Characterization of peptide aptamers to the Anterior Gradient 2: a novel inhibitor of the tumour suppressor protein p53

Anterior Gradient-2 was identified using proteomic technologies as a protein overexpressed in human cancers. We show here properties of AG2 suggesting it has proto-oncogenic properties:(i) the AG2 protein and gene are unregulated in a tamoxifen resistance panel of breast cancer cells lines, (ii) cell lines overproducing AG2 have an elevated clonogenic activity in vitro and also increase cell growth in a xenograft model, (iii) AG2 protein inhibits the tumour suppressor protein p53, and (iv) AG2 localizes to the endoplasmic reticulum, suggesting a proto-onocogenic signalling pathway exists from endoplasmic reticulum to the nucleus to inhibit p53. 
 
To validate the AG2-mediated endoplasmic reticulum pathway as a possible drug target, we developed peptide aptamers to AG2 protein in order to determine whether the oncogenic properties of the protein can be altered by the peptide ligand. 
 
These studies hold promise for developing new types of drugs that can release and reactivate the tumour suppressor p53 in breast cancers.

In order to discover novel pathways that regulate stem cell self-renewal, we have applied functional genomics using an RNAi library targeting 8,000 genes involved in cancer. This has revealed the importance of several pathways not previously associated with stem cell self-renewal. These pathways may represent novel targets for breast cancer therapy aimed at the breast cancer stem cells that survive conventional therapies.  (DOI 10.1186/bcr1551) Background Like many developmental signalling pathways, the Notch pathway has been linked to the aetiology of several different human cancers. The development of focal adenocarcinomas in the murine mammary gland [1] and the transformation of both normal murine and human breast epithelial cell lines following Notch activation [1,2] have long suggested that the pathway may play a role in human breast cancer. However, this question has received little attention. Methods Activation of the Notch pathway in human breast cancer cell lines and breast carcinoma samples was monitored by western blotting with an antibody that recognises the cleaved Notch1 intracellular domain which is produced during signalling. Regulation of apoptosis by Notch was studied in MCF 10A cells transformed by overexpressing the Notch1 intracellular domain. Apoptosis was triggered by treating cells with the kinase inhibitor staurosporine or the DNA damaging agents melphalan and mitoxantrone, and monitored by nuclear fragmentation or cleavage of caspase 3. Changes in the apoptotic machinery were examined by western blotting using a range of antibodies that recognise both total and phosphospecific forms of different components. Results We will present data showing that Notch signalling is activated in a wide range of breast cancer cell lines and in a panel of 20 human breast carcinomas of different pathological grade and prognosis. In addition, we will demonstrate that sustained signalling is required to maintain the transformed phenotype of breast cancer cell lines, as its inhibition by expressing Numb, a natural inhibitor of the pathway, causes both MCF7 and MDA-MB-231 cells to adopt a normal phenotype. Our data with the normal breast epithelial cell line MCF 10A indicate that Notch signalling contributes to the transformed phenotype by inhibiting apoptosis. Activation of Notch signalling in these cells by overexpressing the Notch1 intracellular domain prevents apoptosis in response to growth factor withdrawal, removal from the extracellular matrix and DNA damage. Finally, we will provide evidence that the apoptosis resistance seen in Notch transformed MCF 10A cells is through the activation of the Akt survival pathway. Conclusion Altogether this suggests that targeting Notch signalling may be a novel therapeutic strategy for the treatment of breast cancer. The laminin receptors α 3 β 1 and α 6 β 1 are expressed by endothelial cells, but their direct roles in tumour angiogenesis and especially breast cancer angiogenesis remains unexplored. We show that α 6 β 1integrin is expressed in 80-90% of blood vessels associated with normal breast or ductal carcinoma in situ. However, the proportion of vessels that express α 6 β 1 drops to less than 30% in invasive ductal carcinoma samples, suggesting that loss of this laminin receptor can enhance invasive carcinoma angiogenic events. Furthermore, the deletion of α 6 -integrin or α 3 -integrin in ex vivo angiogenic assays can promote VEGF-mediated microvessel sprouting. Taken together these results implicate these integrins in the negative control of angiogenesis.

S8
Since global deletion of the α 3 -integrin or α 6 -integrin genes in mice is lethal, we have generated mice where these genes are deleted on endothelial cells only.
Our data indicate that mice deficient in individual laminin receptors on endothelial cells in vivo not only support tumour growth but have enhanced tumourigenesis. Moreover, tumour angiogenesis is elevated in these mice, suggesting strongly that laminin receptors are not required for tumour angiogenesis. We also observed that angiogenic responses to hypoxia are enhanced in mice deficient for laminin receptors on endothelial cells and have evidence that, at least in α 3 -null endothelial cells, VEGF-receptor 2 (FLK1) levels are elevated when compared with controls. We provide the first evidence that α 3 -integrin and α 6 -integrin can be differentially expressed in the angiogenic vessels associated with invasive carcinoma of the breast and suggest that these laminin receptors can negatively regulate angiogenesis in vivo and ex vivo. We have previously demonstrated that a functional orthologue of the breast cancer tumour suppressor gene BRCA1 exists in C. elegans (brc-1). Deletion mutants in C. elegans brc-1 or its heterodimeric partner, brd-1, share many of the phenotypic hallmarks of BRCA1deficient human cells, yet are homozygous viable thus permitting extensive reverse genetic analysis. Using a rapid and inexpensive genome-wide screen in C. elegans we set out to identify genes that could be targeted in human patients to selectively kill tumours defective in the BRCA pathway. To this end we have utilized the complete C. elegans RNA-mediated interference library to systematically inactivate all 19,500 C. elegans genes and have identified those genes whose depletion confers synthetic lethality in combination with brc-1 and brd-1 mutations. In total, this screen identified 20 genes including pme-1 and pme-2, the C. elegans counterparts of PARP, a gene whose inhibition selectively kills BRCA defective tumour cells. We are currently using siRNA to knockdown all human homologues to identify those genes whose inactivation specifically kills mammalian cells harbouring mutations in BRCA1. These results and our current progress will be presented.

