with a stem cell origin. Further work has begun to identify patterns of genetic changes and gene expression, which differ between cancer and normal stem cells. From this we are generating a consensus transcriptome, which will assist in both identification and novel targets for therapy of prostate cancers.

(ii) The role of androgen signalling in the regulation of human prostate epithelial stem cell differentiation
Androgens play a pivotal role in the pathogenesis of prostate cancer and benign prostatic hyperplasia (BPH), yet very little is known of the factors or processes that influence the initiation or progression of prostate disease. As stem cells are central to normal homeostasis and may be targeted in carcinogenesis, an understanding of the molecular basis of stem cell fate decisions will lead to insights into their disruption in prostate cancer and possible therapeutic targets. Stroma is the target and mediator of androgenic effects upon the epithelium, thus stem cell fate is likely to be regulated by the stroma. We have already demonstrated that prostatic stroma affect both the differentiation and invasive capacity of epithelial cells. Stroma is a complex mixture of cell types, the majority of which are fibroblasts and smooth muscle cells. We have now successfully cultured and phenotyped stroma from a more than 20 patient's samples. Androgen receptor expression is maintained and the cells respond to androgen through a number of population doublings. Our ability to isolate and maintain the androgen responsive population in stroma will now enable us to identity the androgen responsive, paracrine factors affecting stem cell fate.


Yorkshire Cancer Research P53 Research Laboratory
Director: Professor J. Milner

Disruption of the nucleolus mediates stabilisation of p53 in response to DNA damage and other stresses
Dr. C. P. Rubbi, Prof J. Milner

p53 protects against cancer through its capacity to induce cell cycle arrest or apoptosis under a large variety of cellular stresses. It is not known how such diversity of signals can be integrated by a single molecule. However, the literature reveals that a common denominator to all p53-inducing stresses is nucleolar disruption. We thus postulated that the impairment of nucleolar function might stabilise p53 by preventing its degradation. Using micropore irradiation we demonstrate that large amounts of nuclear DNA damage fail to stabilise p53 unless the nucleolus is also disrupted. Forcing nucleolar disruption by anti-UBF microinjection (in the absence of DNA damage) also causes p53 stabilisation. We propose that the nucleolus is a stress sensor responsible for maintenance of low levels of p53, which are automatically elevated as soon as nucleolar function is impaired in response to stress. Our model integrates all known p53 inducing agents and also explains cell cycle-related variations in p53 levels, which correlate with established phases of nucleolar assembly/disassembly through the cell cycle.
Techniques incorporated through this work: Quantitation of Nucleophosmin translocation; Extension of micropore irradiation to Al -coated filters to eliminate transmittance completely; Statistics of nuclear area coverage by micropore irradiation.

Bcl-2 constitutively suppresses p53-dependent apoptosis in colorectal cancer cells
Dr. M. Jiang, Prof J. Milner

To dissect apoptotic genes governing the survival of colorectal carcinoma cells, we employed RNAi to silence Bcl-2 and Bcl-xL in isogenic clones of p53+/+ and p53-/- cells, and of Bax+/- and Bax-/- cells. We identify a novel proapoptotic function of p53 that does not require activation by genotoxic agents and that appears to be constitutively suppressed by Bcl-2. Silencing of Bcl-2 induced massive p53-dependent apoptosis. The 'Bcl-2/p53 axis' requires Bax and caspase 2 as essential apoptotic mediators. This newly discovered Bcl-2/p53 functional interface represents a key regulator of apoptosis, which can be activated by targeting Bcl-2 in colorectal carcinoma cells.

Loss of p53 Has Site-Specific Effects on Histone H3 Modification, Including Serine 10 Phosphorylation Important for Maintenance of Ploidy
Dr. S.J. Allison, Prof. J. Milner

