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Homeopathy service in an NHS hospital breast cancer clinic: outcome study

May 2008
Breast Cancer Research 10:1-1

DOI:10.1186/bcr1977

Authors:

Clare Relton

Queen Mary, University of London

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Content uploaded by Clare Relton

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Available online http://ccforum.com/supplements/12/S2

Breast Cancer Research Volume 10 Suppl 2, 2008

Meeting Abstracts

Breast Cancer Research 2008

The Royal Society, London, UK

13 May 2008

Published online: 13 May 2008

These abstracts are available online at http://breast-cancer-research.com/supplements/10/S2

KEYNOTE LECTURES

Abstracts not available at time of publication.

Chromosomal instability in the pathogenesis and

treatment of breast cancer

A Venkitaraman

Breast Cancer Res 2008, 10(Suppl 2):L1 (doi: 10.1186/bcr 1877)

Molecular diversity of human breast cancer: clinical and

therapeutic implications

D Slamon

Breast Cancer Res 2008, 10(Suppl 2):L2 (doi: 10.1186/bcr 1876)

Surviving breast cancer: can women expect to ‘get back to

normal’?

P Hopwood

Breast Cancer Res 2008, 10(Suppl 2):L3 (doi: 10.1186/bcr 1878)

SPEAKER PRESENTATIONS

Use of BRCA1 protein:protein interactions to classify

cancer risk

JR Morris

, SV Tavtigian

Department of Medical & Molecular Genetics, King’s College

London, UK;

International Agency for Research on Cancer, Lyon,

France

Breast Cancer Res 2008, 10(Suppl 2):O1 (doi: 10.1186/bcr 1879)

Germline loss-of-function mutations in BRCA1 are associated with

a high lifetime risk of breast and ovarian cancer. Most mutations in

the gene are ‘truncating’: in the main these induce premature termi-

nation codons, resulting in nonsense-mediated decay, loss of the

transcript and/or the entire protein. The improved screening

methods now in use across the UK will identify many carriers of

unclassified BRCA1 variants. These are chiefly missense muta-

tions, introducing an amino acid change in the context of an

expressed protein. Indeed more than one-quarter of entries

recorded in the Breast Cancer Information Core dataset of BRCA1

sequence variants collected from patients worldwide are

unclassified missense alterations (http://research.nhgri.nih.gov/bic/).

Currently, discovery of the majority of missense variants leaves

both variant carriers and their families in an ambiguous position.

These variants remain unclassified because in the majority of cases

it is not possible to follow variants by cosegregation analysis, and

the number of appropriate controls required to be certain that a

variant is absent in unaffected individuals is prohibitive. Currently,

in silico algorithms try to distinguish between missense substi-

tutions that are likely to be pathogenic and those that are not.

These algorithms compile a multicomponent likelihood ratio that

integrates assessment methods ranging from conservation analy-

sis, co-occurrence of a deleterious allele in trans, and immuno-

histochemical analysis [1-3]. What is missing from these analyses

is the relationship between loss of protein function and detriment

to patient health.

We have focused on the N-terminal region of BRCA1. This region

has a high density of missense substitutions, including those of

known pathogenic status, and many currently unclassified variants.

We have shown that experimental missense variants, generated

randomly and selected for loss of interaction with the BRCA1

ubiquitin ligase components, BARD1 and the E2 enzyme UbcH5,

identify variants reported within the Breast Cancer Information

Core database of individuals with a personal or family history of

breast cancer [4]. The E2 component is particularly sensitive to

missense alteration in BRCA1, with the majority of currently

unclassified variants in the region inhibiting interaction, whereas

the BARD1 component is disrupted by a smaller, but overlapping,

subset restricted to substitution of the structurally detrimental zinc-

ligation residues. Variants that inhibited the E2 also prevented the

enzymatic activity. These data strongly suggest that the ligase

activity of BRCA1, through interaction with E2 and BARD1, is

related to breast cancer predisposition.

Using yeast two-hybrid analysis for BRCA1:BARD1 and

BRCA1:E2 interaction, we have tested the most chemically

different substitutions achievable by single nucleotide change in all

of the most highly conserved amino acids of the region (invariant

from human to sea urchin), and have also tested all currently

identified patient missense variants. These data have been

combined with Grantham variation and Grantham deviance scores

(a measure of how conserved an amino acid is, together with how

different the protein change is) to assess the relationship between

protein:protein interaction and measures of disease risk. Risk

measures were based on the results of full sequence tests of

BRCA1 and BRCA2 from 68,000 BRACAnalysis subjects (Myriad

Genetics, Salt Lake City, UT, USA), and used estimates of the

odds of developing breast cancer for a carrier of a BRCA1

missense substitution [2], together with enrichment ratios achieved

by comparing the variants observed in the dataset with the variants

expected on the basis of known substitution rates.

Classification methods in the past have attempted to place variants

in either the pathogenic or the little-clinical significance categories.

The results of this analysis suggest that some classes of variant

may confer an intermediate risk. If so, these data have considerable

implications for the counselling and clinical management of women

found to be positive for missense variants in future.

Breast Cancer Research May 2008 Vol 10 Suppl 2 Breast Cancer Research 2008

Acknowledgement JRM is funded by Breast Cancer Campaign.

References

1. Goldgar DE, Easton DF, Deffenbaugh AM, Monteiro AN, Tavti-

gian SV, Couch FJ: Integrated evaluation of DNA sequence

variants of unknown clinical significance: application to

BRCA1 and BRCA2. Am J Hum Genet 2004, 75:535-544.

2. Tavtigian SV, Samollow PB, de Silva D, Thomas A: An analy-

sis of unclassified missense substitutions in human

BRCA1. Fam Cancer 2006, 5:77-88.