S11
Microarray studies reveal novel genes associated with endocrine resistance in breast cancer S12 Benefits of combined treatments using antiresorptive agents and cytotoxic drugs Background Breast cancer patients often receive a combination of different therapies, but our understanding of how best to utilise such combinations to achieve maximal benefit for the patients is incomplete. We have investigated the ability of the antiresorptive agent zoledronic acid (Zol) and the commonly used chemotherapy agents paclitaxel (Pac) and doxorubicin (Dox) to induce apoptotic breast cancer cell death in vitro. Methods Hormone-dependent (MCF7) and hormone-independent (MDA-MB-436) breast cancer cells were treated with increasing doses of Zol, alone and in sequence or combination with a low dose of Pac (2 nM) or Dox (0.05 µM) for 1-72 hours. The following treatment groups were used: (A) untreated controls, (B) each drug given as a single agent, (C) the drugs given simultaneously, (D) chemotherapy agent followed by Zol, and (E) Zol followed by the chemotherapy agent. In some cases Zol was given together with GGOH, a downstream component of the mevalonate pathway targeted by Zol. The effects of the different treatments on both apoptotic and necrotic cell death were determined at 72 hours, by evaluation of nuclear morphology following staining with Hoechst and PI. The effects of the various treatments on the cell cycle distribution were also determined. Results Our data show that exposing breast cancer cells to the chemotherapy agent prior to Zol results in a synergistic increase in tumour cell death, compared with when the drugs are used as single agents. This was seen both for paclitaxel and doxorubicin, and the effect was found to be associated with changes in the cell cycle distribution following pretreatment with the cytotoxic drug. The synergistic increase in tumour cell death could be reversed by addition of GGOH, a compound that counteracts the effects of Zol on a key metabolic pathway, supporting an essential role of Zol in the toxic effects of the combined treatments. We also show that these effects are significant using clinically achievable doses and exposure times, suggesting that sequential treatments may be relevant also in a clinical setting.
Conclusions We have shown that combining chemotherapy agents and the antiresorptive drug Zol results in a synergistic increase in breast cancer cell death in vitro. We are currently investigating whether the same is seen using more complex in vivo model systems. Our data suggest that in order to achieve maximum benefit from combined treatments, the order and timing of the combinations are crucial. ) ZAC (also known as PLAGL1/LOT1) is a transcription factor gene located on chromosome 6q24, a region that is frequently deleted in solid tumours. ZAC is known to promote cell cycle arrest and apoptosis, and loss of expression has been observed in several different cancers including primary breast tumours and breast cancer cell lines. Due to its antiproliferative properties, the downregulation or loss of this gene would be expected to deregulate cell growth. ZAC has also been shown to act as a transcriptional coactivator of nuclear receptors, including oestrogen receptors which are important as prognostic indicators and therapeutic targets in breast cancer. ZAC is maternally imprinted in most tissues. Its promoter is believed to be located within a differentially methylated CpG island, and it directs transcription exclusively from the unmethylated paternal allele. As this imprinted promoter has been shown to be hypermethylated in ovarian cancer and breast cancer cell lines, similar epigenetic changes may occur in primary breast tumours, and may contribute to altered cell cycle regulation and thus tumour growth. In some tissues, however, ZAC expression is biallelic. We are currently studying the mechanism underlying this tissue-specific phenomenon. A detailed understanding of the way in which the expression of imprinted and nonimprinted transcripts is regulated in normal breast tissue will be required in order to allow analysis of the epigenetic mechanism for ZAC inactivation in breast tumours. inhibit activation in response to DNA damage but not stalled replication forks ( Figure 1). However, the knockdown of TopBP1 alone was insufficient to inhibit activation. Conclusion Inhibition of Chk1 activation in response to ionising radiation requires the loss of both TopBP1 and BRCA1, suggesting redundancy. In addition, as the response to hydroxyurea, or UV, was unaffected, it seems likely that different proteins are involved in Chk1
activation in response to differing stimuli. Analysis of other Chk1 binding proteins continues determining whether they are involved in Chk1 activation in response to stalled replication forks and/or doublestranded DNA breaks. As Chk1 is involved in maintaining tumor cell viability following activation of the replication checkpoint, the Chk1regulated checkpoint(s) may protect cells from ionizing radiationinduced killing. The ability to delineate the control mechanisms of Chk1 is of critical importance in order to target Chk1 with the aim of increasing the selectivity and specificity of anticancer drug treatments. Acknowledgement Breast Cancer Campaign funded the project.

P5
The NEUREGULIN1 gene and breast cancer  apoptotic genes (for example, BIRC5, BCL2, DR4 and DR5) have been implicated, and we reported that a coding single nucleotide polymorphism (SNP) in the caspase 8 gene (CASP8 D302H) is associated with a reduced risk of breast cancer [1]. We hypothesise that CASP8 and other apoptotic genes may play an important role in breast cancer susceptibility. The objectives were to study the functional effect of CASP8 D302H on apoptosis, and to perform a case-control analysis of other CASP8 variants to determine their effect on breast cancer susceptibility. Methods Apoptotic activity in peripheral blood lymphocytes (PBLs) was measured using Annexin-V FITC with propidium iodide and FACs analysis. Genotyping was conducted by TaqMan™ (ABI, UK).

