Differentiation therapy: targeting breast cancer stem cells to reduce resistance to radiotherapy and chemotherapy

Studies have shown that cancer stem-like cells (CSCs) from solid cancers are resistant to both radiotherapy and chemotherapy. We have shown that primary breast cancers (n = 8) and a breast cancer cell line (MCF7) enriched for breast cancer stem cells (BCSC) using mammosphere (MS) clonogenic culture can preferentially survive radiotherapy and chemotherapy treatment in vitro, showing ≥ 50% increase in MS survival compared with non-BCSC enriched cells. The BCSC enriched population, defined by the cell surface markers ESA+/CD44+/CD24-/low, had reduced levels of DNA damage (measured by γH2AX) after 4 Gy irradiation or doxorubicin (1 μM) treatment. This suggests that the BCSC enriched population avoids or repairs the DNA damage significantly more than the whole population. 
 
Differentiating agents have been used to re-sensitise breast cancers to endocrine treatment but effects on BCSC are unknown. All-trans-retinoic acid (ATRA), tricostatin A and vorinostat caused a dose-dependent decrease in the BCSC population using MS culture and FACS analysis after 72 hours of treatment in a monolayer. Vorinostat (100 nM) showed the greatest effect, with 80% reduction in the ESA+/CD44+/CD24-/low population and a 50% reduction in MS formation. Our data suggest that in vitro treatment with differentiating agents reduces the number of the BCSC within the MCF7 cell line. 
 
Combination of ATRA (2 μM) or vorinostat with 6 Gy irradiation caused a significant reduction in MS survival showing a 30% and 70% decrease compared with an irradiated control. Similarly, in combination with paclitaxel (0.5 μM) ATRA and vorinostat caused a significant reduction in MS survival, showing 70% and 60% decrease compared with paclitaxel alone. In primary breast cancers (n = 3), combination of ATRA and 6 Gy irradiation significantly decreased MS formation by ≥ 25% respectively compared with irradiation alone. 
 
These observations suggest that targeting BCSC with agents that eliminate or differentiate BCSC is a promising strategy to overcome resistance to radiotherapy and chemotherapy in the clinic.


Introduction
The response rarely sustains long among the responders for Herceptin (trastuzumab) monotherapy treatment. It is still poorly understood how Herceptin exerts its mechanism of action and how the acquired resistance to this drug occurs. Materials and methods We used a multidisciplinary approach including fl uorescence resonance energy transfer and biochemical methods to assess the eff ects of Herceptin on various signalling pathways and to determine the acquired resistance mechanisms of Herceptin in various HER2-positive breast cell lines and a BT474 xenograft model.

Results
We have shown that Herceptin does not decrease HER2 phosphorylation despite the eff ect on HER2 receptor downregulation. HER2 phosphorylation is maintained by the activation of EGFR, HER3 and HER4 via their dimerisation with HER2 in breast cancer cells. The activation of EGFR, HER3 and HER4 is induced by HER ligand release, including heregulin and betacellulin. The release of HER ligands is mediated by ADAM proteases including ADAM17/TACE. Furthermore, we demonstrated that the feedback loop involving HER ligands and ADAM proteases is activated due to a decrease in PKB phosphorylation induced by Herceptin treatment. The feedback loop is also switched on when PKB phosphorylation is decreased by a PKB inhibitor. We have shown that the feedback loop activates the HER receptors and maintains HER2 phosphorylation in response to Herceptin. Herceptin in combination with a panHER inhibitor also caused a much greater tumour inhibition compared with Herceptin or panHER inhibitor alone in the xenograft model. Conclusions Our data provide evidence that Herceptin as monotherapy may result in poor outcome for patients due to the escape mechanisms through a feedback loop involving the upregulation of ADAM proteases and HER ligands. We have provided a novel mechanism of acquired resistance to Herceptin in HER2-positive breast cancer and have resolved the inconsistencies in the literature regarding the eff ect of Herceptin on HER2 phosphorylation.
The breast and ovarian predisposition protein BRCA1 is a required component of the mammalian response to double-stranded DNA damage. Its conserved BRCT domains are required for BRCA1 accumulation to sites of repair, while the conserved N-terminal RING domain is able to catalyse the conjugation of ubiquitin and act as an E3 ubiquitin ligase. Disruption of either of these domains by missense mutation is associated with disease development. The SUMO conjugation pathway has been implicated in DNA damage response in model organisms, and in Caenorhabditis elegans the Brac1 binding partner Bard1 associates with the SUMO E2 conjugating enzyme Ubc9. In mammalian cells, BRCA1 has been found to be associated with free SUMO-1 resulting in altered transcription. We undertook to examine the potential infl uence of the SUMO pathway on BRCA1 response to genotoxic stress. Using a range of biochemical and cell-biology techniques, we have shown that BRCA1 is modifi ed by SUMO in response to genotoxic stress, and co-localises at sites of DNA damage with SUMO1, SUMO2/3 and the SUMO conjugating enzyme Ubc9. PIAS SUMO E3 ligases co-localise with and modulate SUMO modifi cation of BRCA1, and are required for BRCA1 ubiquitin ligase activity in cells. In vitro SUMO modifi cation of the BRCA1:BARD1 heterodimer greatly increases its ligase activity, identifying it as a SUMO regulated ubiquitin ligase. Further, PIAS SUMO ligases are required for complete accumulation of double-strand DNA damage repair proteins subsequent to RNF8 accrual, and for profi cient double-strand break repair. Because the two features of BRCA1 activity regulated by the SUMO pathway, ubiquitin ligase activity and accumulation at sites of DNA damage, are also inhibited by some BRCA1 mutations that predispose to breast cancer and ovarian cancer, it seems highly likely that the SUMO pathway will be of relevance to cancer predisposition and development. It is now understood that epigenetic alterations occur frequently in sporadic breast carcinogenesis, but little is known about the epigenetic alterations associated with familial breast tumors. We performed genome-wide DNA methylation profi ling on familial breast cancers (n = 33) to identify patterns of methylation specifi c to the diff erent mutation groups (BRCA1, BRCA2 and BRCAx) or intrinsic subtypes of breast cancer (basal, luminal A, luminal B, HER2amplifi ed and normal-like). We used methylated DNA immunoprecipitation (meDIP) on Aff ymetrix promoter chips to interrogate methylation profi les across 25,500 distinct transcripts. Using a support vector machine classifi cation algorithm, we demonstrated that genome-wide methylation profi les predicted tumor mutation status with estimated error rates of 19% (BRCA1), 31% (BRCA2) and 36% (BRCAx), but did not accurately predict the intrinsic subtypes defi ned by gene expression with error rates of 43% (basal) and 54% (luminal A). Furthermore, using unsupervised hierarchical clustering we identifi ed a distinct subgroup of BRCAx tumors defi ned by methylation profi les. Finally, gene expression profi ling and the SNP CGH array previously performed on the same samples allowed full integration of methylation, gene expression and copy number datasets. This integrated analysis revealed frequent hypermethylation of genes that also displayed loss of heterozygosity compared with the tumors that were diploid for that gene. We also observed frequent hypermethylation of genes that show copy number gains compared with diploid tumors providing a potential mechanism for expression dosage compensation. Together these data show that methylation profi les for familial breast cancers are defi ned by the mutation status and distinct from the intrinsic subtypes.

O5
Diff erentiation therapy: targeting breast cancer stem cells to reduce resistance to radiotherapy and chemotherapy R Roy 1 , PM Willan 1 , R Clarke 2 , G Farnie 1 1 Treatment Resistance and Cancer Stem Cell Research,University of Manchester,UK;2 Breast Biology Group, University of Manchester, UK Breast Cancer Research 2010, 12(Suppl 1): O5 (doi: 10.1186/bcr2496) Studies have shown that cancer stem-like cells (CSCs) from solid cancers are resistant to both radiotherapy and chemotherapy. We have shown that primary breast cancers (n = 8) and a breast cancer cell line (MCF7) enriched for breast cancer stem cells (BCSC) using mammosphere (MS) clonogenic culture can preferentially survive radiotherapy and chemotherapy treatment in vitro, showing ≥50% increase in MS survival compared with non-BCSC enriched cells. The BCSC enriched population, defi ned by the cell surface markers ESA + / CD44 + /CD24 -/low , had reduced levels of DNA damage (measured by γH2AX) after 4 Gy irradiation or doxorubicin (1 μM) treatment. This suggests that the BCSC enriched population avoids or repairs the DNA damage signifi cantly more than the whole population. Diff erentiating agents have been used to re-sensitise breast cancers to endocrine treatment but eff ects on BCSC are unknown. All-trans-retinoic acid (ATRA), tricostatin A and vorinostat caused a dose-dependent decrease in the BCSC population using MS culture and FACS analysis after 72 hours of treatment in a monolayer. Vorinostat (100 nM) showed the greatest eff ect, with 80% reduction in the ESA + /CD44 + /CD24 -/low population and a 50% reduction in MS formation. Our data suggest that in vitro treatment with diff erentiating agents reduces the number of the BCSC within the MCF7 cell line. Combination of ATRA (2 μM) or vorinostat with 6 Gy irradiation caused a signifi cant reduction in MS survival showing a 30% and 70% decrease compared with an irradiated control. Similarly, in combination with paclitaxel (0.5 μM) ATRA and vorinostat caused a signifi cant reduction in MS survival, showing 70% and 60% decrease compared with paclitaxel alone. In primary breast cancers (n = 3), combination of ATRA and 6 Gy irradiation signifi cantly decreased MS formation by ≥25% respectively compared with irradiation alone. These observations suggest that targeting BCSC with agents that eliminate or diff erentiate BCSC is a promising strategy to overcome resistance to radiotherapy and chemotherapy in the clinic.