3. Chenevix-Trench G, Healey S, Lakhani S, Waring P, Cum-

mings M, Brinkworth R, Deffenbaugh AM, Burbidge LA, Pruss

D, Judkins T, et al.: Genetic and histopathologic evaluation

of BRCA1 and BRCA2 DNA sequence variants of unknown

clinical significance. Cancer Res 2006, 66:2019-2027.

4. Morris JR, Pangon L, Boutell C, Katagiri T, Keep NH, Solomon

E: Genetic analysis of BRCA1 ubiquitin ligase activity and

its relationship to breast cancer susceptibility. Hum Mol

Genet 2006, 15:599-606.

Dietary patterns across the life course, mammographic

density and implications for breast cancer: results from a

British prospective cohort

G Mishra

, I dos Santos Silva

, S McNaughton

, V McCormack

R Hardy

, A Stephen

, D Kuh

MRC National Survey of Health and Development, Department of

Epidemiology and Public Health, University College London,

London, UK;

Department of Epidemiology and Population Health,

London School of Hygiene and Tropical Medicine, London, UK;

Centre for Physical Activity & Nutrition Research, Deakin University,

Australia;

MRC Human Nutrition Research, Cambridge, UK

Breast Cancer Res 2008, 10(Suppl 2):O2 (doi: 10.1186/bcr 1882)

Background Previous epidemiological studies have investigated

the relationship between individual nutrients such as vitamin D and

vitamin B

and mammographic density, a strong marker of breast

cancer risk [1], with varied results. There has been limited research

on overall dietary patterns and most studies have focused on adult

dietary patterns [2]. We examine prospective data to determine

whether dietary patterns from childhood to adult life affect

mammographic density.

Methods The Medical Research Council National Survey of Health

and Development is a national representative sample of 2,815 men

and 2,547 women followed since their birth in March 1946 [3]. A

wealth of medical and social data has been collected in over 25

follow-ups by home visits, medical examinations and postal

questionnaires. Dietary intakes at age 4 years were determined by

24-hour recalls and in adulthood (ages 36, 43 years) by 5-day food

records. Copies of the mammograms (two views for each breast)

taken when the women were closest to age 50 years were

obtained from the relevant NHS centres. A total of 1,319 women

were followed up since birth in 1946 for whom a mammogram at

age 50 years was retrieved, and the percentage mammographic

density was measured using the computer-assisted threshold

method for all 1,161 women. Breast cancer incidence for the

whole cohort is being ascertained through the National Health

Service Central Register.

Statistical analysis Reduced rank regression analysis, a relatively

new approach to dietary pattern analysis, is being used to identify

dietary patterns associated with mammographic density [4]. This

approach identifies patterns in food intake that are predictive of an

intermediate outcome of the disease process, such as mammo-

graphic density, and subsequently examines the relationship

between the identified dietary patterns and breast cancer risk.

Results Preliminary analyses so far suggest that variations in

dietary patterns in adulthood might explain more than 10% of the

variation in percentage mammographic density at age 50 years

(age 36 years: 13%; age 43 years: 14%), with variations in

patterns in childhood explaining slightly less. Further work is being

carried out on the characteristics of these dietary patterns and

their effects on percentage mammographic density and its two

components (that is, absolute areas of dense and nondense

tissues) and on breast cancer risk, after adjusting for socio-

economic status, anthropometric variables and reproductive factors.

Conclusion The present study will provide for the first time

information on the relationship between dietary patterns across the

life course and mammographic density, and will help to clarify the

pathways through which diet may affect breast cancer risk.

Acknowledgements Breast Cancer Campaign UK–World Cancer

Research Fund International and the Medical Research Council

fund this project. IdSS and VM are members of the Cancer

Research UK Epidemiology and Genetics Group; VM is funded by

a Cancer Research UK training fellowship.

References

1. McCormack VA, dos Santos Silva I: Breast density and

parenchymal patterns as markers of breast cancer risk: a

meta-analysis. Cancer Epidemiol Biomarkers Prev 2006, 15:

1159-1169.

2. Takata Y, Maskarinec G, Park SY, et al.: Mammographic

density and dietary patterns: the multiethnic cohort. Eur J

Cancer Prev 2007, 16:409-414.

3. Wadsworth M, Kuh D, Richards M, et al.: Cohort profile: the

1946 National Birth Cohort (MRC National Survey of

Health and Development). Int J Epidemiol 2006, 35:49-54.

4. Hoffmann K, Schulze MB, Schienkiewitz A, et al.: Application of

a new statistical method to derive dietary patterns in nutri-

tional epidemiology. Am J Epidemiol 2004, 159:935-944.

Loss of oestrogen receptor alpha in long-term

antioestrogen-resistant cells: reversal by a c-src inhibitor

A Bensmail, I Hutcheson, M Giles, J Gee, R Nicholson

Tenovus Centre for Cancer Research, Welsh School of Pharmacy,

Cardiff, UK

Breast Cancer Res 2008, 10(Suppl 2):O3 (doi: 10.1186/bcr 1883)

Background Tamoxifen still remains the most frequently used

antioestrogen for the treatment of breast cancer. However, its

efficacy is often limited by the emergence of acquired resistance

and it has been suggested that, in some instances, this may involve

oestrogen receptor (ER) loss. This study addresses this issue by

examining long-term tamoxifen treatment of breast cancer cells and

identifies that progressive ER loss does occur, leading to greatly

increased aggressive tumour cell behaviour. Encouragingly, even

after 30 months treatment, ER loss is reversible by a c-src inhibitor.

Our data therefore provide a new model to study the cellular

mechanisms associated with antihormone promoted ER loss and

its possible prevention/reversal by signal transduction inhibitors.