Results
We detected a 68% increase in apoptosis in PBLs after treatment with CD95 ligand (R & D Systems, UK) with anti-CD95 antibody (BioLegend, UK), and are currently optimising this assay as a functional screening tool. We identified 50 SNPs in CASP8 by database searching, and 15 more putative SNPs were sequenced, one of which is novel (T51087A in exon 13). Using data from 33 SNPs with a minor allele frequency > 0.05 and various haplotype-tagging SNP (htSNP) selection programs, results suggested that 11 htSNPs (PCA method) need to be genotyped to adequately capture common genetic variation within CASP8. A case-control study of these 11 htSNPs is in progress.
Conclusion These methods will be used to address the hypothesis that apoptotic genes are involved in breast cancer susceptibility and treatment outcome. In the future, this research will help us understand the role of the whole pathway and whether it will be amenable to manipulation by targeted treatments.  [1,2]. In this study, we aim to identify the target genes regulated by PLU-1/ JARID1B and the possible mechanism of PLU-1/JARID1B-mediated transcriptional regulation. Methods Co-immunolocalisation and/or co-immunoprecipitation of PLU-1/JARID1B with HDACs were carried out using anti Myc/HisA antibodies or an antiserum (αPLU-1-C) against PLU-1/Jarid1B after transient transfection of Cos and MCF7 cells with expression vectors coding for Myc or HisA tagged proteins. Direct interactions of PLU-1/ JARID1B expressed from a baculovirus with in vitro translated HDACs were also demonstrated. In vitro mutagenesis and reporter assays were also used. HB2 and MCF7 cells were subjected to microarray using the Affymetrix gene chip HG-U133A after transduction with a recombinant adenovirus or silencing the endogenous gene using a short hairpin RNA (shRNA) expression vector (Imgenex). ChIP assays were carried out using the αPLU-1-C specific antiserum or an antibody against the acetylated form of Histone H3. PCR-assisted DNA binding selection from a random pool of oligonucleotides was carried out using in vitro translated full-length PLU-1/JARID1B and GST-PLU-1-ARID.
Results PLU-1/JARID1B binds to chromatin and the nuclear matrix and localises in MAD bodies when co-transfected with class IIa histone deacetylases (HDACs) or N-CoR. Direct binding to class I and class IIa HDACs is demonstrated using co-immunoprecipitation assays and binding of PLU-1/JARID1B to in vitro translated HDACs. Two PHD domains in PLU-1 were shown to be crucial for binding to a domain in the N-terminal region of HDAC4 and for the transcriptional repression. Approximately 100 target genes were identified by microarray analysis after overexpressing or silencing the human PLU-1/JARID1B gene in human mammary epithelial cells using adenovirus and RNA interference systems, respectively. In mammalian cells, cell cycle progression is governed by distinct cyclin-dependent kinases (cdks) whose activities are regulated by binding of their activating cyclin subunits and through negative regulation by inhibitor proteins such as p21. Cyclin levels oscillate in a phase-dependent manner, ensuring the stage-specific activation of cyclin/cdk complexes. The D-type cyclin levels are thought to act as sensors of the cellular environment: under conditions permissive for proliferation, D-type cyclins accumulate and facilitate the G 1 phase progression; whereas under restrictive conditions, D-type cyclin transcription is attenuated and the protein is destabilised via ubiquitinmediated proteolysis. In addition to the normal cell cycle regulation, a member of D-type cyclins, cyclin D 1 , has been implicated in the DNA damage response. Once activated, DNA damage responses disrupt the function of the cell cycle and can result in a number of outcomes including short-term or long-term cell cycle arrest, apoptosis and necrosis. Cyclin D 1 expression is often found deregulated in cancerous cells, particularly in those of the breast and the head/neck.

Results
Preliminary data showed that the expression of cyclin D 1 responds to the DNA damage induced by an environmental carcinogen, 4-nitroquinoline 1-oxide (4NQO), in a biphasic manner. At a low level (2.5 µM), the cyclin D 1 level is unchanged but p21 is induced strongly after 3 hours; at intermediate levels (10-50 µM), there is a dramatic reduction in the level of cyclin D 1 while p21 fails to accumulate; at high levels (100-200 µM), little change in cyclin D 1 or p21 is observed. The cellular responses associated with different 4NQO doses analysed by flow cytometry will be presented. Conclusion Our findings suggest that the level of cyclin D 1 following the DNA damage induced by 4NQO may play a role in dictating the outcome of the cellular response. Our ongoing research aims to compare and contrast the cellular responses linked to various specific DNA damaging agents in terms of cell cycle regulatory proteins, focusing on cyclin D 1 , and ultimately to understand the molecular mechanisms underlying the regulation of such responses.  [2]. We have identified upregulation of α(v)β 6 integrin on myoepithelial cells in a subset of DCIS; however, the role of α(v)β 6 in this context is not clear. α(v)β 6 is not expressed by normal epithelial cells, but is expressed in some cancers where it promotes tumour cell invasion and enhances MMP expression.

P14 Functional analysis of normal and DCIS modified breast myoepithelial cells M Allen, K Mulligan, S Clark, I Hart, JF Marshall, JL Jones
Methods The purpose of this project is to investigate the hypothesis that DCIS-associated myoepithelial cells lose their tumour suppressor effect and acquire a tumour promoting activity. There are three general aims: (1) to generate a series of myoepithelial cell models to mimic DCIS-associated myoepithelial cells and overexpress α(v)β 6 to assess the contribution of this integrin; (2) to compare tumour suppressor/ promoter properties of normal, α(v)β 6 overexpressing and DCISassociated myoepithelial cells; and (3) to examine the effect of de novo α(v)β 6 expression on the biological activity of myoepithelial cells.