O6
Transforming growth factor-beta co-receptor endoglin suppresses breast cancer invasion and metastasis LA Henry 1 , DJ Johnson 1 , S Lee 1 , PR Quinlan 2 , T Crook 1 , AM Thompson 2 , JS Reis-Filho 1 , CM Isacke 1 1 Institute of Cancer Research, London, UK;2 Ninewells Hospital and Medical School, Dundee, UK Breast Cancer Research 2010, 12(Suppl 1):O6 (doi: 10.1186/bcr2497) Transforming growth factor-beta (TGFβ) signaling in cancer has been implicated in both growth suppression in early lesions as well as enhancing tumor cell invasion and metastasis. However, the cellular mechanisms that determine the signaling output in individual tumors are still largely unknown. In endothelial cells, TGFβ signaling is modulated by the TGFβ co-receptor endoglin (CD105). Here we demonstrate that endoglin is diff erentially expressed in invasive breast cancers and breast cancer cell lines, and is subject to epigenetic silencing by gene methylation. Downregulation of endoglin expression in nontumorigenic MCF10A cells leads to the formation of abnormal acini in 3D culture but does not promote cell migration or result in cell transformation. In contrast, in the presence of an activated oncogene, loss of endoglin in MCF10A cells leads to enhanced migration and invasion into a 3D matrix. Consistent with these data, ectopic expression of endoglin in the endoglin-negative MDA-MB-231 cell line blocks TGFβ-enhanced cell motility and invasion and reduces the ability of cells to successfully colonize the lung parenchyma in an in vivo metastasis model. Unlike endothelial cells, endoglin does not does not modulate canonical TGFβ signaling in breast cells but attenuates the cytoskeletal remodeling to impair cell migration and invasion. Importantly, lack of endoglin expression in clinical samples signifi cantly correlates with ENG gene methylation and poor clinical outcome. Together these data identify endoglin as a key component suppressing the invasive activities of breast cancer cells.

Introduction
The cancer stem cell hypothesis proposes that tumors contain a subset of stem or progenitor cells that are resistant to chemotherapy and have evaded the normally tight control of self-renewal. BRCA1 is known to play an essential role in cancer stem cell maintenance and diff erentiation. In fact, breast tissue from women with germline mutations in BRCA1 show regions of increased ALDH1 positivity, a marker of stem cells. Furthermore, knockdown of BRCA1 in normal tissue from reduction mammoplasty leads to increased ALDH1 and increased stem cell number as assessed by mammosphere formation. Activin B is a secreted protein that is a member of the TGFβ family. It is a homodimer of inhibin βB that is expressed in various tissues and has numerous functions, including regulation of gonadal function, proliferation, metastasis, diff erentiation and stem cell maintenance. Results Inhibin βB was identifi ed as a transcriptional target of BRCA1 in SKBR7 cells on an Almac Breast-specifi c microarray. The target was analysed in several breast cell lines and shown to be repressed in basal-like, but not luminal, cell lines following BRCA1 knockdown or reconstitution. Chromatin immunoprecipitation analysis showed that BRCA1 is present on the INHBB promoter. Furthermore, loss of BRCA1 reduced the amount of secreted protein, which in turn correlated with reduced activation of the TGFβ pathway as shown by reduced phosphorylation of Smad2. We have stably knocked down BRCA1 in MCF10A cells which, as expected, increased stem cell numbers in comparison with control cells as assessed by mammosphere formation and Aldefl uor postivity. This defect can be rescued by addition of recombinant activin B or mimicked by inhibiting activin B activity with follistatin or SB-431542. Conclusions We have identifi ed inhibin βB as a novel transcriptional target of BRCA1 in basal-like cell lines. Preliminary phenotypic studies indicate that the homodimer of inhibin βB, activin B, functions downstream of BRCA1 in regulating stem cell numbers.

P2
FOXM1 is a transcriptional target of ERα and has a critical role in breast cancer endocrine sensitivity and resistance J Millour, EW Lam Imperial College London, UK Breast Cancer Research 2010, 12(Suppl 1):P2 (doi: 10.1186/bcr2499) Previous data have shown that FOXM1 expression is elevated in breast cancer tissues and is strongly correlated with the expression pattern of ERα in breast cancer cells. The expression of ERα is a good prognostic factor in breast cancer, as about two-thirds of these ERα-positive patients respond to treatment with antiestrogens. However, approximately one-half of the patients that initially respond to hormonal therapy develop resistance. Since FOXM1 is critical for the progression of the cell cycle, we investigated the regulation of FOXM1 by ERα and its role in endocrine sensitivity and resistance in breast cancer cells. We fi rstly observed by quantitative RT-PCR a strong and signifi cant positive correlation between ERα and FOXM1 mRNA expression in breast cancer patient samples. We showed that FOXM1 protein and mRNA expression was regulated by ER ligands. We also demonstrated that ectopic conditional expression of ERα, in the presence of estrogens, leads to induction of FOXM1 expression in ER-negative U2OS cells. Using reporter gene assays, we demonstrated that ERα activates FOXM1 transcription through an estrogen-response element site. The direct binding of ERα to the FOXM1 promoter was confi rmed in vitro by mobility shift and DNA pull-down assays and in vivo by chromatin immunoprecipitation analysis. Importantly, silencing of FOXM1 by RNA interference abolishes the estrogen-mediated MCF-7 cell proliferation. Conversely, ectopic expression of a constitutively active FOXM1 can abrogate the cell cycle arrest mediated by the antiestrogen tamoxifen. Taken together, the results clearly demonstrate FOXM1 as a key mediator of the mitogenic functions of ERα and estrogen in breast cancer cells. Our fi ndings that antiestrogens repress FOXM1 expression in endocrine-sensitive but not endocrine-resistant breast carcinoma cell lines and that ectopic expression of an active FOXM1 can abrogate the anti-proliferative eff ects of tamoxifen also suggest that deregulation of FOXM1 may also contribute to antiestrogen insensitivity.

P3
A novel and selective PDK1 inhibitor reduces breast cancer cell invasion and tumour growth C Raimondi 1 , T Maff ucci 1 , BVL Potter 2 , M Falasca 1 1 Queen Mary University of London, UK; 2 University of Bath, UK Breast Cancer Research 2010, 12(Suppl 1):P3 (doi: 10.1186/bcr2500) Impairment of metastasis development is a critical target for cancer therapy. We recently reported that phospholipase Cγ1 (PLCγ1) is involved in regulation of motility and invasion of cancer cells and is required for metastasis development and progression. Experimental metastasis assays in nude mice revealed that inducible knockdown of PLCγ1 strongly inhibits development of MDA-MB-231-derived lung metastasis and reverts metastasis formation. In an eff ort to develop anti-metastatic drugs, diff erent inositol phosphates compounds were tested to identify potential PLCγ1 inhibitors. We found that a synthetic derivative of inositol pentakisphosphate, Ins(1,3,4,5)P5, inhibits cell migration and 3D invasion in MDA-MB-231 and MDA-MB-435 human breast cancer cell lines and in TSA murine mammary adenocarcinoma cells and reduces calcium release upon EGF stimulation indicating a potential inhibition on PLCγ1 activity. Kinase profi le assay, performed in vitro to test the potential inhibitory eff ect of the Ins(1,3,4,5)P5 synthetic derivative on diff erent kinases showed a specifi c inhibition of the 3-phosphoinositide-dependent-protein kinase 1 (PDK1) with an IC 50 of 26 nM. Knockdown of PDK1 using the small interfering RNA technology in breast cancer cell line MDA-MB-231 showed an impairment in cell migration and invasion and inhibition of EGF-induced calcium mobilisation. In addition, it has been recently shown that PDK1 is a critical determinant for resistance to tamoxifen anti-cancer drug. Our experiments show that combined treatment of the Ins(1,3,4,5)P5 synthetic derivative with tamoxifen, paclitaxel, and curcumin in MCF7 and MDA-MB-468 cells results in additive or more than additive eff ects, and therefore suggest that this novel PDK1 inhibitor can be potentially used in combination with other drugs to increase their anti-cancer activity. The PTEN tumour suppressor is a core component of the phosphoinositide 3-kinase (PI3K) signalling pathway. PTEN is mutated, deleted, or otherwise silenced in over one-third of breast cancers, with another one-third carrying other activating mutations in the core PI3K signalling pathway, most of these being activating mutations in the p110α catalytic subunit of PI3K itself. The best characterised tumour suppressor roles for PTEN are the suppression of cell growth, survival, proliferation and metabolic deregulation. However, we have found that in three-dimensional cultured mammary epithelial cells, knockdown of PTEN leads to the loss of cell polarity and tissue architecture. PTEN knockdown also causes a dramatic disruption of normal tight junction formation in adherent mammary epithelial cell cultures. Since the deregulation of cell polarity is becoming recognised as a potential driving force behind the formation of some tumours, we have been further studying the mechanisms by which PTEN controls mammary epithelial cell polarity.
(FFPE) lobular carcinoma in situ (LCIS) tissue and to examine the genetic relationship between LCIS and associated invasive lobular carcinomas (ILC). Introduction LCIS is a risk factor for the development of subsequent invasive breast carcinoma in either breast. Approximately 50 to 70% of these subsequent cancers are ILC. Not all LCIS progresses to invasive disease, and at present there are no biomarkers that predict which cases will develop ILC. Methods LCIS and ILC samples were microdissected from FFPE tissue blocks. Genetic changes were studied using SNP-LOH (Goldengate Assay; Illumina) to assess CN-LOH and copy number changes. Copy number changes were confi rmed using 32k BAC arrays. Ploidy was assessed using Feulgen staining. Results SNP-LOH was successful in 31/35 samples. LOH events were more common in classical LCIS with associated ILC compared with pure LCIS. Commonest changes were on 16q and 1q. Other areas of LOH were more common in LCIS associated with ILC but did not reach statistical signifi cance. Patterns of genetic change were maintained in ILC compared with the associated LCIS and 3/5 cases acquired additional genetic changes. Copy number changes were confi rmed in three cases using BAC arrays. Concordance for copy number gain and loss was 50% and 75%, respectively. Concordance for copy neutral LOH was 100%. Ploidy studies on 20 cases revealed that all cases of classical LCIS were diploid whereas 5/9 pleomorphic LCIS/ILC samples were tetraploid or aneuploid. Conclusions It is feasible to perform SNP-LOH on small amounts of microdissected FFPE LCIS tissue. The pattern of genetic changes confi rms the fi ndings of others that LCIS is a likely precursor of ILC. Further investigation of genetic changes in LCIS associated with ILC is expected to lead to the identifi cation of biomarkers that predict for subsequent invasive transformation.