Histone modification enables the ordered regulation of DNA-related processes. Here, we ask if p53, which interacts with histone modifying complexes in vivo, influences histone H3 modification. For this purpose, we compared isogenic clones of human p53+/+ and p53-/- cells in which it is reasonable to attribute any observed differences in histone modification to p53-related effects. Cell growth and cell cycle analyses indicated equivalent proliferation rates for the p53+/+ and p53-/- cell clones. Modification of histone H3 was determined under normal cell growth conditions and also after UV irradiation and/or treatment with trichostatin A (TSA) or nicotinamide (two inhibitors of histone deacetylation). Site-specific histone H3 modifications were determined by immunoblotting. We provide evidence that p53 influences histone H3 acetylation at lysine 9 (K9) and K14, whereas acetylation of K18 appears to be p53 independent. The most striking p53-related effects are at K9, which is underacetylated in p53-/- cells under normal conditions of growth but which shows a dramatic increase in acetylation after combined treatment with UV plus TSA. Conversely, phosphorylation of serine 10 (S10P) is elevated in p53-/- cells and reduced after UV plus TSA treatment. Similar reciprocity between K9Ac and S10P was not evident in p53+/+ cells. Abnormal S10P in p53-/- cells was also observed under completely different experimental conditions where cells were treated with nocodazole to induce G2-M arrest and elevation of S10P (which is linked with G2-M of the cell cycle). On removal of nocodazole, the p53+/+ cells exhibited rapid reduction in S10P levels and cell cycle recovery. In contrast the p53-/- cells retained elevated S10P levels and failed to show normal cell cycle recovery. Phosphorylation of S10 is known to be linked with the initiation of chromosome condensation in G2 and is also important for proper chromosome segregation at mitosis. Our research indicates that loss of p53, directly or indirectly, perturbs the normal regulation of S10 phosphorylation. We suggest that this effect may contribute towards the development of abnormal chromosomes and aneuploidy in p53-deficient cancers.

P53 represses RNA polymerase III transcription by targeting TBP and inhibiting promoter occupancy by TFIIIB.
D Crighton, A Woiwode, C Zhang, N Mandavia, J.P. Morton, L.J. Warnock, J Milner, R.J. White, D.L. Johnson

The tumour suppressor p53 is a transcription factor that controls cellular growth and proliferation. P53 targets include RNA polymerase (pol) III-dependent genes encoding untranslated RNAs such as tRNA and 5S rRNA. These genes are repressed through interaction of p53 with TFIIIB, a TATA-binding protein (TBP)-containing factor. Although many studies have shown that p53 binds to TBP, the significance of this interaction has remained elusive. Here we demonstrate that the TBP-p53 interaction is of functional importance for regulating RNA pol III-transcribed genes. Unlike RNA pol II-dependent promoter repression, overexpressing TBP can reverse inhibition of tRNA gene transcription by p53. p53 does not disrupt the direct interaction between the TFIIIB subunits TBP and Brf1, but prevents the association of Brf1 complexes with TFIIIC2 and RNA pol III. Using chromatin immunoprecipitation assays, we found that TFIIIB occupancy on tRNA genes markedly decreases following p53 induction, whereas binding of TFIIIC2 to these genes is unaffected. Together our results support the idea that p53 represses RNA pol III transcription through direct interactions with TBP, preventing promoter occupancy by TFIIIB.


Department of Chemistry
Head of Department: Professor R. N. Perutz

Novel anti-tumour agents from marine natural products
Prof. R. J. K. Taylor, Mr. S. Martina

Natural products from the sea are providing many interesting new leads for cancer chemotherapy. The salicylihalamides, oximidines and lobatamides are all potent anti-tumour agents isolated from marine sources. Structurally, these compounds are based on large lactones and contain extremely unusual acyl enamide side chains. These new natural products possess a remarkable pattern of differential cytotoxicity and COMPARE pattern recognition analysis indicates that they have a unique mode of antitumour activity. They inhibit the growth of oncogene-transformed cells with unprecedented selectivity for mammalian versus non-mammalian vacuolar-type (H+)-ATPases (V-ATPases), and higher selectivity than previously known bafilomycin/concanamycin
class of V-ATPase inhibitor. The aim of the project is to prepare a range of these compounds, plus novel synthetic analogues, for biological screening.

Our first target is the synthesis of a racemic truncated version of lobatamide A in order to validate new synthetic methods and to obtain novel analogues for bioassay. We have initially concentrated on a Still-Gennari approach to the synthesis which incorporates a novel use of the Nozaki-Kishi reaction. Novel methodological results have been published. We are currently completing the synthesis of the macrolactone core and will soon have novel analogues. All analogues will be screened as anti-cancer agents by Dr R. M.Phillips at the University of Bradford. These procedures will involve chemosensitivity studies against a panel of cell lines derived from a range of human tumours. The principle aim of this will be to establish the potency of the analogues relative to the parent compounds and to determine structure activity relationships which will drive further chemical synthesis.