Results
We have fully characterised an immortalised myoepithelial cell line, engineered it to overexpress α(v)β 6 and determined that it is functional. We are starting to examine the morphology and phenotype of these cells to determine any differences, and we have been able to show the parental cell line is able to recapitulate the tumour suppressor effect in in vitro systems. We are now looking into what effect the expression of α(v)β 6 has in these systems. We are also in the process of trying to create further myoepithelial cell lines from primary cells isolated from patient tissue. Conclusion Through this work we hope to identify the role α(v)β 6 expression has in DCIS myoepithelial cells with the goal of making this integrin a viable therapeutic target in the future. Acknowledgement This work was funded by Breast Cancer Campaign.  Background In order to provide potential diagnostic markers and to identify potential targets for breast cancer therapy, gene products that are differentially expressed between benign and malignant cells have been isolated and identified by a combination of PCR-selected suppression subtractive libraries [1,2] and inhouse cDNA microarrays, screened using mRNAs from human breast cancer specimens. A number of the cDNAs were differentially expressed by greater than twofold, including the one for AGR2, the secreted human homologue of a Xenopus developmental protein.

Methods and results
In an in vivo model system of metastasis, AGR2 induced metastases compared with no metastases in the control groups [3]. In immunocytochemistry with an inhouse affinity-purified AGR2 antiserum [3], the presence of AGR2 protein in tumour specimens was statistically significantly associated with malignancy, with oestrogen receptor (ER) alpha-positive carcinomas, with low histological grade and with reduced patient survival over a 10-year period of follow-up of a group of ER-positive cases [4].
Conclusions Our results demonstrate that AGR2 is causatively involved in metastasis and associated with poor outcome in patients with breast cancer, indicating that AGR2 might be a valuable new potential diagnostic marker and possible target for breast cancer therapy. Further studies are essential to understand the mechanism of AGR2-induced metastasis.

P16
Insulin-like growth factor signalling in oestrogen nonresponsive breast cancer cells  Conclusion Expression and activation of IGF signalling proteins vary among the oestrogen nonresponsive cells. These differences will affect the response of breast cancer cells to IGF targeted therapy. BT-20 cells provide a useful model for constitutive IRS-1 phosphorylation which is reported to occur in breast tumours [3].

P17
Role of the Brk tyrosine kinase in breast cancer progression Methods To further understand the potential significance of CD44 signalling to breast cancer metastasis, we established a tetracyclineregulated CD44 expression system in the minimally invasive, CD44negative MCF7F cell line. Removal of tetracycline from the growth media resulted in time-dependent increases in CD44 expression in MCF7F cells, promoting increased cell invasion and migration responses in addition to potentiating the adhesion of MCF7F cells to BMECs. Subsequent microarray analysis was conducted using this expression system to identify CD44/HA regulated genes in breast cancer cells.

Results
The expression and activation of CD44 was associated with increased expression of a subset of genes implicated in metastasis including proteolytic enzymes, growth factors and cytoskeletal proteins (for example, cortactin). Interestingly, the cysteine protease cathepsin K and the matrix metalloprotease MT1MMP were identified as CD44/HA regulated genes. These proteases target collagen I, a major component of the bone matrix whose degradation is a major consequence of osteolytic metastasis of breast cancer. Four transmembrane domain proteins of the tetraspanin superfamily are the organisers of specific microdomains at the membrane (tetraspaninenriched microdomains (TERM)) that incorporate various transmembrane receptors and modulate their activities. Tetraspanin CD82 is frequently downregulated or absent in the metastatic cancers. In human prostatic cancer, downregulation of CD82 has been correlated with tumour progression, providing evidence for its role as a metastasis suppressor. We have shown recently that the overexpression of metastasis suppressor tetraspanin CD82/KAI1 led to the attenuation of epidermal growth factor receptor (EGFR) signalling, to an increased internalisation rate of the receptor and to the redistribution of EGFR at the plasma membrane [1]. Moreover, our latest data suggested that the effect of CD82 on the EGFR signalling is mediated by gangliosides [2]. Gangliosides (a subclass of glycosphingolipids) are essential structural components of distinct microdomains at the membrane. In addition, these glycosphingolipids are also involved in the regulation of signalling and tumour progression. We currently demonstrate that inhibition of the glycosphingolipid biosynthetic pathway with specific inhibitors of glucosylceramide synthase (NB-DGJ and PPMP) resulted in specific weakening of the interactions involving tetraspanin CD82, including CD82-EGFR association. Furthermore, ectopic expression of the plasma membranebound sialidase Neu3 in mammary epithelial cells destabilised CD82containing complexes. The destabilisation of these complexes correlated with the redistribution of the proteins within the plasma membrane. Importantly, depletion of gangliosides affected EGF-induced signalling only in the presence of CD82. Taken together our results provide strong evidence that gangliosides play an important role in supporting the integrity of CD82-enriched microdomains [3]. Furthermore, these data demonstrate that the association between different proteins in TERM in mammary epithelial cells is controlled by distinct mechanisms.
In further experiments we are going to investigate the role of TERM, and specifically CD82-enriched microdomains, in the signalling through the ErbB3 receptor. The ErbB3 receptor is considered a major partner for the ErbB2 receptor and is involved in the progression of breast cancer.