P6
Suppression of the NF-κB co-factor, Bcl3, delays the metastatic progression of breast cancer AM Wakefi eld, RWE Clarkson Cardiff University, Cardiff , UK Breast Cancer Research 2010, 12(Suppl 1):P6 (doi: 10.1186/bcr2503) A large proportion of breast cancers overexpress the HER receptors, HER1 or HER2. Generally these patients have a poor prognosis, exhibit resistance to fi rstline anti-cancer drugs, and frequently develop metastatic disease -the most common cause of patient death. NF-κB transcription factors lie downstream of HER1/2 signalling pathways and are aberrantly activated in the majority of these breast tumours. We have found that a constitutive defi ciency in Bcl3 (an NF-κB co-factor that modifi es NF-κB signalling) delayed HER2 (ErbB2) tumour onset and inhibited metastasis of mammary tumours in mice while growth of primary tumours was unaff ected. In those Bcl3-null animals that did acquire metastases, the size of secondary tumours was signifi cantly reduced compared with controls. Critically, Bcl3 defi ciency did not aff ect normal mammary function other than having a transitory eff ect on apoptosis of epithelial cells in the post-lactational mammary gland. Therefore, unlike other NF-κB regulators, Bcl3 exhibited tumour-specifi c eff ects in vivo. The pro-metastatic properties of Bcl3 were confi rmed in several human breast cancer cell lines exhibiting elevated HER2 and/or HER1 levels, including aggressive basal-like tumour cell types. These observations are signifi cant because they suggest it may be possible to target Bcl3 in established tumour cells to reduce metastatic behaviour, and furthermore that Bcl3's eff ects are not restricted to ERBB2-positive breast tumours, consistent with the observed increase in NF-κB activity seen in both ERBB1-positive and ERBB2-positive breast tumours. BRCA1 was identifi ed in 1994 as one of the genes predisposing to early-onset breast and ovarian cancer. It is currently estimated that 5 to 10% of all breast and ovarian cancer cases are inherited and the breast cancer susceptibility genes, BRCA1 and BRCA2, have been identifi ed as being responsible for up to 21 to 40% of these cases. Although the exact function of BRCA1 remains to be defi ned, roles in DNA damage repair, cell cycle checkpoint control, transcriptional regulation and, more recently, ubiquitination have been inferred. p63 was identifi ed as a positively regulated BRCA1 target gene through microarray analysis, and the functional signifi cance of the BRCA1/p63 signalling axis was investigated. Knockdown of BRCA1 and p63 leads to enhanced proliferation of breast cancer cell lines and increased stem cell numbers as assayed for by mammosphere culture and Aldefl uor assay. Expression of BRCA1 or p63 in a background of low BRCA1 and p63 results in decreased cell proliferation. We therefore examined co-regulated targets of BRCA1 and p63 mediating growth control. S100A2, a tumour suppressor, is a known p63 target. Knockdown of BRCA1 and p63 leads to the loss of S100A2 expression. BRCA1 and p63 were found to be localised to the S100A2 promoter by chromatin immunoprecipitation assay. Loss of p63 resulted in recruitment of BRCA1 to the S100A2 promoter. In a p63 and BRCA1 null background, expression of S100A2 results in a reduction of cell proliferation. Conversely, loss of S100A2 in a BRCA1 and p63 expressing background leads to increased proliferation. We have explored the regulation of signalling pathways by p63 and BRCA1 that are involved in growth control, diff erentiation and stem cell regulation. We will identify potential regulators of these pathways using microarray analysis to elucidate p63 and BRCA1 co-regulated targets. We recently reported that the human p53 gene encodes at least nine diff erent p53 isoforms, two of which (p53β and Δ133p53) can modulate p53 transcriptional activity and apoptosis. In the present study, we aimed to investigate the regulation of Δ133p53 isoform expression and the physiological role of Δ133p53 in modulating p53 activities. We report that in response to genotoxic stress, p53 transactivates directly the human p53 internal promoter inducing Δ133p53 protein expression, which by diff erentially modulating p53 target gene expression prevents p53-mediated apoptosis without inhibiting cell cycle arrest. This indicates that Δ133p53 does not simply act at physiological level in a dominant-negative manner towards any p53 targets, but rather modulates p53 transcriptional activity in a promoter and stress-dependent manner. Hence, we have established a novel feedback pathway that modulates the p53 response, which might have an impact on p53 tumour suppressor activity. These observations may provide some explanations for the diffi culties in many clinical studies of associating p53 status with cancer treatment and clinical outcome. Therefore, it would be interesting to determine whether Δ133p53 expression is associated with tumour markers, clinical outcome and cancer treatment in human cancers.
Introduction CUB and Sushi multiple domain protein 1 (CSMD1) is a candidate tumour suppressor gene of unknown function. CSMD1 maps to chromosome 8p23, a region deleted in 50% of breast cancers (BC). We have examined the contribution of CSMD1 to the tumorigenic phenotype of mammary acini and evaluated its prognostic value in BC patients. Materials and methods A shRNA CSMD1 MCF10A three-dimensional matrigel model was established. Moreover, functional assays were performed using shCSMD1 cell lines. CSMD1 was tested by immunohistochemistry in 275 BC samples. Results Loss of CSMD1 in the MCF10A three-dimensional model resulted in an increased number of acini (P = 0.001), which are also larger in size (40%, P = 0.02) and misshapen relative to the control. Although expressing a high level of active caspase 3, shCSMD1 acini failed to form lumen. Loss of CSMD1 expression caused a 56% (P = 0.001) increase in proliferation and a 44% (P = 0.0006) decrease in adhesion. shCSMD1 cells migrate much faster than control cells and showed 33% (P <0.001) increase in invasion. These results were confi rmed in two other cell lines.
Loss of CSMD1 expression was identifi ed in 79/275 (28.7%) of BC cases, which was associated with high tumour grade (P = 0.003) and low overall survival (HR = 0.607, 95% CI = 0.4 to 0.91, P = 0.018). Moreover, CSMD1 is an independent predictor of overall survival (HR = 0.607, 95% CI = 0.4 to 0.91, P = 0.018) [1]. Conclusions Loss of CSMD1 aff ects cell adhesion, proliferation, migration and invasion, which lead to disruption of mammary duct formation. Loss of CSMD1 is associated with poor prognosis in BC, suggesting its use as a new prognostic biomarker. . Here we show that PI3K-C2β is overexpressed in several human breast cancer cell lines as compared with normal breast cells. Downregulation of PI3K-C2β expression by shRNA inhibits oestradioldependent and heregulin-dependent growth of MCF-7 and T47D cells and softagar colony formation. Immunohistochemistry analysis of breast cancer tissues from 90 patients revealed that PI3K-C2β is not expressed in normal portions of breast tumour specimens (used as internal controls) and follicular breast tissues, whereas it is highly expressed in infi ltrating ductal carcinoma breast cancer tissues. Interestingly, we found a highly positive signifi cant (Spearman's rho test, P = 0.002) association between PI3K-C2β expression and the proliferative status (Ki67) of tissues analysed. In addition, we compared the expression levels of PI3K-C2β in 20 primary-metastasis pairs from breast cancer patients. We found that PI3K-C2β expression is signifi cantly increased in lymph node metastasis with primary tumours (Wilcoxon-Mann-Whitney test, P = 0.001). Taken together these data suggest a correlation between PI3K-C2β expression and activation and breast cancer progression, and identify a novel molecular target. ERβ1 is often downregulated in cancer compared with normal cells, suggesting that it may function as a tumour suppressor and could play an important role in carcinogenesis. The expression of ERβ1 is regulated by multiple mechanisms such as methylation. Five splice variants of ERβ mRNA have been identifi ed, ERβ1 to ERβ5. However, it is unclear whether and how the full-length version, ERβ1, is regulated post-transcriptionally.