Methods Using quantitative PCR based on SYBR Green fluores-

cence, the expression of total ERα mRNA and its constituent

mRNA variants were quantified in MCF7 cells and in our in vitro

developed tamoxifen-resistant breast cancer cells (TamR), which

have been cultured in the presence of tamoxifen for 30 months.

Specific PCR amplification of all ERα mRNA variants was possible

using forward primers designed to bind specifically to the 5′

untranslated regions of ERα mRNA and used separately with a

common reverse primer that anneals to the 5′ end of the protein

Available online http://breast-cancer-research.com/supplements/10/S2

encoding region of exon 1 of ERα cDNA. Expression of ERα

protein was assessed by western blot and immunohistochemistry.

Results In MCF7 cells, the ERα mRNA isoforms A, B and C were

detected as the most predominant variants, with C ERα mRNA

showing the highest expression level. In TamR cells, about a 40%

fall in total ERα mRNA was observed in comparison with MCF7

cells and was most apparent for the C variant. Extension of the

tamoxifen treatment period to 30 months produced a further

dramatic decrease in ERα mRNA (all variants) and protein levels,

resulting in ER negativity being recorded in >90% of the cells by

immunohistochemistry. These cells show increased levels of

phosphorylated Erk 1&2, AKT, PKCα and src, and are highly

aggressive in their growth behaviour, with increased cell motility

and invasiveness. Treatment of the cells with the demethylating

agent 5-azacytidine did not restore ERα expression, suggesting

that epigenetic alterations are unlikely to be responsible for the

reduced ER levels. However, Affymetrix data in the TamR cells

showed that some positive regulators of ER expression, such as

p53 and Foxo3A, are downregulated during the development of

the resistant phenotype and their continued absence may

contribute to the progressive ER loss. Significantly, pathway

inhibitor studies revealed c-src to be an important regulator of ER

loss, since its inhibition rapidly restored ER levels.

Conclusion Our data indicate that considerable ER loss can occur

during antihormonal treatment of breast cancer cells and that this

can lead to a more aggressive phenotype. Encouragingly, however,

even after 30 months exposure to tamoxifen, the process is

reversible by inhibition of c-src. These data suggest that

combinations of antihormones with signal transduction inhibitors

could retain ER functions in treated cells and prevent a drift

towards more aggressive cancer cell behaviour.

Suppression of the NF-

κκ

B cofactor Bcl3 inhibits mammary

epithelial cell apoptosis and, in breast tumours, correlates

with poor prognosis

A Wakefield

, L Piggott

, D Croston

, WG Jiang

, R Clarkson

Life Sciences, School of Biosciences, University of Cardiff, UK;

Angiogenesis and Metastasis Group, School of Medicine,

University of Cardiff, UK

Breast Cancer Res 2008, 10(Suppl 2):O4 (doi: 10.1186/bcr 1884)

Background Several transcription factors have been shown to

play important roles in the regulation of apoptosis at the onset of

murine mammary involution. These include LIF-activated STAT3,

c/ebpdelta, Ap-1 and IKK/NF-κB-mediated regulation of death

receptor ligands. A study of STAT3 and STAT5 transcriptional

targets in mammary epithelial cells in vitro showed that both

c/ebpdelta and c-fos (a component of Ap-1) were upregulated by

STAT3, suggesting a degree of interdependence between these

transcription factor pathways in mediating their apoptotic effects.

Interestingly, while no NF-κB or IKK genes were significantly

regulated by STATs, the NF-κB cofactor gene, Bcl3, was found to

be a principal transcriptional target of STAT3. This factor plays a

role in altering the transcriptional capacity of specific NF-κB

subunits and has previously been described as an oncogene in B-

cell lymphomas. In this study we set out to establish whether Bcl3

had a role in regulating the cell fate of mammary epithelial cells

either in the normal mammary gland or in mammary/breast cancer.

Methods Archived material representing a range of tumour grades

and types was collected from breast cancer patients immediately

after surgery (tumour tissues = 122, normal tissues = 32). The

median follow-up of the patients was 120 months (range 12 to

156 months). QRT-PCR for Bcl3 was performed and this infor-

mation was used to determine statistically significant correlations

with the clinical data on breast pathology. MCF7, T47D and MDA-

MB231 human breast cancer cell lines were subjected to Bcl3-

specific siRNA knockdown and subsequently assessed for cell

motility characteristics using ECIS technology. Bcl3-knockout mice

were assessed histologically for alterations in apoptosis rate during

the adult pregnancy cycle. Western blots, quantitative PCR and

DNA binding assays were used to determine the activity of

molecular markers of apoptosis in these animals. Bcl3-deficient

animals were crossed with mmtv-neu (c-erbB2) mice to establish

the role of Bcl3 in primary (neu-dependent) mammary tumour

growth, and magnetic resonance imaging was performed on

tumour-bearing animals, to establish metastasis rates in the

presence/absence of Bcl3.

Results An analysis of 122 human breast cancer tissues showed

that Bcl3 gene expression was suppressed in a significant

proportion of invasive tumours, which correlated with poor

prognosis. This also correlated with a significant decrease in Bcl3

gene expression in human breast cancer cell lines exhibiting

increased motility characteristics. The effects of siRNA-mediated

knockdown of Bcl3 are ongoing. In the mouse mammary gland,

Bcl3 expression was restricted to epithelial cells during the first 24

hours of involution. Bcl3 deficiency resulted in a transient delay in

the appearance of apoptotic bodies in the early involuting

mammary gland in Bcl3

–/–

mice, while pSTAT3 levels were

unchanged compared with equivalent timepoints in control

animals. The activities of initiator/executor caspases of both

intrinsic and extrinsic pathways were significantly decreased in

Bcl3

–/–

tissues at this time, which correlated with decreases in the

expression of key regulators of intrinsic/extrinsic apoptosis. Results

from the ongoing magnetic resonance imaging study of tumour

incidence/progression in mmtv-neu/Bcl3

–/–

mice will be presented.

