research report

University of York

2007/08


Department of Biology
(Current address: Northern Institute for Cancer Research, Newcastle University)  
Y249  

The Role of DNA Mismatch Repair in Therapy Induced Gastrointestinal Cancer  
Dr. James M. Allan

Working with Dr. Lois Travis (National Cancer Institute, United States of America),
Professor Matthew Seymour, Dr. Susan Richman (University of Leeds) and
Dr. Daniel Scott (Harrogate NHS Trust) we have undertaken a study to investigate the role of DNA mismatch repair in the aetiology of therapy-induced gastrointestinal cancer.  

The occurence of independent second cancers has emerged as a serious long-term side-effect of chemotherapy and radiotherapy for a previous primary cancer. Understanding how and why such cancers develop could allow for preventive or surveillance measures to be implemented. Evidence suggests that defective DNA mismatch repair is a common feature of therapy-induced myeloid leukaemia, and abrogation of this pathway may also be a feature of other therapy-induced cancers.  

We have shown that loss of DNA mismatch repair is a relatively common feature of therapy-related gastrointestinal cancer, and that this is associated with concomitant genomic instability in some cases. Specifically, we have shown that epigenetic silencing of the MLH1 DNA mismatch repair gene via promoter methylation is a very common occurrence in radiogenic gastrointestinal cancers (occuring in approximately 50% of cases). Moreover, we have identified a common constitutional genetic variant in the promoter of MLH1 (carried by 25% of United Kingdom Caucasians) that predisposes to loss of MLH1 protein expression and confers risk of developing colorectal cancer with low to moderate penetrance. We are using the information from this study to identify patients at high risk of developing radiation-induced and sporadic gastrointestinal cancer.    


Department of Biology
Mammalian DNA Replication Research Group
Principal Investigator: Dr. Dawn Coverley        

Functional consequences of cancer-associated Ciz1 splice variant expression  
Faisal Abdel Rahman, Katherine Roper and Dawn Coverley  

Initiation of DNA replication is a major control point in the mammalian cell cycle and a prime target for intervention in cancer therapy. We identified a novel DNA replication protein, Cip1-interacting zinc finger protein 1 (Ciz1) and showed that it is part of the sub-nuclear framework (nuclear matrix) that organises DNA replication in time and space. Our functional analysis suggests that Ciz1 is a mediator between the DNA replication machinery and the nuclear matrix and that it plays a positive role in the replication process. Withdrawal of Ciz1 restrains DNA replication and cell proliferation in normal and cancer cells. Therefore we are studying the function and expression of alternatively spliced forms of Ciz1 that occur in some cancers, as they offer potentially selective targets for intervention. We have shown that one splice-variant, lacking exon 4 (ΔE4), is mis-expressed as a consequence of mutations in intronic splicing regulators in Ewings Tumour (ET) cell lines. Functional assays show that conditional exclusion of exon 4 does not abolish activity but does influence the spatial distribution of Ciz1 within the nucleus, and possibly therefore the spatial organization of DNA replication. We have now designed and produced a Ciz1-specific splice-variant microarray, using probes that detect all predicted and observed exons and exon-exon junctions. This will be used to systematically survey Ciz1 splice-variant expression in normal tissues, in two paediatric cancers and in a range of lung cancers.  Initial results have validated the array, using ET cell lines, and shown that it is capable of differential detection of Ciz1 splice variant expression.      


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

Stem cell hierarchies in human prostate tumours  

Dr A.T. Collins, Mr P. Berry, Mrs K. Hyde, Prof. N.J. Maitland  

Collaborators:
Mr M. Stower - York Hospital NHS Trust
Dr R. Birnie - Pro-Cure Therapeutics Ltd, York
Dr J. Lewis - YCR Cancer Research Unit
Dr Y-J. Lu, St Bartholomews Hospital, London
Prof. C. Cooper, Dr J. Clark - Institute of Cancer Research, London
Prof. F. Hamdy, Prof. N. Brown, Dr K. Linton - University of Sheffield
Dr D. Hudson - ICR Sutton, Surrey
Dr G. Van Leenders,
Dr G. Jenster - Erasmus University, Rotterdam
Dr G. van der Pluijm - Leiden University Medical Center
Dr M. Cecchini - BERN
Dr J. Shalken - RUNMC  

The tumour-initiating capacity of many cancers is thought to reside in a small subpopulation of cells. i.e. cancer stem cells.  We have previously shown that rare prostate epithelial cells with a CD133+/a2ß1hi  phenotype have the properties of prostate cancer stem cells, in particular, self-renewal, extended lifespan, a high invasive capacity, a primitive epithelial phenotype and an ability to differentiate to recapitulate the phenotypes seen in prostate tumours  (Collins et al., Cancer Res. 2005, 65:10946-10951).   