Reference
MicroRNAs are a class of nonprotein coding small RNAs that regulate expression of genes at post-transcriptional levels. Using rapid amplifi cation of 3΄ complementary DNA ends (3΄RACE), we have confi rmed 3΄ untranslated region (3΄UTR) expression and sequences of ERβ1 mRNA in MCF-7 cells. Based on miRNA expression profi ling of human breast cancer studies, we found that miR-92 is upregulated in malignant breast. In silico analysis using the miRGen database and RNA hybrid predicted that there are two putative miR-92 target sequences within the 3΄UTR of human ERβ1 mRNA. Firstly, we profi led the expression of ERβ1 mRNA and miR-92 in breast cancer tissue and cell lines. miR-92 levels were higher in ERβ1-negative MDA-MB-453 cells than ERβ1-positive MCF-7 cells. We observed miR-92 upregulation in breast tumours while ERβ1 mRNA expression was decreased compared with matched adjacent normal tissues. We found a signifi cant negative correlation between miR-92 and ERβ1 mRNA and protein in breast tumour (r = -0.5, P = 0.001 and r = -0.39, P = 0.037), respectively. Transfection of MCF-7 cells with anti-miR-92 increased endogenous ERβ1 mRNA and reduced cell proliferation. EGFP report experiment also confi rms that the 3΄UTR of ERβ1 carries the directly binding sites of miR-92. Finally, we showed that miR-92 expression is modulated by the ER ligands 17β-estradiol and tamoxifen in MCF-7 cells. These fi ndings prove that ERβ1 expression is negatively regulated at a post-transcriptional level by miR-92. This miRNA could be considered a potential therapeutic target in breast cancer. CTCF is a highly conserved and ubiquitous transcription factor with versatile functions. We previously demonstrated that elevated protein levels of CTCF in breast cancer cells were associated with the specifi c anti-apoptotic function of CTCF. We used proteomics and microarray approaches to identify regulatory targets of CTCF specifi c for breast cancer cells. Among the CTCF identifi ed targets were proteins involved in the control of apoptosis. A proapoptotic protein, Bax, negatively regulated by CTCF, was chosen for further investigation. Repression of the human Bax gene at the transcriptional level by CTCF in breast cancer cells was confi rmed by real-time PCR. Two CTCF binding sites within the Bax promoter were identifi ed by electrophoretic mobility shift assay and footprinting. In reporter assays, the Bax-luciferase reporter construct, containing CTCF-binding sites, was negatively regulated by CTCF. In vivo, CTCF occupied its binding sites in breast cancer cells and tissues, as confi rmed by chromatin immunoprecipitation assay. Our fi ndings suggest a possible mechanism of the specifi c CTCF anti-apoptotic function in breast cancer cells whereby CTCF is bound to the Bax promoter, resulting in repression of Bax and inhibition of apoptosis; depletion of CTCF leads to activation of Bax and apoptotic death. CTCF binding sites in the Bax promoter are unmethylated in all cells and tissues inspected. Therefore, specifi c CTCF interaction with the Bax promoter in breast cancer cells, and the functional outcome, may depend on a combination of epigenetic factors characteristic for these cells. Interestingly, CTCF appears to be a negative regulator of other proapoptotic genes (for example, Fas, Apaf-1, TP531NP1). Conversely, stimulating eff ects of CTCF on the anti-apoptotic genes (Bcl-2, Bag-3) have been observed. Taken together, these fi ndings suggest that specifi c mechanisms have evolved in breast cancer cells to protect them from apoptosis; regulation of apoptotic genes by CTCF appears to be one of the resistance strategies. We have previously reported that induction of EGFR and erbB2 in response to antihormones may provide an early mechanism allowing breast cancer cells to evade the growth inhibitory action of such therapies and ultimately drive resistant growth. More recently, another member of the erbB receptor family, erbB3, has been implicated in antihormone resistance in breast cancer. In the present study we have investigated whether induction of erbB3, and related family member erbB4, may provide an alternative resistance mechanism to antihormonal action in a panel of four ER-positive breast cancer cell lines. MCF-7, T47D, BT474 and MDAMB361 cell lines were exposed to fulvestrant (100 nM) for 7 days, and eff ects on erbB3/4 signalling and growth were assessed. Eff ects of the erbB3/4 ligand heregulin-β1 were also examined in the absence and presence of fulvestrant. Fulvestrant potently reduced ER expression and transcriptional activity and signifi cantly inhibited growth in all four cell lines. However, alongside this inhibitory activity, fulvestrant also consistently induced protein expression and activity of erbB4 in the four cell lines and also promoted erbB3, erbB2 and EGFR protein expression and activity in MCF-7 and T47D cells. Consequently, fulvestrant treatment sensitised each cell line to the actions of heregulin-β1 with enhanced erbB3/4-driven signalling activity and signifi cant increases in cell proliferation being observed when compared with untreated cells. Indeed, in T47D and MDAMB361, heregulin-β1 was converted from a ligand having negligible or suppressive growth activity into one that potently promoted cell proliferation. Consequently, fulvestrant-induced growth inhibition was completely overridden by heregulin-β1 in all four cell lines. In conclusion, these fi ndings would suggest that although antihormones, such as fulvestrant, may have potent acute growth inhibitory activity in ER-positive breast cancer cells, their ability to induce and sensitize cells to growth factors, such as heregulins, may serve to reduce and ultimately limit their inhibitory activity. Methods Three hundred and seventy-four BRCA1 mutation carriers and 346 BRCA2 mutation carriers were followed up for up to 30 years following breast cancer diagnosis. The incidence of a contralateral breast cancer and the eff ect of tamoxifen use and oophorectomy on this were observed. Results Follow-up over a 25-year to 30-year period shows a constant 2% annual risk of contralateral breast cancer in BRCA1/BRCA2 mutation carriers. This risk is not aff ected by age at diagnosis of fi rst breast cancer. Over the follow-up period, oophorectomy, if performed below the age of 45 years, led to a reduction in contralateral breast cancer risk of 40%. Tamoxifen use was shown to reduce the risk of a contralateral breast cancer in the fi rst 6 years following initial diagnosis but no eff ect was seen after this period. Over the full follow-up period, tamoxifen use did not signifi cantly reduce the risk.

Risks of contralateral breast cancer in
Conclusions Women who carry a BRCA1/BRCA2 mutation who have had breast cancer have a constant increased risk of a second contralateral breast tumour.
Oophorectomy has a greater impact on reducing this risk than tamoxifen use. The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif ) family are a group of 19 extracellular, secreted proteases whose known functions include processing of procollagen molecules, cleavage of extracellular matrix proteoglycans and anti-angiogenesis. Our previous studies have shown that ADAMTS15 is a novel predictor of good prognosis in breast cancer; patients whose tumours had high levels of ADAMTS15 expression had an increased relapse-free survival compared with those with lower levels [1]. ADAMTS15 has also emerged as a candidate cancer gene from cancer genome sequencing, and its tumour suppressive function has recently been documented in colorectal cancer [2]. Our study has focused on the cellular eff ects of overexpression in MCF7 and MDA-MB-231 breast cancer cell lines of full-length wild-type ADAMTS15 and an E-A mutant that lacks metalloproteinase activity. We have generated stable transfectants carrying either an inducible (lentivirus tet-off system) or constitutive vector system. The eff ects on cell adhesion, migration and proliferation have subsequently been analysed. Proliferation (MTT assay) and adhesion to various matrix components (including collagen, fi bronectin and laminin) was not altered with the addition of ADAMTS15. However, ectopic expression (inducible and constitutive) of either full-length ADAMTS15 or the catalytically dead mutant signifi cantly reduced migration in both cell lines.

ADAMTS15 metalloproteinase inhibits breast cancer cell migration
Wild-type ADAMTS15 also enhanced the aggregation of MCF7 cells. These data suggest that ADAMTS15 may exert tumour suppressive eff ects via modulation of the interactions of breast carcinoma cells with their environment independent of its metalloproteinase activity. Objective There is evidence to support the view that infl ammatory processes are important in the development of local progression and metastases in patients with breast cancer. The sphingosine kinase-1/sphingosine-1-phosphate (SK1/ S1P) pathway, which is a known mediator of infl ammation, is critically implicated in breast cancer progression and chemotherapy resistance and is linked with poor prognosis. In this study we have investigated the implication of the SK1/ S1P pathway in the interaction between tumour-associated macrophages and breast cancer cells.

References
Methods We have used modifi ed Boyden chambers to perform macrophagetumour cell co-culturing. Cytokine production and alterations in gene expression were measured by quantitative RT-PCR. Proteome profi ler assays were used to identify secreted cytokines. Cell motility and chemotaxis were assayed in 96-well plates of Dunn chambers respectively using high-throughput video time-lapse scanning microscopy.
Results MDA-MB-231 breast cancer cells were pretreated with docetaxel and subsequently co-cultured with THP-1 macrophages. Macrophages exhibited increased chemotaxis towards apoptotic tumour cells (aTCs) or aTC conditioned media. Co-culturing with aTCs has transiently increased macrophage SK1 activity. Proteome profi ling of media from macrophages revealed that aTCs induced an SK1-mediated secretion of IL-6 and siCAM-1. Interestingly, co-culturing with macrophages increased aTC chemoresistance. Incubation of untreated cancer cells with macrophages pretreated with conditioned media from aTCs induced an IL-6-mediated upregulation of cancer cell SK1 expression in cancer cells, which has lead to an increase in cancer cell motility and chemotaxis in gradients of macrophage conditioned media. These enhanced migratory phenotypes were reversed following treatment of cancer cells with SK1 siRNA. Conclusions Our results suggest a novel IL-6/SK1-dependent mechanism of macrophage-induced breast cancer chemoresistance and metastasis.

P17
PLU-1/Jarid1B contributes to estrogen-induced cell proliferation in the normal mammary gland and in breast cancer, and is required for embryonic survival S Catchpole 1 , B Spencer-Dene 2 , D Hall 1 , A Scibetta 1 , J Burchell 1 , J Taylor PLU-1/Jarid1B is a nuclear protein that is widely expressed in breast cancer, with higher levels being seen in ER + cancers. Expression in normal adult issues is largely restricted to testis and the diff erentiating mammary gland. Through the JmjC domain the protein can demethylate H3K4me3, which correlates with its function as a transcriptional repressor. PLU-1/Jarid1B contains a DNA binding domain, and can be recruited to DNA through binding to transcription factors. We now fi nd that the protein interacts with the ERα receptor and contributes to estrogen-induced survival of MCF-7 cells in in vitro culture and when grown as tumours in nude mice.
To investigate the function of Plu-1/Jarid1B in vivo, transgenic mice expressing defective Plu-1/Jarid1B have been developed. The systemic KO is an embryonic lethal with no homozygote embryos being detected at day 7.5. Another strain expressing the protein missing the ARID AT-rich DNA binding domain (which is also required for demethylase function) shows a mammary phenotype. In these ΔARID mice, the development of the mammary tree at puberty and early pregnancy is delayed, but the gland recovers by late pregnancy. The inhibition of development of terminal end buds at puberty, which is crucially dependent on ERα signalling, suggests an involvement of Plu-1/Jarid1B in this signalling that is impaired in the ΔARID mouse. Confi rming this, levels of expression of downstream targets of ERα (progesterone receptor and Wnt4) are reduced in the ΔARID mouse. The development of spontaneous mammary tumours in the ΔARID mouse is delayed compared with wild-type mice, suggesting that Plu-1/ Jarid1B contributes to tumour growth, and that this action is impaired when the ARID domain is deleted.
The data suggest that PLU-1/JARID1B is involved in estrogen-induced growth of normal and malignant mammary epithelial cells. We are interested in fi nding novel therapeutic targets for breast cancer, particularly focusing on C-terminal binding proteins (CtBPs) as when they are inhibited, cells show an increased sensitivity to apoptotic stimuli. Using siRNA to inhibit CtBP expression, we have found that CtBPs are essential for the survival of breast cancer cells, and in particular those with a more aggressive p53-mutant phenotype. To direct our future studies into therapeutic strategies targeting CtBPs in breast cancer, we need to know more precisely how their loss results in death, and which of their functions are required for this prosurvival role.
Here we show that loss of CtBP function through siRNA treatment suppresses proliferation through a combination of p53-independent apoptosis, reduction in cell-cycle progression into mitosis, and aberrations in transit through mitosis itself. This third phenotype includes errors in mitotic chromosome segregation, activation of, but failure to sustain, the spindle assembly checkpoint, decreased expression of Aurora B, and a high rate of failure to complete cytokinesis. We showed that loss of CtBP in breast cancer cells with a functional p53 response pathway resulted in a marked upregulation of the p53 protein. Here p53 appears to be providing a protective role by arresting aberrant cells in G 1 , thus preventing them from entering S-phase with incorrectly segregated DNA.
CtBPs are known to act in the nucleus as transcriptional co-repressors and in the cytoplasm as regulators of Golgi fi ssion. Using a series of dominant negative CtBP mutants microinjected into either the cytoplasm or nucleus, we show that localisation of CtBPs to the nucleus is critical for its function in ensuring the correct division of breast cancer cells. This suggests that CtBPs function in maintaining mitotic fi delity, and thus in the continued proliferation and survival of breast cancer cells through their actions as a transcriptional co-repressor within the nucleus. and RhoBTB1 (ca. 50%) found across a wide range of carcinomas is suffi cient to explain the observed frequency of downregulation of CXCL14. We propose that downregulation of RhoBTB1/2 represents the causative mechanism for altered CXCL14 expression in cancer cells. Previous work has shown that CXCL14 expression is lost from mammary epithelial cells in ductal carcinoma in situ, but is upregulated in the surrounding myoepithelial cells. This is suggestive of an autocrine to paracrine switch. In keeping with this, we fi nd that exogenous CXCL14 disrupts the organisation of mammary cell acini grown in threedimensional culture. Similar switching in CXCL14 production from epithelium to stroma has been reported in prostate and oral carcinoma. We are currently investigating the contribution of CXCL14 on invasive behaviour.