Conclusion These observations suggest that Bcl3 promotes

apoptosis in the mammary gland and provides preliminary evidence

of cross-talk between STAT3 and NF-κB pathways, both of which

have been implicated in breast cancer. Our current data on Bcl3 in

primary breast tumours and breast cancer cell lines contrasts with

other studies, to suggest that Bcl3 suppresses the metastatic

progression of primary breast cancer and has a neutral role in

breast cancer incidence or primary tumour growth.

Acknowledgement Funded by the Breast Cancer Research Trust.

Activation of TGF-beta signalling in breast cancer

metastatic cells

S Giampieri, E Sahai

Tumour Cell Biology Laboratory, London, UK

Breast Cancer Res 2008, 10(Suppl 2):O5 (doi: 10.1186/bcr 1880)

Background The onset of metastasis in organs such as the lung,

bone and brain is a major cause of mortality in breast cancer patients.

Many signalling pathways have been implicated in mediating

progression to metastatic disease, including the transforming

growth factor beta (TGFβ) signalling pathway. In many tissues

TGFβ results in a growth inhibitory signal. This is mediated by

transducers of the Smad family, which translocate to the nucleus

and activate transcription. In tumour cells, however, TGFβ-

dependent antiproliferative control is lost and cells acquire the

ability to replicate in TGFβ-rich environments. Despite molecular

and clinical evidence pointing to a role for TGFβ signalling in

cancer progression and metastasis, 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 either GFPSmad2

or a Smad3 responsive promoter driving the expression of ECFP

(CAGA::ECFP). This allowed the monitoring of Smad-dependent

TGFβ signalling in vivo using multiphoton confocal microscopy.

Results Our results indicate that TGFβ signalling, measured by

cytoplasmic to nuclear translocation of GFPSmad2 and by

activation of CAGA ECFP, does not occur ubiquitously within the

primary tumour. In particular, cells that have acquired a motile

phenotype display active TGFβ signalling. As nuclear localisation

of GFPSmad2 and activation of CAGA ECFP are also observed in

nonmotile cells, however, TGFβ signalling may be necessary but

not sufficient to elicit cell motility in primary tumour cells.

Furthermore, activation of TGFβ signalling in motile cells is

transient, as lymph node metastasis display little activation of the

pathway. In addition, we have uncovered a second role for TGFβ

signalling in the metastatic process. After intravenous injection in

mouse tail vein, TGFβ pretreated cells colonise the lungs more

efficiently than untreated controls and this results from the ability of

these cells to survive clearance from the lungs.

Conclusion Together these data suggest that TGFβ signalling may

positively influence two distinct steps of the metastatic cascade,

first by enabling cells to become motile and second to enhance

their survival during the lung colonisation.

Acknowledgement Supported by Breast Cancer Campaign.

Development of breast cancer immunotherapy using

MUC1-retargeted T lymphocytes

S Wilkie

, G Picco

, J Foster

, D Davies

, S Julien

, L Cooper

S Arif

, S Mather

, J Taylor-Papadimitriou

, J Burchell

, J Maher

1,4

Breast Cancer Biology Group, King’s College London, Guy’s

Hospital, London, UK;

Centre for Cancer Imaging, Institute of

Cancer and the CR-UK Clinical Centre, Barts and The London,

Queen Mary’s School of Medicine and Dentistry, Department of

Nuclear Medicine, St Bartholomew’s Hospital, London, UK;

Department of Immunobiology, King’s College London, Guy’s

Hospital, London, UK;

Department of Allergy and Clinical

Immunology, King’s College Hospital, London, UK

Breast Cancer Res 2008, 10(Suppl 2):O6 (doi: 10.1186/bcr 1881)

Background MUC1 is a highly attractive target for immunotherapy

of breast cancer owing to its overexpression, altered glycosylation

and loss of polarity in over 90% of tumours. To exploit this, we are

developing genetic approaches to retarget T-cell specificity to

MUC1 using chimeric antigen receptor (CAR) technology.

Methods A panel of CARs have been generated using scFv

derived from the SM3 and HMFG2 hybridomas. Using the SFG

oncoretroviral expression vector, gene transfer was achieved in up

to 75% of human T cells.

Results Two parameters proved crucial in engineering an optimized

CAR ectodomain. First, we found that MUC1-mediated activation of

engineered human T cells is subject to steric hindrance. This was

observed using anchored but not soluble MUC1 and was

independent of MUC1 glycosylation status. To circumvent this, we

increased the flexibility and reach of CAR binding arms using the

elongated hinge found in IgD. Second, CAR function was highly

dependent upon strong binding capacity across a broad range of

tumour-associated MUC1 glycoforms, including MUC1 Tn, T and

sialylated derivatives. This was realized using an scFv cloned from

the HMFG2 hybridoma. To optimize CAR signalling, tripartite

endodomains were constructed that contain modules derived from

TNF receptor family members in addition to CD28 and CD3ζ.

Ultimately, this iterative design process yielded a potent MUC1-

specific CAR termed HOX that contains a fused CD28/OX40/CD3ζ

endodomain. HOX-expressing T cells proliferate vigorously in vitro

upon repeated encounter with soluble or membrane-associated

MUC1, mediate production of proinflammatory cytokines (IFNγ and

IL-17) and elicit brisk antigen-dependent killing of MUC1

tumour

cells. To test function in vivo, a human breast cancer xenograft

model has been established using MDA MB 435 tumour cells

engineered to coexpress MUC1 and firefly luciferase. When

introduced into SCID/Beige mice by intraperitoneal injection, rapid

tumour growth occurs that can be monitored longitudinally and

noninvasively by bioluminescent imaging. Mice bearing established

tumour have been treated intraperitoneally with a single dose of

human T cells grafted with HOX, two control CARs (DOX: lacking

the HMFG2 scFv; HDFTr: lacking a functional endodomain) or

medium alone. We observed that treatment with HOX-expressing T

cells resulted in a significant delay in tumour growth, as measured by

bioluminescent imaging, compared with control mice (Figure 1). In

addition, HOX-grafted T cells confer a significant survival advantage

upon mice bearing MCF7 breast cancer xenografts.