P22 Expression of adrenomedullin in long-term oestrogen-deprived human breast cancer cells A Sadler, P Darbre University of Reading School of Biological Sciences, Reading, UK Breast Cancer Research 2006, 8(Suppl 2):P22 (DOI 10.1186/bcr1577)
Oestrogen is a major requirement for the growth of human breast cancer cells. Current treatments are aimed at reducing the action of oestrogen with antioestrogen therapy. However, many patients are able to progress to a state where they no longer respond to antioestrogen therapy. Long-term growth of breast cancer cell lines in the absence of oestrogen leads to the development of acquired resistance where the cells are able to grow without the addition of oestrogen, they can still be inhibited by antioestrogens and there is no loss of oestrogen receptor alpha. The aim of this work was to identify novel molecular markers that could indicate impending failure to endocrine therapy. Adrenomedullin is a 52-amino-acid peptide which may play a role in tumour survival and angiogenesis. Microarray data comparing oestrogen-maintained MCF7 cells with long-term oestrogen-deprived MCF7 cells showed that the expression of adrenomedullin mRNA was 12-fold upregulated after more than 1 year of culture in the absence of oestrogen. Real-time RT-PCR data were able to confirm the increase in the levels of adrenomedullin mRNA in long-term oestrogen-deprived cells. Immunocytochemistry using a monoclonal antibody specific for adrenomedullin was also able to show an increase in the amount of adrenomedullin protein in long-term oestrogen-deprived cells. Furthermore, long-term treatment of oestrogen-maintained cells with tamoxifen and fulvestrant led to an increase in the level of adrenomedullin mRNA which was not observed in long-term oestrogen deprived cells. Further validation with tumour samples is required to examine the importance of adrenomedullin as a possible marker of endocrine resistance in human breast cancer. A protein called protein kinase B (PKB, also called Akt) is frequently elevated in breast cancers and has been implicated as a key player in breast cancer development and progression. The activation level of PKB is also thought to correlate with patient outcome. However, the function of the three isoforms of PKB in mediating the crucial responses is unknown. We have developed a set of antisense phosphorothioate oligonucleotide probes that target antisense-active regions in PKB and that enable > 90% knockdown of all three known PKB isoforms (alpha, beta and gamma), either individually or in various combinations, including removal of all three isoforms together. We have demonstrated that these agents specifically and potently prevent the growth of breast cancer cells. Application of these antisense agents offers a unique opportunity to understand how PKB works and contributes to breast cancer, and to provide insight into the role of signalling by individual PKB isoforms in breast cancer cells. Such work may also identify clinically relevant markers of disease, thereby enabling better predictors of patient outcome, and provide the necessary intellectual framework for the development of PKB-isoform selective inhibitors (for example, antisense oligonucleotides, small chemical inhibitors) as novel therapeutic agents. Oestrogen receptor (ER) alpha remains the only reliable biological prognostic marker in breast cancer. A sister molecule, ERβ, has been described, but while ERα predicts a favourable disease outcome, the utility of ERβ as a clinical prognosticator is unclear. ERβ exists as five isoforms (ERβ1-ERβ5), each with a unique exon 8. The aim of our research is to understand the function of ERβ and its isoforms in the normal mammary gland and in breast cancer. We have previously shown high expression of total ERβ in normal gland with declining expression in the transition to breast tumours. LOH analysis in 27 paired samples of tumours and normal breast showed no correlation between LOH and loss of total ERβ expression by immunohistochemistry, indicating the latter was not a mutational event. Instead this was due to methylation as treatment of ERβ-negative cell lines resulted in re-expression of total ERβ at the protein and mRNA level. Furthermore, using methylation-specific PCR, ERβ was methylated in up to 50% of all tumours but not in matched normal gland. Recent immunohistochemical analysis of isoforms ERβ1-ERβ5 using specific well-validated antibodies in 777 invasive breast cancers with long-term clinical follow-up showed nuclear expression of ERβ2 was significantly correlated with tumour size, grade, NPI, overall survival, distant metastasis, death from breast cancer and ERα, PR, AR and BRCA1 expression. ERβ5 was predominantly expressed in high-grade cancers and showed a significant positive correlation with ERβ1. ERβ1, however, was not associated with any other pathological parameters.

P24 Progress towards unlocking the secrets of oestrogen receptor beta in breast cancer
Using an antibody to detect total ERβ, positive tumours were more likely to develop distant metastasis. Notably, this study also highlighted the importance of cytoplasmic expression of ERβ in dictating outcome, a feature that had previously been reported but the significance of which had not been elucidated. In our study cytoplasmic staining, whether alone or in combination with nuclear staining, was associated with decreased overall survival. In summary, ERβ and its variants do seem to influence the breast cancer outcome. The data accumulated thus far and the importance of its sib ERα in directing breast cancer therapy create an imperative for us to continue to unlock its secrets. Both the proportion and intensity of expression was recorded for the total and subpopulations of CXCR4 recognised by ARP4016 and the two cytoplasmic antibodies, respectively. For immunofluroscence the average fluorescence intensity/unit area of cells stained with the respective antibodies were plotted and quantified.

Results
The proportion and intensity of invasive cells expressing CXCR4 was significantly less in Grade III infiltrating ductal carcinoma compared with Grades I, II and lobular types (P < 0.0001 by Kruskal-Wallis). There is a complex relationship between survival and total CXCR4 expression, with a subset of high CXCR4 expressing, Grade III tumours showing a trend towards poor prognosis. This association will be further elucidated by results of the CXCR4 cytoplasmic antibody staining. Conclusions CXCR4 was expressed uniformly across a spectrum of normal, and a panel of invasive breast tumour cells but only a subset of Grade III tumours expressing high CXCR4 correlated with poor prognosis. It may be that only highly invasive cells that are metastatic and very poorly differentiated express functional CXCR4 receptors. CXCR4 function is subject to complex and potentially tightly controlled regulation in breast cancer cells via differential G protein receptor complex formation and this regulation may play a role in the transition from non metastatic to malignant transformation [1]. The application of new antibody tools and optical technologies to these pathological samples will assist the discovery of new biomarkers that will report on the function of CXCR4 in situ. We demonstrated that expression of these P450s altered the effects of estrogens and antiestrogens on cell cycle and apoptotic markers. Currently, the MCF-7-derived cell lines are being grown in xenografts. P450 expression will be induced by doxicycline in the drinking water, and animals will be treated with or without tamoxifen. Subsequently, the effects of P450 expression on tumour growth, angiogenesis and apoptosis will be measured. It is anticipated that the results of these investigations will greatly enhance our understanding about the aetiology of breast cancer and may provide strategies to improve treatment. Methods The first 1,200 cases from the study in whom diagnostic pathology reports were submitted were analysed. We looked at the distribution of the reported tumour phenotype (major prognostic histopathological features) in women aged ≤ 35 years (43% of the total cohort) compared with women diagnosed age 35-40 years in order to further explore biological explanations for the known worse clinical prognosis for women aged under 36 years compared with older women. The χ 2 statistic was used to compare groups; genetic risk for each recruit was derived using software that incorporates a general genetic model rather than a gene specific model. The highest genetic risk groups are likely to harbour most of the BRCA1 and BRCA2 gene carriers.