To date, DNA and tissue microarrays have failed to account for cellular heterogeneity and differences in the proliferative potential of the distinct populations within tumours.  By directing expression analysis to enriched populations of tumourigenic cells, we have identified a gene expression signature for the cancer stem cell (CSC) which is distinct from normal stem cells and differentiated progenitors.  By further analyzing this expression signature in the context of biologically connected genes, key pathways have been identified that are associated with inflammation, extracellular matrix interactions and stem cell self-renewal.  This work has recently been accepted for publication (Genome Biology 2008) and is the subject of several patent applications.  

Further research is centred on the pathways identified above and identifying the tumour-initiating population from prostate tumours.  We are currently determining whether JAK-STAT signalling is involved in cancer stem cell self-renewal, and studying which ligand (IL6, LIF) is responsible for constitutive activation of the pathway in prostate tumours.  

Although we have identified the clonogenic cells in prostate tumours, we have not fully determined their tumour-initiating capacity.  We are accumulating data from a series of prostate tumour samples and our preliminary data suggests that tumour incidence is greater from the α2ß1hi/CD133+ stem cell population, whilst differentiated phenotypes form only rare tumours, even with an input of 10⁶ cells.   

Development of lentiviral vectors to track the lineage and differentiation of prostate cancer stem cells  

Dr F. Frame, Ms S. Hager, Dr J. Burns, Dr A.T. Collins, Prof. N.J. Maitland  

Collaborators:
Dr G. van der Pluijm - Leiden University Medical Center
Dr M. Coles - University of York  

Our aim is to develop a method of visually tracking the differentiation of prostate cancer stem cells. Our strategy is to (i) generate lentiviruses containing differentiation stage-specific promoters driving expression of fluorescent genes and (ii) drug selection of cells containing the lentivirus and expression cassette.  

Our findings indicate that the inclusion of a polyA signal leads to more efficient and stable expression of the fluorescent gene, resulting in increased brightness of the cells, a desirable feature for the purpose of tracking. We have investigated the use of DsRed, EGFP, tdTomato, mOrange and Citrine fluorescent genes. By analysing microarray results from the laboratory that compare prostate cancer stem cell expression patterns to those of the more differentiated cancer cells we have selected a number of genes whose expression marks both differentiation and carcinogenesis. The promoters of the candidate genes have been cloned into the lentivirus transduction system. We have candidates that are upregulated in stem cells, committed basal cells and terminally differentiated luminal secretory cells.  

We have generated cell lines using LNCaP, P4E6 and PC3 cells containing the lentivirus constructs and eight different promoters.  To test the functionality of these promoters the cells have been stimulated with treatments to induce differentiation including the addition of calcium, stroma, serum and androgens. Some promoters show the expected reponse to these stimuli and we are assessing promoter activation in cells grown as 3D spheroids. Further validation of this promoter activity will indicate which of the lentiviruses is the most useful tool in tracking stem cell differentiation. Also, P4E6 cells contain a CD133 (stem cell) population of cells and we have shown that this subpopulation of cells can be infected by the lentiviruses. In addition, work has been initiated to optimise infection of primary prostate cancer cells, obtained from patient tumour samples, with lentiviruses.  

The role of androgen signalling in the regulation of human prostatic stem cell differentiation  
Dr A.T. Collins, Mr P.A. Berry  

There is now increasing evidence that stem cells are targeted in carcinogenesis.  We have embarked upon a systematic study to determine the molecular basis of stem cell fate decisions in the prostate.  We knew, from published data, that the extrinsic factors influencing prostate growth and differentiation are regulated by androgens and are secreted by the stromal cells in the prostate.  