RhoBTB2 in breast cancer
Introduction AP-2 transcription factors constitute a family of sequence-specifi c DNA-binding proteins encoded by fi ve highly homologous yet functionally distinct genes, AP-2α to AP-2ε. AP-2γ appears to play a major role in breast cancer, being expressed in a large proportion of primary tumours. In this study we have analysed in more detail the mechanism of transcriptional regulation of the p21/cyclin-dependent kinase inhibitor 1A (p21/CDKN1A) gene by AP-2γ.

Materials and methods
Silencing of AP-2γ was carried out in MCF-7 cells using siRNA or doxycycline inducible shRNA. Chromatin immunoprecipitation (ChIP) assays were performed using specifi c antibodies against AP-2γ (H77), AP-2α, Myc, histone demethylase PLU1/JARID1B, histone H3 and trimethyl dimethyl and monomethyl histone H3 followed by quantitative PCR. Electrophoretic mobility shift assay (EMSA) competition assay and reporter assays were used to identify the AP-2 binding site.

Results
Silencing of AP-2γ by either siRNA or inducible shRNA inhibits cell proliferation and results in upregulation of p21/CDKN1A expression with no induction of apoptosis. ChIP assays demonstrated binding of AP-2γ, PLU1/ JARID1B and Myc to a region adjacent to the transcription start site of the p21/CDKN1A gene. Reduction in the binding of cMyc and PLU1/JARID1B and increased levels of histone H3 trimethyl-K4 were observed at the proximal region of p21/CDKN1A promoter after silencing of AP-2γ. Treatment of MCF-7 cells with the antimitotic drug vinblastine but not with hydroxyurea reduced the CDKN1A binding of AP-2γ, PLU-1/JARID1B and Myc. H3396 cells treated with the oestrogen receptor inhibitor Faslodex, which upregulates p21/CDKN1A, decreased AP-2γ binding but increased binding of AP-2α at the p21/CDKN1A promoter. EMSA competition assays and reporter assays showed that AP-2γ and AP-2α bind to a new site (GCC N3 GGG) at position -105 of the p21/CDKN1A promoter.
Conclusions The repression of the p21/CDKN1A gene by AP-2γ may contribute to the activation of proliferation associated with this transcription factor in breast cancer. The ability of a tumour to adapt and overcome targeted therapies has been recognised clinically for some time, but the molecular mechanisms driving this metamorphosis remain unclear. The steroid receptor coactivator protein, SRC-1, is a strong predictor of reduced disease-free survival in breast cancer patients. SRC-1 can also interact with nonsteroidal transcription factors, and defi ning these new transcriptional networks will uncover fresh strategies for managing endocrine resistance.

P21
Here we employed a mass spectrometry-based screen to identify proteins that are specifi c to the endocrine-resistant phenotype. The developmental protein, HOXC11, was identifi ed and functionally validated as an interaction partner of SRC-1. We provide evidence that HOXC11 and SRC-1 cooperate to regulate expression of the calcium binding protein S100β in resistant breast cancer cells. Moreover, both nuclear HOXC11 and S100β were found to be strong predictors of poor disease-free survival in breast cancer patients (n = 560; hazard ratios = 5.79 and 5.82, respectively; P <0.0001). Elevated serum levels of S100β detected in patients also predicted reduced disease-free survival (n = 80; hazard ratio = 5.3; P = 0.004). This translational study identifi es a biomolecular interaction network central to the adaptive response to endocrine therapy with clear clinical applications. Introduction Insulin-like growth factor binding protein-2 (IGFBP-2) is often elevated in breast tumours and the presence of IGFBP-2 has been shown to correlate with malignancy. Previously we have shown that IGFBP-2 reduces PTEN abundance [1] and thus helps to maintain the activity of the phosphoinositide 3-kinase signalling cascade, a key mitogenic and survival pathway.
Objective We therefore investigated whether IGFBP-2 could act as a survival factor for breast cancer cell lines. Using MCF7 and T47D cells, we tested the ability of exogenous IGFBP-2 to alter apoptosis induced by various chemotherapeutic agents. As both these cell lines produce large amounts of IGFBP-2, we also examined the eff ect of silencing IGFBP-2 using siRNA.
Conclusions These data show that the production of IGFBP-2 by breast cancer cells enhances their survival and protects them against chemotherapy. Thus in breast tumours the increase in IGFBP-2 production could be a survival mechanism making IGFBP-2 a legitimate target for intervention.

Rationale and hypothesis
Mutations in high-penetrance genes such as BRCA1 and BRCA2 predispose to breast cancer, and recently a number of lowpenetrance breast cancer genes have also been identifi ed. We reported that a coding SNP in the caspase-8 gene (CASP8 D302H) is associated with a reduced risk of breast cancer. More recently we identifi ed a CASP8 4-SNP haplotype associated with an increased risk of breast cancer [1]. A CASP8 promoter indel has been associated with breast cancer in an Asian population, although this has not been confi rmed in Europeans. Our hypothesis is that these CASP8 variants may infl uence breast cancer susceptibility via eff ects on the apoptotic response.
Objective Our objectives are to study the functional eff ects of six relevant CASP8 variants on caspase-8 activity and apoptosis induction in peripheral blood lymphocytes (PBLs). Methods We recruited 68 healthy women attending the Sheffi eld Mammography Screening Service and measured the ability of their PBLs to undergo drug-induced apoptosis. Levels of apoptosis and caspase-8 activity were determined by fl uorescence-activated cell sorting analysis (Annexin V-FITC with PI and Red-IETD-FMK (Calbiochem), respectively). The six SNPs were genotyped using TaqMan assays (Applied Biosystems). Data were analysed using parametric and nonparametric analysis of variance.

Results
The median levels of induction of caspase-8 activity and apoptosis were 70.03% (28.19 to 94.65) and 78.11% (18.57 to 92.99), respectively. The rare alleles of rs3834129 (promoter indel), rs7608692 (intron 2) and rs1861269 (intron 4) were associated with reduced caspase-8 activity (P ANOVA = 0.03, 0.005 and 0.048, respectively). In addition, rs1861269 was signifi cantly associated with reduced apoptosis (P ANOVA = 0.036). Although these results need to be confi rmed, they suggest that SNPs in the caspase-8 gene may infl uence breast cancer susceptibility via eff ects on caspase-8 activity and apoptosis. suggest that levels may be partly genetically determined. Quantifi cation of hormone levels in premenopausal women, however, is diffi cult because of their cyclical nature. In particular, oestrogen has a marked peak in the follicular phase and a further wider peak in the luteal phase. Methods We developed a protocol to capture peak follicular phase urinary oestrone glucuronide (E1G), and luteal phase E1G in healthy premenopausal women. Repeated measurements of creatinine-adjusted E1G levels, in 789 women, were used to describe features of the E1G curve such as mean and peak follicular E1G, and luteal E1G. A total of 691 tagging SNPs capturing common variation in genes within the oestrogen synthesis and metabolism pathways were successfully genotyped. Geometric mean urinary E1G levels and endogenous plasma hormone levels were estimated and tested for an association with the genotype of each SNP.

Results
We identifi ed a rare SNP (minor allele frequency 7%), in which the minor allele was associated with a 20% reduction in circulating levels of E1G in healthy premenopausal women (P <10 -8 ). We are currently genotyping this SNP in 12,000 breast cancer cases and 12,000 controls to test whether the reduction in circulating oestrogen levels is also associated with a reduction in breast cancer risk. Objective To evaluate the psychoneuroimmunological eff ects of refl exology in women with early breast cancer. Methods One hundred and eighty-three women with early breast cancer were randomised 6 weeks post surgery to self-initiated support (SIS) (comparator intervention), SIS plus refl exology, or SIS plus scalp massage (control for physical and social contact). Patients randomised to refl exology and massage had eight sessions at weekly intervals. Primary and secondary endpoints were 18 and 24 weeks post surgery, respectively. Mood, coping and quality of life were assessed pre-randomisation, and at the two endpoints. Blood was also taken at these three time points to enumerate lymphocyte subsets (CD profi les), cytokine production (Th1, Th2), and hormones (prolactin, cortisol, growth hormone). Results At week 18, massage, but not refl exology, was signifi cantly better than SIS in terms of the primary outcome measure, the Trial Outcome Index (TOI) of FACT-B. At the secondary endpoint (week 24), refl exology, but not massage, was better than SIS in terms of the TOI. Lymphocyte phenotyping found that CD25 + cells were signifi cantly higher in the massage group compared with the SIS group at week 24. The percentage of T cells, more specifi cally the T-helper subset expressing IL-4, decreased signifi cantly in the massage group compared with the SIS group at week 24. An accompanying increase in the percentage of CD8 + cytotoxic T cells expressing IFNγ in the massage group showed that the immunological balance of patients can be altered in a potentially benefi cial manner by massage. Neither refl exology nor massage aff ected any of the lymphocyte subsets, or hormones. Conclusions Refl exology and massage have modestly benefi cial eff ects on quality of life. Massage showed statistically signifi cant eff ects on immunological parameters, although the clinical signifi cance of these eff ects will require further investigation. Acknowledgements Funding from the NHS R&D cancer programme.