Results
The majority of women at all ages were treated with anthracycline-based adjuvant chemotherapy and there was no difference in the choice of immediate surgical management between either age groups or between genetic risk groups. Significantly more women in the ≤ 35 years age group had grade 3 (P < 0.01) and ER-negative (P < 0.02) tumours compared with women diagnosed in the older age group (> 35 but ≤ 40 years). There was no significant difference in tumour size or lymph node status based on age categories. Compared with women with no family history, women falling into the 10% of the cohort estimated from family history to be most likely to carry BRCA1 or BRCA2 gene mutations, high genetic risk women had significantly more grade 3 tumours (P < 0.001) and a nonsignificant trend towards more ER-negative tumours. Conclusion These data are from a preliminary pending systematic pathology review but bear out the observations by others that very young age of onset and host genotype affect the tumour phenotype and are therefore likely to have an impact on prognosis. Longer followup of this cohort is planned and outcome data based on age and based on genetic risk category and genetic mutation status will be available in a further 12 months time. TGFβ acts as a suppressor of primary tumour initiation but is implicated as a promoter of the later malignant stages. A hypothesis explaining this dual action proposes that TGFβ acts through the ubiquitous ALK5/SMADs2&3 signalling pathway to inhibit proliferation of primary tumour cells, but acts through the endothelial-specific ALK1/ SMADs1&5 pathway to promote angiogenesis, which is required for progression to malignancy. In a previous study we showed that a SNP generating a leucine to proline substitution in the signal peptide of the TGFB1 protein was associated with a 1.24-fold increase in the risk of invasive breast cancer, with increased levels of the protein in human serum and with a 2.8-fold increase in amount of the protein secreted in vitro.
It is also plausible that other genes in the TGFβ signalling pathways might be associated with altered risk of breast cancer.  [1] and there is evidence that energy restriction may reduce risk [2]. Animal studies indicate that intermittent energy restriction (IER) reduces risk and may be superior to continuous energy restriction (CER) [3]. We have shown chronic energy restriction reduces biomarkers of breast cancer in women at risk but is hard to maintain. We hypothesise that IER may be superior to CER in reducing biomarkers of breast cancer risk and may also be more acceptable to women. Methods We are undertaking a 6-month randomised trial to compare the two approaches in 104 premenopausal women aged 30-45 years at high risk of breast cancer because of adult weight gain >7 kg. Women will be randomly assigned to either CER (75% estimated energy requirements: ~1,500 kcal 7 days/week) or IER (75% estimated energy requirements: 650 kcal for 2 days and ~1,800 kcal 5 days/week) over 6 months. Study end points will be measures of insulin sensitivity (HOMA, SHBG and testosterone), potential breast cancer growth factors (IGF axis, leptin and adiponectin), inflammatory markers (C-reactive protein and sialic acid), oxidative stress marker (urinary F2 isoprostane), weight and body composition (waist/hip circumference, fat free and total fat mass). The relative acceptability of IER and CER will be assessed using a quality of life questionnaire (RAND SF-36) and scales of behaviour change and adherence. The relative efficacy and acceptance of intermittent and chronic calorie restriction will inform future weight loss programmes to prevent breast cancer. Methods FOXP3+ T R were detected by immunohistochemistry with our new FOXP3 monoclonal antibody, 236A/E7. Numbers of FOXP3+ lymphocytes in tissue microarray cores from pure ductal carcinoma in situ (DCIS) (n = 62), from invasive breast cancer (n = 237) or from comparable areas of normal terminal duct lobular breast tissue from patients without cancer (n = 10) were determined. A median cutoff value of 15 defined patients with high numbers of T R . Results T R numbers were significantly higher in DCIS and invasive breast carcinomas when compared with normal breast, with invasive tumours having significantly higher numbers than DCIS (P = 0.001).
High numbers of FOXP3+ T R identified patients with DCIS at increased risk of relapse (P = 0.04) and patients with invasive tumours having both shorter relapse-free (P = 0.004) and overall survival (P = 0.007). High T R numbers were present in high-grade tumours (P < 0.001), in patients with lymph node involvement (P = 0.01) and in estrogen receptor alpha (ER)-negative tumours (P = 0.001). Importantly, quantification of FOXP3+ T R identified a group at high risk of relapse, within the so-called good prognostic group of ER-positive patients (P = 0.005) and these patients have a prognosis as poor as those that lack ER expression. Multivariate analyses, in ER-positive patients, demonstrated that greater T R numbers independently conferred a significantly higher hazard ratio than that of tumour grade and nodal status for relapse-free and overall survival, respectively. Unlike conventional clinicopathological factors, high numbers of FOXP3+ T R identified patients at risk of late relapse after 5 years disease-free survival.
Conclusion These findings indicate that quantification of FOXP3+ T R in breast tumours is valuable for assessing disease prognosis and progression, and represents a novel marker for identifying late-relapse patients who may benefit from aromatase therapy after 5 years of tamoxifen treatment. Furthermore, tumour vaccination approaches in combination with targeting T R cells are just entering clinical trials and our data strongly suggest that such therapy would be beneficial for a significant proportion of breast cancer patients. Acknowledgement The authors would like to thank Breast Cancer Campaign for their research support.