We firstly enriched for androgen receptor-positive (AR+) stroma by sorting cells on the basis of cell surface expression of integren α1ß1, whose expression correlated well with AR expression.  Using an Affymetrix gene expression array we then looked for androgen-regulated genes as potential candidates regulating stem cell fate decisions in prostate epithelia.  This study was conducted in cells from patients with benign prostatic hyperplasia (n=12).  

After treatment with androgen, 193 probes were found to be significantly differentially expressed after 14h, and 165 probes after 6h.  We are currently validating two candidate genes from the 6h and three from the 14h time point, by RT-PCR. These genes have putative androgen-response elements suggesting androgen receptor binding ability. We have evaluated two androgen-responsive element constructs in prostate stromal cells by gene transfection to use as a reporter system for analysis of androgen stimulation and candidate gene expression analysis studies.  Further validation will be carried out at the protein level and future work will determine the functional effects of these proteins on stem cell self-renewal and differentiation.  

Development of lentiviral vectors to express suicide genes in prostate cancer cells  

Dr J.E. Burns, Ms H.F. Walker, Prof. N.J. Maitland  

Collaborators:  Dr P. Searle - University of Birmingham  

Escherichia coli nitroreductase (NTR) converts the inactive prodrug CB1954 into an active toxic metabolite that causes DNA crosslinks. NTR has been trialled in prostate cancer gene therapy using an adenovirus delivery vector and has also been used in developmental studies to selectively kill specific populations of cells. We aim to use NTR to selectively kill cells derived from cancer stem cells at different stages of tumour development.  

We are using the Gateway lentivirus system to generate lentiviruses that express NTR under constitutive and prostate-specific promoters. The advantage of using lentiviruses is that they are uniquely able to infect slow-growing and quiescent cells and must integrate into host cell DNA in order for transgenes to be expressed.  

We have made lentiviruses expressing wild type NTR or a mutant, F124N, which has 5x enhanced activity, under the control of the hCMV or EF1α constitutive promoters or PSA/Probasin prostate-specific promoter. As "proof-of-principle", virus-transduced clones were generated in prostate cell lines P4E6 and LNCaP and tested for CB1954 sensitivity. Most CMV- and some EF1α clones were susceptible to CB1954, although promoter inactivation was seen, particularly with the EF1α promoter, and we are now making viruses with the weaker cellular ß-actin promoter in place of these. The PSA/Pb-driven NTR was expressed in LNCaP cells but not in basal undifferentiated P4E6 clones, in which this differentiation-specific promoter is inactive. We are now testing the extent of the bystander effect of NTR expression in prostate cells.  

We are also constructing double transgene lentiviruses containing a constitutively expressed marker gene and NTR under constitutive or prostate-specific promoter control. Using these we hope to be able to track NTR-transduced cells within a cell population or tumour before exposure to CB1954. Marker genes are fluorescence (mCitrine, mOrange), luciferase or a fluorescence-luciferase fusion.  

Retargeting of baculoviral gene therapy vectors to infect the prostate  

Ms S. Swift, Dr J. Burns, Prof. N.J. Maitland  

Collaborators:
Members of the EU Funded GIANT Program (co-ordinated by York)
Dr L. Stanbridge, Ms H. Evans, Ms R. Nugent - YCR Cancer Research Unit, University of York
Prof. C. Bangma, Dr R. Kraaij, Dr E. Schenk-Braat - Erasmus University, The Netherlands
Dr A. Antson - York Structural Biology Laboratory, University of York
Pro-Cure Therapeutics, York -        
10 further collaborating laboratories
Members of the EU funded BACULOGENES Programme  

We are developing the baculovirus Autographa californica MNPV as a vector for prostate cancer gene therapy. The baculovirus represents an excellent and novel gene therapy vector due to its capacity to accept large inserts (>50kb), the ability to transduce slow-growing tumours and the lack of immune response upon initial inoculation into a patient. Baculovirus attachment to, and infection of, mammalian cells is achieved via the major envelope protein, gp64, which binds to heparan sulphate residues found on most cell types. We are re-targeting baculovirus to the cell surface Neurotensin Receptor (NTR), which we have shown to be upregulated in prostate cancer cell lines and primary malignant prostate tissue samples. These studies have also shown a relationship between NTR expression profile and cellular differentiation status in advanced prostate cancer cells, with NTR1 upregulated in basal, undifferentiated cells and NTR2 and 3 upregulated in well-differentiated luminal cells.  