P27
An integrated informatics platform to facilitate transforming tissue into knowledge PR Quinlan 1 , A Ashfi eld 1 , L Jordan 2 , C Purdie 2 , AM Thompson 1 Tissue banks provide a core facility that enable translational research to be undertaken on quality-assured patient-donated samples. These samples require clinical and pathological annotations to allow comparisons to be drawn between the research-generated data and the conventional pathological and clinical outcome parameters. The challenge is to develop a fl exible database system to capture these parameters and to provide a statistical framework for automated analysis. Various database solutions have been developed to capture the data at the numerous stages of research. A tissue banking platform allows complete tracking of samples as they are frozen, extracted and released, combined with an independent pathology database to capture the pathological and clinical outcome data for all breast cancer patients. This web-based interface allows the appointed research nurse to submit data as they are approved at the Multi Disciplinary Team Meetings. The patient records are also routinely checked to capture any data relating to disease recurrence, treatments and surgical procedures. A digital pathology database is also used to capture and store the tissue microarray results. All of these databases are then combined using another locally designed system, INSPIRE, which brings all the data together to perform statistical analyses and present the results via a user-friendly, webbased interface. The various systems developed allow tissue-banked samples to be tracked throughout the research process and to be annotated with high-quality pathology and research-derived data. INSPIRE then completes the cycle by aggregating these data to perform statistical analyses and present these in a user-friendly, web-based interface. This allows the researcher to focus purely on generating high-quality research and enhancing our knowledge in breast cancer.

Introduction
The problems of adherence to energy restriction in humans are well known. Animal data suggest intermittent energy restriction (IER) is comparable with or better for preventing breast cancer than continuous restriction (CER).
Objective To compare the feasibility and eff ectiveness of IER with CER for weight loss and improving insulin sensitivity and other markers of breast cancer risk. Methods Randomised comparison of a 25% energy restriction as IER (~2,266 kJ/ day for 2 days/week) and CER (~6,276 kJ/day for 7 days/week) in 107 overweight or obese (mean ± SD body mass index = 30.6 ± 5.1 kg/m 2 ) premenopausal women over 6 months. Weight, anthropometrics, blood markers for risk of breast cancer and other metabolic diseases, insulin resistance, oxidative stress markers, leptin, adiponectin, lipids, infl ammatory markers (high-sensitivity C-reactive protein and sialic acid), insulin-like growth factor-I and insulin-like growth factor binding proteins, androgens, prolactin and menstrual cycle length were assessed at baseline, 1, 3 and 6 months. Results Eighteen subjects withdrew before 6 months (11 IER, seven CER). Last observation carried forward analysis showed IER and CER are equally eff ective for weight loss: mean (95% CI) weight change for IER was -6.4 (-7.9 to -4.8) kg vs. -5.6 (-6.9 to -4.4) kg for CER (P for diff erence between groups = 0.4). Both groups had signifi cant and comparable improvements in disease risk markers; however, IER was signifi cantly better than CER in reducing insulin resistance: mean (95% CI) change for IER was -28 (-37 to -17)% vs. -15 (-24 to -4)% for CER.
Conclusions IER is as eff ective as CER for weight loss and biomarker change. Its additional benefi cial eff ect on insulin sensitivity indicates that it may be an alternative approach for weight loss. Redox systems are often deregulated in cancer. To investigate whether altered expression predicted response to therapy, core biopsies from 80 locally advanced breast tumours (pre-six cycles of fl uorouracil epidoxorubicin cyclophosphamide/fl uorouracil adriamycin cyclophosphamide chemotherapy) were stained, using standard immunohistochemistry, to examine members of the Trx system (Trx1, TrxR and TxNIP), GST-π, GST-θ, catalase and MnSOD, and results were correlated with clinicopathological criteria. Signifi cant results were obtained between TxNIP and progression-free survival (P = 0.008) and overall survival (P = 0.05), with low expression predicting a worse prognosis. A redox protein profi le was developed, using an artifi cial neural network approach, with four of the proteins (catalase, GST-θ, GST-π and TxNIP), that stratifi es patients into good/poor prognostic groups for overall survival with an 88% sensitivity and 87% specifi city. Conventional in vitro studies show that, using MCF-7 cells, targeting the Trx system by pretreatment with novel inhibitors (PMX464, PMX290 or IV-2) sensitises resistant cells to conventional C/T but that sensitivity of the parental line remains unchanged. Initial results, using single agents in novel threedimensional (3D) systems, shows diff erential chemosensitivity, between normal and malignant cells, that is not apparent using conventional two-dimensional (2D) systems. Parental cell lines (MCF-7, MDA-MB-231) maintain or become more sensitive when exposed in 3D to conventional chemotherapy and Trx inhibitors (doxorubicin, PMX464/PMX290, IV-2 that is IC 50 2D and 3D 0.01 μM, 0.5 μM, 25 μM -paclitaxel IC 50 3D <0.01 μM, 2D 0.01 μM), assessed by clonogenic survival. Normal breast epithelial cells (MCF10As), however, show increased resistance to drugs (paclitaxel, doxorubicin, PMX464/PMX290, IV-2 IC 50 3D 0.1 μM, 1 μM, >10 μM, 200 μM -IC 50 2D 0.05 μM, 0.01 μM, 0.5 μM, 25 μM, respectively). The reasons for such altered chemosensitivity in 3D matrices are a focus of current work.

Thioredoxin and related redox systems as targets in breast cancer
Results from the immunohistochemistry and in vitro studies suggest the suitability of targeting redox proteins, particularly the Trx system, in breast cancer to improve the effi cacy of conventional therapies. There is increasing evidence that recurrent metastatic breast cancer arises in part due to the presence of long-lived, slow-cycling, and drug-resistant stem cells. Adult stem cells, known as side population (SP) cells, whose phenotype has been demonstrated to be due to the expression of ABCG2, are known to be resistant to a number of structurally unrelated compounds. In the present study we have observed that while both oestrogen-responsive and non-oestrogenresponsive breast cancer cell lines contain SP, exhibit multidrug resistance and express elevated levels of ABCG2, the non-oestrogen-responsive more highly metastatic MDA-MB-231 SP cells exhibit higher levels of mitoxantrone resistance than the other cell populations examined. These SP cells would therefore have a higher survival capacity when exposed to many currently utilised therapeutic regimes. Importantly, we have shown there is a statistically signifi cant relationship between the presence of these SP cells in fi ne needle aspirates associated with ER-negative palpable breast lesions, and that these cells are more frequently associated with triple-negative breast tumours. Novel treatments directed against SP cells should be sought to off er patients better treatment strategies in these triple-negative tumours that fail to respond to conventional targeted therapy. Further analysis of this small population of potentially important cells is warranted.

P31
A novel tumour-based test to identify breast cancer due to BRCA1 and BRCA2 mutations Q An 1 , L Jones 2 , W Tapper  Conclusions This tumour-based predictor for BRCA1 and BRCA2 carriers may prove useful to identify gene carriers at low a priori chance of having a mutation, to direct BRCA1/2 targeted treatment approaches and to identify familial non-BRCA1/2 cases that may be suitable for new gene discovery studies.

P32
Melanoma-associated antigen family protein-D4: clinical signifi cance and functional relevance in breast cancer S Germano 1 , S Rani 1 , S Kennedy 2 , J Crown 3 , M Clynes 4 , L O'Driscoll 1 Melanoma-associated antigen (MAGE) family genes are broadly expressed during development and are involved in the regulation of cell survival, cell cycle progression and apoptosis. MAGE family proteins are generally described as tumour-specifi c antigens and as representing ideal targets for cancer immunotherapy. In the current study, we identifi ed melanoma-associated antigen protein-D4 (MAGE-D4), a recently characterised MAGE family member, as a new prognostic biomarker and potential therapeutic target for breast cancer. Specifi cally, in a whole genome microarray analysis of 103 cases of invasive breast tumours, MAGE-D4 expression was observed in 43.8% of tumours, while undetectable in normal breast tissue. Multivariate and univariate analyses also indicated MAGE-D4 expression to be associated with tumour grade, spread to lymph nodes and shortened times to relapse (P = 0.0369) and death (P = 0.0133) from time of cancer diagnosis; suggesting a role for MAGE-D4 in tumour progression. To further investigate the involvement of MAGE-D4 in breast cancer cell biology, the phenotypic eff ects of this gene were characterised in vitro. We observed a marked upregulation of MAGE-D4 expression -at both mRNA and protein levels -in the breast cancer cell line Hs578T compared with the syngenic Hs578Bst breast cell line. Interestingly, RNA interference-mediated knockdown of MAGE-D4 expression in Hs578T cells signifi cantly reduced cell migration and invasion and correlated with inhibition of STAT3 and NF-κB p65 subunit phosphorylation, thus aff ecting two common signalling pathways involved in regulating cancer progression. Moreover, monolayer cell growth rate was not aff ected by MAGE-D4 gene knockdown, while growth in soft agar was signifi cant compromised. Our results indicate that MAGE-D4 contributes to the tumorigenesis of breast cancer cells by regulating migration, invasion and anchorage-independent growth, and therefore may represent a novel target for the detection and treatment of breast cancer. Acknowledgements Funding support from Ireland's Health Research Board (RP/2006/77) and Science Foundation Ireland (08/SRC/B1410). Breast cancer is not a single disease but is highly heterogeneous at both the molecular and clinical levels. Gene expression profi ling of breast tumours by multiple independent groups and technologies have revealed fi ve major molecular subtypes of breast cancer. These molecular diff erences result in distinct clinical outcomes and responses to treatment. The gene expression profi ling studies that have defi ned the molecular subgroups of breast cancer to date were performed using fresh frozen tissue. Routine clinical practice dictates the preservation of surgical specimens via paraffi n embedding of formalin-fi xed tissue. Therefore, derivation of molecular subgroups of breast cancer from formalin-fi xed, paraffi n-embedded (FFPE) preserved tissue would have more application in the clinical setting as such profi les could be applied to routinely collected specimens. The Almac Diagnostics' Breast Cancer DSA™ has been optimised for analysis of FFPE samples enabling the use of these valuable archived tissue banks. We have demonstrated the ability to identify the molecular subgroups previously defi ned within a cohort of sporadic, BRCA1 mutant and BRCA2 mutant FFPE breast tumours as well as defi ning two novel subgroups within this tumour set. This study demonstrates that it is possible to derive biologically meaningful data from a cohort of archived FFPE tumour samples using the Almac Breast DSA™. We demonstrate that there is considerable molecular diversity within BRCA mutant and sporadic breast tumours, suggesting that traditional assumptions of the behaviour of tumours based on their immunohistochemistry status may not always be correct. At present, a number of clinical trials are stratifying patients for poly(ADP-ribose) polymerase 1 (PARP-1) inhibitor therapy based on BRCA mutation and triple-negative status. The data presented here would suggest that not all BRCA1 mutant, BRCA2 mutant and indeed triple-negative patients are similar at the molecular level and as such will not respond equally to PARP-1 inhibitor or indeed other therapeutics in the same manner.