Conclusion Despite its role in tumorigenesis and immune evasion,

we show that the near-ubiquitous breast cancer antigen MUC1

can be targeted using CAR grafted T cells.

Breast Cancer Research May 2008 Vol 10 Suppl 2 Breast Cancer Research 2008

Figure 1 (abstract O6)

*P < 0.05, HOX versus DOX, HOX versus medium if present; **P < 0.05, HOX versus DOX, HOX versus medium, HOX versus HDFTr.

Acknowledgements Supported by a Health Foundation/Royal

College of Pathologists Senior Clinician Scientist Research Fellow-

ship and a Project Grant awarded by Breast Cancer Campaign.

POSTER PRESENTATIONS

Role of the Hsp90 cochaperone, FKBPL, in oestrogen

receptor signalling and breast cancer growth and survival

H McKeen, C Byrne, A Valentine, M O’Rourke, A Yakkundi,

K McClelland, K McAlpine, DG Hirst, T Robson

Molecular Therapeutics Group, School of Pharmacy, Queens

University Belfast, UK

Breast Cancer Res 2008, 10(Suppl 2):P1 (doi: 10.1186/bcr 1885)

Hsp90 chaperone complexes are involved in maintaining the

stability and signalling of Hsp90 client proteins such as the

oestrogen receptor (ER). The ER is the primary mediator of breast

cancer proliferation in response to oestrogen. Since increased ER

levels and transcriptional activation are associated with over 50%

of breast cancers, the ER is an attractive target for cancer

treatment strategies. Hsp90 inhibitors such as 17AAG are known

to destabilize these complexes by promoting proteasome-mediated

degradation of the steroid hormone receptor leading to tumour

growth inhibition [1] and sensitization to chemotherapy [2] and

radiotherapy [3]. Using protein interaction assays, we have

identified FKBPL, a novel gene that codes for an immunophilin-like

protein, as an Hsp90 cochaperone associated with the ER and

dynein motor protein complex. Overexpression studies have

demonstrated that FKBPL modulates ER signalling and affects

breast cancer growth and survival. Since most tumours become

refractory to current hormonal therapies within a year of starting

treatment, FKBPL represents a novel drug target that would enable

the disruption of signalling pathways integral in maintaining ER-

mediated tumour growth and survival.

Acknowledgements Funded by Breast Cancer Campaign, Action

Cancer and DEL.

References

1. Solit DB, Scher HI, Rosen N: Hsp90 as a therapeutic target

in prostate cancer. Semin Oncol 2003, 30:709-716.

2. Arlander SJ, Eapen AK, Vroman BT, McDonald RJ, Toft DO,

Karnitz LM: Hsp90 inhibition depletes Chk1 and sensitizes

tumor cells to replication stress. J Biol Chem 2003, 278:

52572-52577.

3. Bisht KS, Bradbury CM, Mattson D, Kaushal A, Sowers A,

Markovina S, et al.: Geldanamycin and 17-allylamino-17-

demethoxygeldanamycin potentiate the in vitro and in vivo

radiation response of cervical tumor cells via the heat

shock protein 90-mediated intracellular signaling and

cytotoxicity. Cancer Res 2003, 63:8984-8995

Two functionally distinct epithelial progenitors exist within

the luminal cell compartment of the mouse mammary gland

J Stingl

, CJ Eaves

1,2

, CJ Watson

Department of Pathology, University of Cambridge, Cambridge,

UK;

Terry Fox Laboratory, British Columbia Cancer Research

Centre, Vancouver, Canada

Breast Cancer Res 2008, 10(Suppl 2):P2 (doi: 10.1186/bcr 1886)

Background The organization of the mammary epithelial cell

hierarchy is poorly understood.

Methods To determine the cells that make up this hierarchy and

the relationship between them, we used fluorescence-activated

cell sorting in combination with in vitro colony-forming cell assays

to examine the growth and differentiative properties of pheno-

typically distinct subsets of mouse mammary epithelial cells.

Results Our results indicate that >95% of all colony-forming cells

present within the mammary epithelium are localized within the

luminal cell compartment and that >90% of these have a

CD45

–

Ter119

–

CD31

–

(Lin

–

)CD24

high

CD14

phenotype. This

progenitor cell population can be further resolved into two

functionally distinct subpopulations based on the expression of

Sca1. The Lin

–

CD24

high

CD14

Sca1

–

progenitors, which express

low levels of estrogen receptor alpha and intermediate levels of

keratin 14 (K14), are perceived to be progenitors that produce

Lin

–

CD24

high

CD14

–

Sca1

–

alveolar cells during pregnancy. The

Lin

–

CD24

high

CD14

Sca1

progenitors, which express intermediate

levels of estrogen receptor alpha and are K14

–

, are perceived to

be precursors of the steroid receptor expressing cells, of which the

vast majority are terminally differentiated and have a

Lin

–

CD24

high

CD14

–

Sca1

phenotype.

Conclusion These results demonstrate the existence of two

functionally distinct progenitor cells within the luminal compartment

of the mammary gland and provide a framework for interpreting

breast tumour gene expression profiles and the possible origins of

breast tumours.