P32
Development of anti-MUC1 DNA aptamers for the imaging and radiotherapy of breast cancer Background Aptamers are novel oligonucleotide-based recognition molecules which can bind to almost any target, including extracellular proteins, antibodies, peptides and small molecules. Aptamers can be rapidly generated, and offer reduced immunogenicity, good tumour penetration, rapid uptake and clearance, and can thus be used as alternatives to monoclonal antibodies in molecular targeted radiotherapy and diagnostic imaging.
Methods We have previously reported the isolation of high affinity and specificity DNA aptamers against the protein core of the MUC1 glycoprotein as a tumour marker on breast cancer cells. Once conjugated with a chelating agent and labelled with a radionuclide ( 99m Tc or 188 Re), such aptamers can be particularly useful in the diagnosis and targeted radiotherapy of breast cancer. The conjugation is achieved using standard peptide coupling reactions between an amino modification on the aptamer and the carboxylic group on the ligands.

Results
We have coupled the aptamer with the highest affinity for the MUC1 glycoprotein to different ligands (MAG2 or meso-2,3-dimercaptosuccinic acid) and labelled it with 99m Tc and 188 Re to obtain stable complexes. An efficient and convenient labelling of the aptamer with short half-life radioisotopes was achieved as the last step of the synthesis (postconjugation labelling).

Conclusions
The selected ligands have strong 99m Tc and 188 Re binding properties and the resulting complexes are highly stable in vivo both in terms of nuclease degradation and leaching of the metal. The presence of more than one molecule of aptamer per complex or the conjugation of the aptamer to high molecular weight polyethylene glycol modifies the pharmacokinetic properties of the radiolabelled products, allowing the complex to remain longer in circulation and thus offering improved tumour imaging properties and further possibilities for development into a targeted radiopharmaceutical for breast cancer therapy. Acknowledgement The authors thank Breast Cancer Campaign for financial support. However, the expression and prognostic significance of modified histones in breast cancer has not been previously explored. Methods Global histone modification in a large well-characterised series of breast carcinomas (n = 880) with long-term follow-up was therefore assessed using immunohistochemistry and tissue microarray. Specific antibodies were used to detect acetylation of H3 (Lys9 and Lys18) and H4 (Lys12), and dimethylation of histone H4 (Arg3) and H3 (Lys4). The presence of these chromatin 'marks' was correlated with clinicopathological variables and patients' outcome.
Results Reduced levels of histone acetylation/dimethylation were observed in medullary-like carcinomas, whereas they were readily detected in lobular and tubular carcinomas. Reduced global histone acetylation/dimethylation was significantly associated with established poor prognostic variables; larger tumour size, higher stage, recurrence, distant metastases and higher mortality rate. Survival analyses showed low detection of the histone modifications, with the exception of acetylated H3K9, was associated with shorter overall survival and shorter disease-free interval. Conclusion Our results show, for the first time, that global changes in specific histone modification patterns may play an important role in breast cancer development and progression and their reduced expression is associated with poor prognosis and shorter survival.

P34
The Methods The primary question is: does correction of dose homogeneity using forward-planned IMRT improve the cosmetic outcome in patients with early breast cancer? Patients with significant dose inhomogeneities with 2DRT are randomised to IMRT or standard 2D RT. High-quality normal tissue toxicity and cosmesis data are being collected, including a novel analytical method of breast volume measurement using a 3D laser camera.
Results Eight hundred and eighty-five patients have been recruited to date, and accrual of 1,000 patients is on target for January 2007. A high-quality radiographer-led 3D breast radiotherapy service has developed as a direct result of the trial. Blood DNA samples from trial patients will enable investigation of individual genetic variation in normal tissue radiosensitivity within a multicentre translational radiogenomics study.

Conclusion
The results from this trial could provide impetus to improve the quality of breast radiotherapy for many women worldwide. The DNA database will greatly contribute to the ultimate aim of individualised radiotherapy based on genetics. Results These preliminary studies indicate that Herceptin induces receptor internalization. Further studies are planned whereby cells will be co-transfected with both c-erbB2-YFP and EGFR-GFP and exposed to an anti-EGFR antibody as well as Herceptin. Confocal microscopy will be utilized in mapping the fate of receptors and their antibodies. It may be that this dual targeting will exaggerate receptor internalization and degradation.

Conclusion
We demonstrate that both constructs can be expressed in mammalian cells and receptor trafficking can be observed using digital fluorescent microscopy. In addition, we have fluorescently labeled Herceptin and its ability to bind c-erbB-2 is retained. This study of receptor and antibody trafficking may lead to further knowledge of Herceptin's mechanism of action as well as that for drug resistance and the possible effects of the use of combined therapies. , with the aim to improve the current knowledge and understanding of DCIS from the patient's perspective. DCIS is a preinvasive breast condition increasingly detected by mammogram screening and has an uncertain natural history (some DCIS cells may develop into invasive cancer, but there is no marker to determine which DCIS cells will). Although DCIS is not an invasive condition, many women undergo extensive surgery (including mastectomy); therefore, this is a paradoxical situation -these women are reassured that it is noninvasive, caught early and not lifethreatening, but they are offered similar treatment as women with invasive breast cancer. The presentation aims to disseminate the initial findings of an exploratory qualitative study. Methods In-depth semistructured interviews with 16 women previously diagnosed with DCIS explored their experience. Thematic analysis highlighted the important issues from the women's own perspective. Results This study identified seven themes, which included two subthemes relating to appearance that are presented here. The paradox of DCIS and concerns about appearance were clearly evident in several participants.