P34
Evaluating gene expression in formalin-fi xed, paraffi n-embedded breast cancer tissues using DASL® T Burr 1 , R Dixon 1 , A Green 2 , I Ellis 2 , C Murray 1 The study of gene expression in conventionally processed tissues is hampered by degradation of mRNA. Expensive, low-multiplex, quantitative PCR methods can be unreliable due to the limited template sizes. One way to overcome this problem is to use array-based methods. DASL® technology relies on random priming for production of cDNA, in concert with universal bead arrays to allow the detection and relative quantitation of expression of specifi c gene subsets. Using the Illumina DASL® Cancer Panel (500 cancer-associated genes on one array), we evaluated the expression of key genes in archival formalin-fi xed, paraffi n-embedded tissue samples from 80 breast cancer patients with wellcharacterised pathological and clinical features. We fi rst assessed transcript integrity in the samples on the basis of levels of mRNA encoding RPL13A, prior to running the Cancer Panel. A subset of genes of interest was then assessed by quantitative PCR to confi rm the relative levels observed using the DASL® assay. Finally the expression of the same subset of genes was evaluated at the protein level by immunohistochemistry. We were able to predict, with good accuracy based upon RPL13A assays, those samples unsuitable for DASL® analysis. Furthermore, the results of DASL® analysis showed good correlation with protein levels, as measured by immunohistochemistry, for a number of key genes including ERBB2 (HER2) and ESR1 (ER). We conclude that DASL® represents a powerful tool for assessing expression of multiple genes in archival tissue. Paclitaxel is a microtubule inhibitory chemotherapeutic drug that is increasingly used for the treatment of solid tumours. In vitro studies have demonstrated that attenuating the spindle assemble checkpoint (SAC) alters the post-mitotic responses to paclitaxel. Furthermore, the aberrant expression of a number of the SAC proteins, MAD2, BUBR1, and Aurora A kinase, are associated with poor patient prognosis. We have identifi ed a microRNA, miR-433, that regulates the expression of MAD2. Overexpression of miR-433 in Hela cells induced downregulation of MAD2 mRNA and protein expression. We have also shown that Hela cells overexpressing miR-433 and treated with paclitaxel are no longer capable of cyclin B stabilisation, and thus have lost the ability to activate the SAC in response to paclitaxel. In addition, cell viability assays showed that Hela cells overexpressing miR-433 and treated with paclitaxel have an attenuated response to paclitaxel compared with microRNA scrambled controls. We have characterised the levels of miR-433, MAD2 gene expression and MAD2 protein levels in a cohort of ovarian cancer cell lines. Cell viability assays on this cohort revealed that responsiveness to paclitaxel is associated with high MAD2 protein expression and lower miR-433 expression. We hypothesise that the expression of miR-433 when deregulated in cancer leads to altered MAD2 expression and a compromised SAC, a key feature underlying drug resistance to paclitaxel. In a pilot study of paired human breast tumour and normal breast tissue samples we have shown that expression levels of miR-433 are elevated in cancer tissue. Targeting this microRNA in cancer may improve the effi cacy of paclitaxel in treating breast cancer and ovarian cancer.

Introduction
There is currently extensive diagnostic use of breast tissue calcifi cations through their diff erential mammographic appearance, albeit with relatively low specifi city. However, the details of the calcifi cation chemical and structural composition remain somewhat vague. Thus any associated clinical signifi cance, such as indications of tumour type, grade and stage, have not previously been explored. Methods The biochemical composition and incorporation of carbonate into the hydroxyapatite lattice of type II microcalcifi cations was studied by infrared microspectroscopy, allowing spectral information to be directly correlated with associated histopathology of the surrounding tissue. Results It was shown that the chemical characteristics of calcifi cations asso ciated with benign, in situ and invasive pathologies are signifi cantly diff erent. For the fi rst time, a relationship between grade of pathological breast disease and chemical nature of associated microcalcifi cations has been demonstrated. In particular we have found signifi cant correlations between distinct pathology grades and physiochemical features such as the carbonate content of micro calcifi cations and protein to mineral ratios. Further, such correlations were also demonstrated within carcinoma in situ and invasive cancer subgrades. Quantifi cation of the calcifi cation carbonate content indicated that the degree of carbonate substitution followed a monotonic trend between benign, ductal carcinoma in situ (DCIS) and invasive pathologies (see Figure 1). This suggests that benign tissue calcifi cation (consisting Introduction The existence of breast cancer-initiating cells was initially demonstrated by Al-Hajj and colleagues [1] using antigen expression, and subsequent studies have employed several methodologies to identify and isolate these cells. However, there are limited data describing whether similar cell populations are recognized by the diff erent approaches. Materials and methods Using breast cancer cell lines MCF7, MDAMB231 and MDAMB468, we have compared the antigen expression profi le (CD44 + CD24 −/low ) against the side population and the ability to form tumour spheroids. Immunostaining on cells and xenografts was also performed to search for expression of potential stem cell markers. Results Our data showed increased CD44 + CD24 −/low population in both MCF7 and MDAMB468 spheroids, but growth advantage was only observed in sorted MDAMB468 CD44 + CD24 −/low cells. In contrast, analysis of the antigen profi le of the side population did not demonstrate any correlation and no growth advantage was found in sorted MCF7 and MDAMB468 cells. Immunostaining of MCF7-derived tumour xenografts showed two potential markers, p63 and sox2, in addition to CD44; both MDAMB231 and 468-derived xenograft expressed strong CD44, and the latter was also stained for p63 and aldehyde dehydrogenase (ALDH). In addition, comparison between the antibodies only demonstrated partial overlap between CD44 and p63/ALDH in MCF7 and MDAMB468 xenografts. Therefore, in MCF7/MDAMB468-like breast tumours, p63 and sox2/ ALDH recognize diff erent stem/progenitor cell populations, and the combination of CD44 and p63/ALDH further clarifi es the boundary of these cells.

Conclusions
Our results indicate that each breast cancer is unique, and therefore tumour-initiating cell markers and methodologies should be applied specifi cally. Introduction Dysfunctional mitochondria contribute to the onset of malignant transformation and growth. Molecules that regulate mitochondrial homeostasis are therefore the object of great attention to identify novel therapeutic strategies. The mitochondrial translocator protein (mTSPO) stands in a critical position for mitochondrial homeostasis and is involved in the physiology of breast cancer where it is overexpressed and positively associated with aggressiveness [1]. mTSPO ligands are therefore exploited for cancer imaging and chemotherapy, such as PK11195. mTSPO is associated with the voltagedependent anion channels (VDACs), which regulate the metabolites' fl ux into mitochondria [2]. mTSPO expression is driven by the oncogene protein kinase Cε, suggesting a fundamental crosstalk for malignant transformation and uncontrolled proliferation. We hypothesized that mTSPO by regulating VDAC performance impinges on metabolism and pharmacologically induced cell death in breast cancer cells.

Results
In human breast adenocarcinoma MCF-7 and in cervical cancer cells (HeLa) we found, via imaging and luminescent-based approaches, that a decreased mTSPO/VDAC ratio of expression uperegulates mitochondrial Ca 2+ uptake and ATP generation whilst reducing the rate of reactive oxygen species generation calling for a metabolic switch via an improvement of mitochondrial function. mTSPO suppression also impairs protein kinase Cε activation and facilitates Ca 2+dependent apoptosis triggered by C 2 -ceramide. Nevertheless, mTSPO targeting with PK11195 -which impinges on Ca 2+ homeostasis [3] -raises C 2 -ceramide cell death in MCF-7 and in the more aggressive line of adenocarcinoma MDA10characterized by an increased mTSPO/VDAC ratio of expression. Introduction Previously we identifi ed MCPH1, a DNA damage response protein involved in the regulation of BRCA1 and BRCA2, as the defective protein in one form of microcephaly. BRCA1 mutations are associated with basal-like breast cancer, which are often also negative for oestrogen receptor (ER), progesterone receptor (PR) and HER2. Our data indicate that MCPH1 plays a role in response to chemotherapeutic agents used in the treatment of breast cancer due to its role in DNA repair and the spindle checkpoint.
Methods MCPH1 immunohistochemistry was performed on 320 breast cancers and correlated with pathology, survival, ER, PR and HER2 data. Drug assays were performed on the breast cell lines MCF10A, MCF7 and HCC193 with diff erent MCPH1 and BRCA1 backgrounds created using siRNA.

Results
We identifi ed reduced MCPH1 expression in 23% (74/320) of breast cancers. After performing continuous data analysis, the mean MCPH1 expression decreased with increasing grade, grade 1 and 2 versus grade 3 (P <0.006). Interestingly, mean MCPH1 expression was also lower in ER/PR-negative (P <0.001) and triple-negative cancers (P <0.004). An association with HER2positive cancers was also identifi ed (P <0.03). While no association with survival was identifi ed initially, the longer term survival was better in patients with higher mean MCPH1 expression. Our cell line drug assays indicate that MCPH1 plays a role in resistance to Taxol and sensitivity to cisplatin and doxorubicin. Conclusions MCPH1 expression is reduced in 23% of breast cancers, particularly in higher grade tumours. Interestingly, reduced mean MCPH1 expression was associated with the triple-negative phenotype often seen in basal-like cancers. Further basal cell markers are currently under investigation. Aggressive basallike breast cancers have a poor prognosis; MCPH1 expression may potentially improve treatment of these cancers.