Re-targeting of baculovirus is achieved by inserting the short coding sequence for the natural ligand for NTR - neurotensin (NT) - into a copy of the gp64 gene. The virus is also equipped with a reporter gene cassette to allow functional infectivity studies. We have shown that this NTgp64 targeting protein is expressed on the baculovirus surface in an active binding configuration. We have also shown that NTR-targeted baculovirus shows highly efficient attachment to prostate cancer cells, but binds to a much lower degree in normal prostate cell models. In future experiments we plan to replace the fluorescent reporter gene with a therapeutic gene, such as a prodrug-activating gene, under the control of a prostate-specific promoter, to effectively kill the prostate tumour.


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

Basal expression of c-Jun involves selective use of the proximal AP-1 site and is subject to up- and down-regulation by JNK1 and JNK2 respectively  
Dr. S.U. Ahmed and Prof. J. Milner  

JNK1 and JNK2 are regulators of stress-induced apoptosis in mammalian cells.  Here we have used RNA interference to investigate the basal functions of JNK1 and JNK2 in the absence of applied stress.  Selective silencing of JNK2 in epithelial cancer cells induced apoptosis via increased expression of a sub-set of AP-1 regulated genes, namely c-Jun, Jun-B and Fos-B.  Co-silencing JNK1 with JNK2 revealed that up-regulation of c-Jun, Jun-B and Fos-B requires JNK1 and rescued cells from apoptosis. Thus, under basal conditions, JNK2 suppresses apoptosis via inhibiting JNK1-dependent expression of c-Jun, Jun-B and Fos-B pro-apoptotic genes.  These effects were independent of p53 and also appeared independent of stress-related modifications of JNK1, JNK2 and c-Jun, indicating distinct mechanisms for their regulation under basal versus stress-induced conditions.  For c-Jun this was confirmed by chromatin immunoprecipitation (ChIP) which revealed differential usage of proximal and distal AP-1 sites on the c-Jun gene under basal versus stress-induced conditions.  Under basal conditions c-Jun expression involved a JNK1-dependent auto-regulatory loop operating via the proximal AP-1 site and normally selectively suppressed by JNK2.  Thus we identify and characterise a novel JNK1/JNK2 conduit in which JNK1 and JNK2 serve opposing pro- and anti-apoptotic functions in the basal regulation of apoptosis.  

SIRT3 is Pro-Apoptotic and Participates in Distinct Basal Apoptotic
Pathways. Dr. S.J. Allison and Prof. J. Milner  

SIRT3, one of seven mammalian sirtuins, is a NAD-dependent deacetylase. SIRT3 localizes to mitochondria where it deacetylates and thus activates acetyl-CoA synthetase 2 (AceCS2), indicating a role for SIRT3 in metabolism. Here we provide evidence that SIRT3 also impacts upon apoptosis and cell growth control. Using RNAi under basal (non-stress) conditions we show that SIRT3 is required for apoptosis induced by selective silencing of Bcl-2 in HCT116 human epithelial cancer cells. Identical treatment of ARPE19 epithelial non-cancer cells induces G₁ growth arrest which also proved to be SIRT3-dependent. Previously we have identified SIRT1 and JNK2 as constitutive suppressors of apoptosis in HCT116 cells. We now demonstrate that SIRT3 functions in JNK2-regulated apoptosis but is dispensable for SIRT1-regulated apoptosis. SIRT3 is also dispensable for stress-induced apoptosis. Thus the pro-apoptotic functioning of SIRT3 is selectively coupled with defined pathways regulating cell survival under basal conditions.  