Interactions between BRCA2 protein and the meiosis-

specific recombinase DMC1

T Thorslund, F Esashi, SC West

Cancer Research UK, London Research Institute, South Mimms, UK

Breast Cancer Res 2008, 10(Suppl 2):P3 (doi: 10.1186/bcr 1887)

Homologous recombination has a dual role in eukaryotic

organisms. Firstly, it is responsible for the creation of genetic

variability during meiosis by directing the formation of reciprocal

crossovers that result in random combinations of alleles and traits.

Secondly, in mitotic cells, it maintains the integrity of the genome

by promoting the faithful repair of DNA double-strand breaks. In

vertebrates it therefore plays a key role in tumour avoidance.

Mutations in the tumour suppressor protein BRCA2 are associated

with predisposition to breast and ovarian cancers, and loss of

BRCA2 function leads to genetic instability, as BRCA2 is required

for regulation of double-strand break repair by homologous

recombination. BRCA2 protein regulates recombinational repair by

interacting directly with RAD51 recombinase via a series of

degenerate BRC repeat motifs encoded by exon 11 (BRCA2996-

2113), and an unrelated C-terminal domain (BRCA23265-3330).

Recent observations show that BRCA2 is also required for

homologous recombination at meiosis. We show that human

BRCA2 binds directly to the meiosis-specific recombinase DMC1

and define the primary DMC1 interaction domain to a 26 amino

acid region located at BRCA22386-2411. This region is highly

conserved in BRCA2 proteins from a variety of mammalian

species, but is absent in BRCA2 from Arabidopsis thaliana,

Caenorhabditis elegans, and other lower eukaryotes. Within this

region, we demonstrate the critical importance of Phe2406,

Pro2408, and Pro2409 at the conserved motif 2404KVFVPPFK2411,

and define this novel DMC1 interaction domain the PhePP motif.

The PhePP motif promotes specific interactions between BRCA2

and DMC1, and no interactions take place between this region of

BRCA2 and RAD51. Thus, the RAD51 and DMC1 interaction

domains on BRCA2 are distinct from each other, allowing

coordinated interactions of the two recombinases with BRCA2 at

Available online http://breast-cancer-research.com/supplements/10/S2

meiosis. These results lead us to suggest that BRCA2 is a

universal regulator of RAD51/DMC1 recombinase actions.

Lineage commitment in mammary epithelium is regulated

by type 2 cytokines and Stat6

WT Khaled

, SE Nicholson

, FO Baxter

, N Sprigg

, JP Stingl

ANJ McKenzie

, CJ Watson

Department of Pathology, University of Cambridge, UK;

Division

of Cancer and Haematology, The Walter and Eliza Hall Institute of

Medical Research, Parkville, Victoria, Australia;

Medical Research

Council Laboratory of Molecular Biology, Cambridge, UK

Breast Cancer Res 2008, 10(Suppl 2):P4 (doi: 10.1186/bcr 1888)

Naïve T-helper cells differentiate into Th1 and Th2 subsets that

have unique cytokine signatures, activators, and transcriptional

targets. The Th1/Th2 cytokine milieu is a key paradigm in T-cell

lineage commitment and IL-4/IL-13 and Stat6 are known to be

important mediators of Th2 development. We have now

demonstrated that this paradigm applies also to mammary

epithelial cells, which undergo a switch from Th1 to Th2 cytokine

production upon the induction of differentiation. Thus, the Th1

cytokines IL-12, IFNγ, and TNFα are downregulated concomitantly

with the upregulation of the Th2 cytokines IL-4, IL-13 and IL-5 as

epithelial cells commit to the luminal alveolar lineage. Moreover, we

show that Th2 cytokines play a crucial role in mammary gland

development in vivo, because differentiation and alveolar morpho-

genesis are reduced in both Stat6 and IL-4/IL-13 doubly deficient

mice during pregnancy. This unexpected discovery demonstrates a

role for immune cell cytokines in epithelial cell fate and function,

and adds an unexpected tier of complexity to the previously held

paradigm that steroid and peptide hormones are the primary

regulators of mammary gland development.

Acknowledgements Supported by the BBSRC, Breast Cancer

Campaign, and the National Health and Medical Research Council,

Australia (Program grant #257500).

Topoisomerase II expression as a determinant of

chromosomal radiosensitivity and possible susceptibility in

breast cancer

PE Bryant

, AC Riches

, S Terry

, O Shovman

, D Adamson

Bute Medical School, University of St Andrews, UK;

Ninewells

Teaching Hospital, University of Dundee, UK

Breast Cancer Res 2008, 10(Suppl 2):P5 (doi: 10.1186/bcr 1889)

Background Elevated chromosomal radiosensitivity in lymphocytes

of breast cancer patients is thought to be an indicator for the

presence of one or more as yet unidentified genes of low

penetrance that promote susceptibility to the disease in up to 60%

of cases [1,2]. One such gene may be TOPO2A, encoding for the

DNA processing enzyme topoisomerase IIa. The involvement of

topoisomerase IIa is predicted from the author’s model for formation

of chromatid breaks [3]. In the model the DNA double-strand break

is not directly involved in the chromatid break, but acts as an initiator

in a sequence of events leading to a chromatid break. It is thought

that a chromatid break may be formed by a misjoining of chromatin

ends during topoisomerase IIa decatenation of chromatids as the

cell progresses through G

towards mitosis. Topoisomerase IIa is

known to be vulnerable to perturbation by oxidative stress during

the precise process of cutting and joining DNA strands [4].

Methods Gamma-radiation-induced chromatid breaks are scored

for chromatid breaks in colcemid-blocked chromosome spreads of

metaphase HL60 and mitoxantrone-resistant variants: MX1 and

MX2 cells with reduced topoisomerase II expression. Topoiso-

merase IIa expression levels were measured using western

blotting. SiRNA was used to knock down expression in normal

exponentially growing human cells that are irradiated with a low

dose of γ-rays and scored for the presence of chromatid breaks.