Introduction
Overexpression of human epidermal growth factor receptor 2 (HER2) tyrosine kinase cell surface receptor occurs in 25 to 30% of all breast cancer and is linked to aggressive phenotype and high-mortality disease. HER2 is a clinically important target in diagnosis and treatment of breast cancer but despite its pivotal role there are no established tools for quantitative clinical imaging of the extent and location of HER2-positive (HER2 + ) tumours in patients. Such a tool could provide important clinical diagnostic information by early detection of subclinical HER2 + disease, optimal management of current anti-HER2 therapies and response assessment of novel therapeutics. We aimed to generate recombinant proteins that would achieve sensitive and specifi c detection of HER2 + tumours in the clinic using radioimmunoimaging. Materials and methods Two diff erent HER2-binding molecules were investigated: C6.5 a small dimeric antibody fragment (diabody), which is approximately 1/3 of the size of an antibody; and G3, a small monomeric designed ankyrin repeat protein (DARPin) that is 1/10 the size of an antibody. The agents were generated in the yeast Pichia pastoris system using processes compliant with good manufacturing practice (GMP). C6.5 and G3 production strains were constructed to allow methanol-inducible, soluble expression. The expressed proteins were purifi ed using expanded-bed adsorption-immobilized metal affi nity chromatography. Results and conclusions For C6.5 the fi nal product was homogeneous, stable and free of host cell and other relevant contaminants. The protein was stable during storage, with no evidence of aggregation. In addition, affi nity for HER2, as measured by Biacore analysis, was not compromised by storage at either 4 or -80°C. Preliminary results with the G3 DARPin indicate that this protein is also amenable to GMP production in P. pastoris. The relative effi cacy of these agents for specifi c radioimmunoimaging of HER2 + tumours in vivo is currently under investigation. Methods Extensive gene expression profi ling and data analysis has been performed on a cohort of 70 formalin-fi xed, paraffi n-embedded-derived BRCA1 mutated breast tumours and matched sporadic controls using the Almac Breast Cancer DSA™ research tool. Validation of gene targets has been performed by quantitative RT-PCR and western blotting. Results A list of diff erentially expressed transcripts has been derived from the comparison of these BRCA1 mutant breast tumours to matched sporadic controls. Functional analysis of this gene list was performed to identify the main pathways and processes that are deregulated by these transcripts. BRCA1 defi ciency was associated with deregulation of pathways involved in: immune response, metastasis and invasion, cytoskeletal remodelling, spindle assembly and chromosome separation, and apoptosis and survival. We have now validated several panels of genes that characterise this BRCA1-defi cient breast cancer profi le. A high-throughput siRNA-based screening strategy will now be performed to identify functionally relevant BRCA1-associated gene targets involved in cell growth, diff erentiation and chemotherapy response.

P43
Conclusions This approach has identifi ed a set of transcripts that could be used to identify both hereditary and sporadic breast cancer patients with BRCA1 defi ciency. Insulin-like growth factor receptor (IGF-IR) signalling classically involves phos phory lation of insulin receptor substrate-1 (IRS-1) to recruit key downstream signalling pathways eff ecting breast cancer cell proliferation and survival. Recently, we have shown a further capacity for IRS-1 to associate with the epidermal growth factor receptor (EGFR/erbB1), with activation of EGFR promoting recruitment and phosphorylation of IRS-1 in an oestrogen receptor (ER)-positive tamoxifen-resistant breast cancer cell line. In this study, we examined recruitment of IRS-1 by another member of the erbB receptor family, erbB3, in three ER-positive breast cancer cell lines. Our studies revealed an interaction between erbB3 and IRS-1 in MCF-7, T47D and BT474 cells with HRGβ1 treatment signifi cantly enhancing this recruitment and promoting IRS-1 phosphorylation at tyrosine (Y) 612, a specifi c phosphoinositide 3-kinase (PI3K) binding site. IRS-1 appears to play a key role in erbB3 signalling in MCF-7 and T47D cells as its knockdown using siRNA greatly impaired HRGβ1 signalling via PI3K/AKT in these cell lines. This novel interaction may have clinical relevance as immunohistochemical analysis of ER-positive breast cancer patient samples revealed IRS-1 Y612 expression positively correlated with total erbB3, p-AKT and Ki67 expression. Importantly, we found that recruitment of IRS-1 by erbB3 impaired IRS-1 recruitment by IGF-IR in both MCF-7 and T47D cells, whilst blockade of IGF-1R enhanced erbB3/IRS-1 interaction and sensitised both cell lines to HRGβ1. Consequently, blockade of erbB3 signalling enhanced the eff ects of IGF-IR inhibition in these cells. In conclusion, these and previous fi ndings suggest that IRS-1 can be recruited to IGF-1R, EGFR and erbB3 in ER-positive breast cancer cells and this may provide an adaptive resistance mechanism when these receptors are targeted individually. Consequently cotargeting of IGF-IR and erbB receptors may prove to be a more eff ective strategy for the treatment of ER-positive breast cancer. Based on protein expression profi les of core regulatory proteins involved in the G 1 -S and G 2 -M phase transitions, we have identifi ed three distinct cell cycle phenotypes in a series of 200 breast cancers: a G 0 out-of-cycle state (18% of cases); a G 1 arrested/delayed state (24% cases); and accelerated S-G 2 -M phase progression (58% of cases). The accelerated cell cycle progression phenotype had a higher risk of relapse when compared with G 0 and G 1 -delayed/arrested phenotypes (HR = 3.90 (95% CI = 1.81 to 8.4), P <0.001) and was associated with Her2 and triple negative subtypes (P <0.001). High-grade tumours with the G 1delayed/arrested phenotype showed an identical low risk of relapse compared with well-diff erentiated G 0 tumours. In addition to its prognostic signifi cance, the cell cycle phenotype also impacts on individualised therapeutic decisions. Only patients showing the actively cycling, aggressive cell cycle phenotype are likely to benefi t from conventional chemotherapeutic S-phase-directed or M-phase-directed agents or from the new generation of targeted cell cycle inhibitors that are now entering clinical trials. The DNA replication initiation factor Cdc7 is an emerging anticancer target. Cdc7 inhibition results in an abortive S phase and potent cancer cell killing. Specifi city is based on normal cells undergoing a reversible G 1 arrest following Cdc7 inhibition due to activation of a novel cell cycle checkpoint that is lost or impaired in cancer cells. Our analysis of the molecular circuitry underlying this replication origin activation checkpoint reveals that G1 arrest is dependent on three nonredundant checkpoint axes coordinated through the Forkhead transcription factor FoxO3a and p53. We show that only breast cancers displaying the accelerated cell cycle phenotype express elevated Cdc7 levels and are therefore highly represented in p53 mutant Her2-subtype and triple negative tumours. Breast cancers of the luminal subtype expressing low levels of Cdc7 undergo a cytostatic G 1 arrest after Cdc7 inhibition due to their p53 included the TRAIL-resistant breast cancer cell line, T47D. In addition, a modifi ed approach of culturing primary breast tumour explants ex vivo to maintain their three-dimensional architecture provided a more clinically relevant breast tumour model. Importantly, all TRAIL-sensitive breast tumour cell lines responded only to a TRAIL-R1-specifi c form of TRAIL. Despite expressing TRAIL-R1/TRAIL-R2, the T47D cell line required initial sensitization by doxorubicin and again exhibited selectivity towards apoptosis induced by a TRAIL-R1-selective ligand. Crucially, we show that doxorubicin can also sensitize TRAIL-resistant primary breast tumour explants to TRAIL-induced apoptosis, while having no eff ect on normal breast tissue. Furthermore, in this ex vivo model, TRAIL combined with doxorubicin induced signifi cantly more apoptosis via TRAIL-R1 than TRAIL-R2. Conclusions Our results have important implications for the potential treatment of breast cancer with TRAIL-based therapeutic agents. We propose that using a TRAIL-R1-specifi c ligand/mAb combined with subtoxic concentrations of doxorubicin will selectively target tumour cells and minimise potential side eff ects, such as triggering of TRAIL-induced prosurvival pathways in TRAILresistant primary tumour cells or cardiotoxicity induced by higher concentrations of doxorubicin used in monotherapy.

P59
Modelling breast cancer in a three-dimensional Microenvironmental factors are fundamental in the regulation of normal and tumour breast tissue. Two cell types have been implicated in having opposing eff ects on breast tumour cell behaviour: myoepithelial cells exhibit broad tumour-suppressor activity, whilst fi broblasts frequently promote tumour growth and invasion. Previous work has described the development of a physiologically relevant three-dimensional heterotypic culture system containing tumour, myoepithelial and fi broblast cells. The data showed organisation of the cells into co-unit structures recapitulating ductal carcinoma in situ breast, with homing of myoepithelial cells around luminal cells, and highlighted a central role for tumour-associated fi broblasts in disrupting ductal carcinoma in situ structures. This study describes further manipulation of the model to include tumour cells that represent the heterogeneity of breast cancer. MCF-7 (ER + ), MDA-MB-468, MDA-MB-231 (basal) and MDA-MB-453 (Her2 + ) were cultured in collagen for 7 days in the presence or absence of normal myo epithelial cells. Gels were fi xed in formalin, paraffi n embedded and immunohistochemistry was performed for a series of markers recognising the cell types along with basal polarity and basement membrane proteins. Initial morphological analysis of the cultures has been performed to assess the degree of co-unit formation, based on a visual description of the size and shape of the co-units. Co-unit formation has been employed as a representative measure of tumour progression as it is known to be a key feature in early breast cancer invasion. When cultured alone, MCF-7 and MDA-MB-468 cells formed spherical co-unit structures and this was maintained in the presence of myoepithelial cells. In contrast, MDA-MB-231 and MDA-MB-453 cells show a more scattered appearance. The presence of myoepithelial cells induced polarity in the MDA-MB-231 cells and a more ordered appearance. This study is the fi rst time that the co-culture of tumour cell populations with myoepithelial cells has been investigated in three-dimensional collagen gels showing diff erences in morphology that may relate to tumour progression.