Basal compared with stress-induced regulation of p53.
Dr. J.R. Ford and Prof. J. Milner  

The pro-survival sirtuin SIRT1 is a protein deacetylase that has diverse functions in homeostasis, development, disease and repair of damaged DNA. Additionally, it regulates the function of transcription factors involved in the cellular response to stress, prominent amongst which is the pro-apoptotic tumour suppressor p53. Here, we have examined regulation of p53 under basal conditions (absence of applied stress). In HCT116 cancer cells p53 status is regulated by SIRT1 and the transcription factor FoxO4, in which stabilisation and activation of p53 is suppressed by SIRT1 and induced by FoxO4. SIRT1 also suppresses p300-mediated acetylation of p53. However, induced stress by-passes regulation of p53 by SIRT/FoxO4. Basal p53 stability is not regulated by HDM2 in HCT116 cells nor in ARPE-19 non-cancer cells, and p53 status is refractory to the SIRT1/FoxO4 axis in ARPE-19. Therefore, basal regulation of p53 differs between the cancer and non-cancer cells. Additionally we observe distinct patterns of p53 post-translational modifications in response to stress in HCT116 and ARPE-19 cells, with different consequences for induced expression of p53 target genes p21 and HDM2. Thus we identify SIRT1 and FoxO4 as crucial regulators of p53 in cancer cells under basal conditions, and key differences in the regulation of p53 in cancer and non-cancer cell lines.  

Crosstalk between site-specific modifications on p53 and histone H
Dr L.J. Warnock, Dr R.E. Adamson, Dr. C.J. Lynch and Prof. J. Milner  

Previously, we have observed a link between p53 expression and histone H3 post-translational modifications. Here, we ask if specific post-translational modifications of p53 impact upon histone H3 modifications in a selective manner.  We have also screened for internal co-operative effects within the repertoire of p53 modifications. Exogenous p53 constructs were expressed in HCT116 p53^-/- cells. Four mutant p53 constructs were used, with single 'phosphorylation' mutants at serines 15 and 17 (S15A, S15D, S37A and S37D) and compared with exogenously expressed wild-type p53. The results showed that the replacement of serine 15 with either alanine (S15A) or aspartic acid (S15D) induced phosphorylation at S33P, S37P and S46P. In contrast, phosphorylation mutants p53(S37A) and p53(S37D) were not phosphorylated on S33. S46 phosphorylation appeared specifically enhanced by p53(S37D) relative to p53(37A). Distal induction of S392 phosphorylation was observed for each of the p53 N-terminal phosphorylation mutants. Analysis of endogenous histone H3 (from the transfected cells) revealed loss of di-methylated K9 following expression of wild type and mutant p53 constructs. Expression of p53 (S15A), (S15D) and (S37A) selectively induced acetylation at K9 and K14. In contrast, wt p53 and p53(S37D) had no effect upon K9 or K14 acetylation. K18 acetylation status was unaffected throughout.  

Tetramerisation and site-specific post-translational modifications of the tumour suppressor protein p53.
Dr. L.J. Warnock, Ms R.E. Adamson, Dr C.J. Lynch and Prof. J. Milner  

The tumour suppressor protein p53 is considered the "Guardian of the Genome", crucial for cell cycle control and mutated in over 50% of human cancers.  In non-stressed cells, p53 levels are under tight regulation, altering throughout the normal cell cycle. However, in response to cellular stress, post-translational modifications such as phosphorylation and acetylation stabilise and activate p53 for cell cycle arrest, DNA repair, apoptosis or senescence. P53 protein functions as a tetramer, (dimer of dimers) and we have previously demonstrated that loss of tetramerisation and changes at the N-terminus influence the recovery of wild type p53 'status'. To investigate the relationship between tetramerisation and post-translational modifications we examined a range of site-specific modifications in wild type and dimeric mutant (M340Q/L344R) murine p53 expressed in MEFs p53^-/- and in wild type, monomeric (L344P) and dimeric (M340Q/L344R) human p53 expressed in HCT116 p53^-/- cells. Using site-specific antibodies we demonstrate that in murine p53, S15 is phosphorylated in a tetramerisation-dependent manner.  In contrast, human p53 S15 phosphorylation is not tetramerisation-dependent. However, inability to form tetramers in human p53 proteins reduced N-terminal phosphorylation at S6, S9 and S46 and in addition reduced C-terminal phosphorylation and acetylation at S315 and K382 respectively.