Conclusion
The results emphasise that women may have posttreatment concerns and appearance issues following surgery for DCIS; these women may require specific support and advice in order to adjust for and accept the impact that the treatment may have on their appearance and feelings following surgery. Further research is needed to explore this area. The research team plans to address this by following a group of DCIS patients prospectively in order to identify how women's feelings and concerns (including appearance) change during the diagnosis and treatment for DCIS. Acknowledgement Funded by Breast Cancer Campaign. Before 2004, the recommended minimum 'threshold' for significant familial risk was set by a number of guidelines issued, which broadly required one first-degree relative diagnosed with breast cancer before age 40 or two close relatives both diagnosed before age 60. In 2004, NICE issued detailed guidelines in which the age requirement for two affected relatives was removed. However, it is widely recognised that the evidence base for any specific minimum threshold is limited and that there is a need for empirical studies to validate current and future recommendations. That is the object of the present study. Methods Records of the four Scottish Breast Cancer Family clinics were scrutinised for the period January 1994-December 2003 to identify any women referred but discharged because the level of familial risk was judged to fall below the (pre-NICE) threshold. From dates of birth and dates of discharge, the number of women-years of observation (to December 2003) within each 5-year age group (35-39 years, 40-44 years, and so on) was calculated. With permission from the Privacy Committee, the list was then checked against Scottish Cancer Registry records and any breast cancers recorded were rechecked from hospital notes. Expected cancer rates for an age-matched Scottish population were derived from Cancer Registry Statistics. Results A total of 2,074 'low risk' women were identified, giving over 8,000 woman-years of observation. Twenty-eight invasive breast cancers were recorded while 14.4 would have been expected (relative risk = 1.9 assuming complete ascertainment). A further eight invasive breast cancers have been recorded since 2003 (records incomplete). One-third of the cancers were in women who would have met the new NICE criteria for surveillance, whereas only some 10% of the total cohort had 'NICE moderate' family histories. The great majority of the cancers occurred in women between age 45 and 56. For them the relative risk approached 2 even when 'NICE moderate' women were excluded. Conclusion The new NICE family history guidelines are more accurate than previous ones in identifying women who should be included in breast surveillance programmes, but consideration should be given to making some provision particularly for women between age 45 and 56 with 'limited' family histories of breast cancer. The cohort we have identified should continue to be followed up since cancers are continuing to accrue and each year provides a further 2,000+ womanyears of observation. Methods Human breast tumour spheroids (600 µm) were infiltrated with human monocytes in vitro, allowed to differentiate into macrophages, coated with alginate to isolate from the host (murine) cells and implanted into dorsal skin-fold chambers on nude mice. The resultant angiogenesis surrounding the spheroids infiltrated with human macrophages prior to implantation was quantified using image analysis (Angiosys), and compared with that induced by spheroids consisting of tumour cells alone.

Results
The presence of macrophages resulted in at least a threefold upregulation in the release of vascular endothelial growth factor (VEGF) in vitro when compared with spheroids composed only of tumour cells. A homogeneous distribution of macrophages surrounding the hypoxic centre was observed in the majority of spheroid sections assessed. The angiogenic response measured around the spheroids 3 days after in vivo implantation was significantly greater in the spheroids infiltrated with macrophages; the number of vessels increased (macrophages vs no macrophages, 34 ± 1.9 vs 26 ± 2.5, P < 0.01), and were shorter in length (macrophages vs no macrophages, 116 ± 4.92 vs 136 ± 6.52, P < 0.008) with an increased number of junctions (macrophages vs no macrophages, 14 ± 0.93 vs 11 ± 1.25, P < 0.025), all parameters indicative of new vessel formation. By day 7 no significant differences were seen. Viable human but no murine macrophages were identified in the tumour spheroids at the end of the study, using immunohistochemistry. Conclusions This is the first in vivo study to demonstrate that macrophages modulate breast tumour angiogenesis, in the early stages of development, with an increased number of vessels and branches.

P44
Is transforming growth factor beta signalling required for breast cancer metastatic cell motility? Background In many cell types, transforming growth factor beta (TGFβ) results in a growth inhibitory signal, which is mediated by transducers of the Smad family. In tumour cells, however, TGFβdependent antiproliferative control is lost and cells acquire the ability to replicate in TGFβ-rich environments. Furthermore, molecular and clinical evidence points to a role for TGFβ signalling in cancer progression and metastasis; however, it is unclear at which points of the metastatic process TGFβ signalling occurs and whether it is necessary and/or sufficient to elicit cancer cell motility. Methods To address these questions, MTln3E rat breast cancer cells were used as a relevant model system. When injected into the mammary fat pad of nude mice, these cells form a primary tumour from which motile cells will depart to form metastasis in the lymph nodes and the lungs. To gain insight into TGFβ signalling in vivo, MTln3E cells were engineered to express GFPSmad2. This allowed monitoring Smad-dependent TGFβ signalling in vivo by imaging the primary tumour and in lymph-node metastasis using multiphoton confocal microscopy.

Results
The results indicate that TGFβ signalling, measured by cytoplasmic to nuclear translocation of GFPSmad2, does not occur ubiquitously within the primary tumour. On the contrary, TGFβ signalling appears most prominent in movement-rich areas. Within these areas, all the cells that have acquired a motile phenotype display active TGFβ signalling. Furthermore, none of the motile cells display nuclear exclusion of GFPSmad2.
Conclusions Together these data suggest that TGFβ signalling may be required in metastatic cells, possibly to enable acquisition of the motility phenotype. However, as nuclear localisation of GFPSmad2 is observed also in nonmotile cells, TGFβ signalling alone may not be sufficient to elicit cell motility in primary tumour cells.