The chromatid break frequency and topoisomerase IIa expression

(ELISA assay) are being compared in 3-day-stimulated peripheral

blood T lymphocytes from a group of breast cancer patients and

control individuals exposed to a low dose of γ-radiation.

Results We show that chromatid radiosensitivity (based on the

frequency of metaphase chromatid breaks in irradiated G

cells) is

significantly lower in a topoisomerase IIa underexpressing variant

cell lines [5], and preliminary results show that reducing expression

with SiRNA also reduces chromatid radiosensitivity. In a pilot study

we are currently comparing the chromatid radiosensitivity and

topoisomerase IIa expression in stimulated peripheral blood

lymphocytes of a group of Tayside breast cancer patients and a

similar number of normal noncancer control individuals.

Conclusion Our data support the hypothesis that topoisomerase

IIa expression is a determinant of chromatid break frequency in

irradiated G

cells, and thus could be an underlying cause of the

observed variability of chromatid radiosensitivity among both

sporadic breast cancer cases and normal control individuals.

Acknowledgements Supported by the CSO and Breast Cancer

Campaign.

References

1. Scott D, Barber JBP, Spreadborough AR, Burrill W, Roberts

SA: Increased radiosensitivity in breast cancer patients: a

comparison of two assays. Int J Radiat Biol 1999, 75:1-10.

2. Riches AC, Bryant PE, Steel CM, Gleig A, Robertson AJ,

Preece PE, Thompson AM: Chromosomal radiosensitivity in

-phase lymphocytes identifies breast cancer patients

with distinctive tumour characteristics. Br J Cancer 2001,

85:1157-1161.

3. Bryant PE: Repair and chromosomal damage. Radiother

Oncol 2004, 72:251-256.

4. Chen LT-K, Yu C, Mao Y, Wang H, Liu LF: Activation of

topoisomerase II mediated excision of chromosomal DNA

loops during oxidative stress. Genes Dev 1999, 13:1553-

1560.

5. Terry S, Riches AC, Bryant PE: A role for topoisomerase IIa

in the formation of radiation-induced chromatid breaks. Br

J Cancer 2008, submitted.

Chromosome translocations in breast cancer

K Howarth

, K Blood

, B Ng

, J Beavis

, Y Chua

, S Cooke

J Pole

, S Chin

, K Ichimura

, VP Collins

, I Ellis

, C Caldas

N Carter

, PAW Edwards

Department of Pathology, Hutchison/MRC Research Centre,

University of Cambridge, UK;

The Wellcome Trust Sanger

Institute, Cambridge, UK;

Cancer Research UK Cambridge

Research Institute, Cambridge, UK;

Division of Molecular

Histopathology, Department of Pathology, University of Cambridge,

UK;

Department of Histopathology, School of Molecular Medical

Sciences, University of Nottingham, UK

Breast Cancer Res 2008, 10(Suppl 2):P6 (doi: 10.1186/bcr 1890)

Background Genome rearrangement is a major mechanism of

gene alteration in cancer. Chromosome translocations and

inversions can result in gene fusion, promoter insertion or gene

inactivation. In the past it has been assumed that such rearrange-

ments are not significant players in the common epithelial cancers,

Breast Cancer Research May 2008 Vol 10 Suppl 2 Breast Cancer Research 2008

as they are in leukaemias and sarcomas. However, this view is now

being challenged. In particular, Tomlins and colleagues found that

around 70% of prostate cancers have translocations or inversions

of the ETS family of transcription factors [1]. In breast cancer, we

have shown that the NRG1/heregulin gene is translocated in 6%

of primary cases [2] and Soda and colleagues described fusions of

ALK in 7% of lung cancers [3].

Methods and results We present a comprehensive analysis by

array painting of the chromosome translocations of breast cancer

cell lines HCC1806, HCC1187 and ZR-75-30. In array painting,

chromosomes are isolated by flow cytometry, amplified and

hybridized to DNA microarrays [4]. A total of 200 breakpoints were

identified and all were mapped to 1 Mb resolution on BAC arrays,

then 40 selected breakpoints, including all balanced breakpoints,

were further mapped on tiling-path BAC arrays or to around 2 kb

resolution using oligonucleotide arrays. Many more of the

translocations were balanced than expected, either reciprocal

(eight in total) or balanced for at least one participating

chromosome (19 paired breakpoints). Many breakpoints were at

genes that are plausible targets of oncogenic translocation,

including CTCF and P300. Two gene fusions were also demon-

strated, TAX1BP1-AHCY and RIF1-PKD1L1.

Conclusion Our data establish that array painting is a very

effective way to map substantial numbers of translocation

breakpoints and support the emerging view that chromosome

rearrangements that fuse, activate or otherwise alter genes at their

breakpoints may play an important role in common epithelial

cancers.

References

1. Tomlins SA, et al.: Recurrent fusion of TMPRSS2 and ETS

transcription factor genes in prostate cancer. Science

2005, 310:664-668.

2. Huang HE, et al.: A recurrent chromosome breakpoint in

breast cancer at the NRG1/neuregulin 1/heregulin gene.

Cancer Res 2004, 64:6840-6844.

3. Soda M, et al.: Identification of the transforming EML4-ALK

fusion gene in non-small-cell lung cancer. Nature 2007,

448:561-566.

4. Fiegler H, et al.: Array painting: a method for the rapid

analysis of aberrant chromosomes using DNA microar-

rays. J Med Genet 2003, 40:664-670.

ZNF366 is a novel corepressor for estrogen receptor alpha

that mediates its effects through interaction with CtBP

S Ali

, M Periyasamy

, J Lopez-Garcia

, RS Thomas

M Christian

, MG Parker

, L Buluwela

Department of Oncology, Imperial College London, UK;

Institute

of Reproductive and Developmental Biology, Imperial College

London, UK

Breast Cancer Res 2008, 10(Suppl 2):P7 (doi: 10.1186/bcr 1891)

Regulation of gene expression by the estrogen receptor (ER)

requires the coordinated recruitment and dissociation of trans-

criptional coactivator complexes and concomitant chromatin

remodelling and histone modification. In addition to the well-

characterised recruitment of coactivator proteins, a number of

corepressor proteins can also be recruited to the liganded ER,

including RIP140 and L-CoR.

We have recently identified a new ER interacting protein, ZNF366,

which is recruited to the liganded ER, through interactions

involving the zinc finger domains of both proteins. We show that

repression of ER-regulated genes by ZNF366 involves recruitment

of the well-described corepressor CtBP. This interaction is

mediated by two sequence motifs in ZNF366, conforming to the

consensus CtBP-binding motif (PXDLS). Mutation of these motifs

in ZNF366 reduces, but does not abolish, the corepressor activity

of ZNF366. Additionally, ZNF366 interacts with RIP140, raising

the possibility that RIP140 and ZNF366 may act synergistically in

regulating ER activity [1].

Finally, we show that although ZNF366 is expressed in normal

breast epithelial cells, its expression is not detected in breast

cancer cells. This raises the possibility that regulation of ER activity

by ZNF366 may be important in breast cancer development.

Reference

1. Lopez-Garcia J, Periyasamy M, Thomas RS, Christian M, Leao

M, Jat P, Kindle KB, Heery DM, Parker MG, Buluwela L,

Kamalati T, Ali S: ZNF366 is an estrogen receptor corepres-

sor that acts through CtBP and histone deacetylases.

Nucleic Acids Res 2006, 34:6126-6136.

A novel role for C-terminal binding proteins in the

regulation of mitotic fidelity in breast cancer cells

LM Bergman

, CN Birts

, M Darley

, B Gabrielli

, JP Blaydes

Somers Cancer Research Building, University of Southampton,

MP824 Southampton General Hospital, Southampton, UK;

Centre

for Immunology and Cancer Research, University of Queensland, R

Wing Princess Alexandra Hospital, Brisbane, Queensland, Australia

Breast Cancer Res 2008, 10(Suppl 2):P8 (doi: 10.1186/bcr 1892)

C-terminal binding proteins (CtBPs) (CtBP1 and CtBP2) are dual-

function proteins that act in the nucleus as transcriptional co-

repressors and in the cytoplasm as regulators of mitotic Golgi

fissioning. They have been implicated in the process of cellular

transformation through their physical and functional interactions

with the viral oncoproteins adenovirus E1A, and EBNA3C. Studies

in which the expression or function of CtBPs has been suppressed

in mammalian cells have independently identified both a role in

suppressing apoptosis, through their regulation of transcription of

proapoptotic genes, and a requirement for cell-cycle progression,

dependent on their role in the Golgi. Here we have undertaken a

holistic analysis of the phenotypic consequences of ablating CtBP

expression in breast cancer-derived cell lines. We find that loss of

CtBPs suppresses the proliferation of these lines through a

combination of induction of 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, diminished localisation of spindle checkpoint

proteins at kinetochores, and a high rate of failure to complete

cytokinesis. These represent novel roles for CtBPs in the regulation

of critical stages of the cell division cycle.

Available online http://breast-cancer-research.com/supplements/10/S2

RASSF2 can suppress the growth of breast cancer cell

lines and is epigenetically inactivated in breast tumours

WN Cooper

, RE Dickinson

, A Dallol

, LB Hesson

, I Bieche

GJ Clark

, ER Maher

, ER Zabarovsky

, F Latif

Department of Medical and Molecular Genetics, Institute of

Biomedical Research, University of Birmingham, Edgbaston,

Birmingham, UK;

Laboratoire d’Oncogenetique-INSERM E0017,

Centre Rene Huguenin, St-Cloud, France;

JG Brown Cancer

Center, Department of Medicine, Molecular Targets Group,

University of Louisville, KY, USA;

MTC, Karolinska Institute,

Stockholm, Sweden

Breast Cancer Res 2008, 10(Suppl 2):P9 (doi: 10.1186/bcr 1893)

Background RASSF2 is located at 20p13, a region frequently lost

in human cancers. RASSF2 is a recently identified member of the

ras association domain of family tumour suppressor genes, and

many other members of this family are inactivated in human

tumours by promoter methylation.

Methods Methylation-specific PCR and combined bisulphite and

restriction analysis were used to analyse the methylation status of

the RASSF2 promoter CpG island in a series of breast tumours

and cell lines. Bioinformatic approaches were used to study

RASSF2 and a highly conserved putative bipartite nuclear localisa-

tion signal (NLS) was identified. Colony formation, growth in soft

agar and growth in immunocompromised mouse assays were used

to assess the tumour suppressive activities of RASSF2.

Results RASSF2 was frequently methylated in breast tumour cell

lines, 65% (13/20), and in primary breast tumours, 38% (15/40).

In the 10 samples for which corresponding normal DNA was

available this methylation was tumour specific. RASSF2 expression

could be switched back on in methylated breast tumour cell lines

after treatment with 5-aza-2dC. Endogenous RASSF2 localised to

the nucleus and mutation of the putative nuclear localisation signal

abolished the nuclear localisation. RASSF2 suppressed breast

tumour cell growth in vitro and in vivo, while the ability of NLS-

mutant RASSF2 to suppress growth was much diminished.

Conclusion These data indicate that RASSF2 is frequently

methylated in breast tumours, and thus RASSF2 is a novel

methylation marker that has the potential to be developed into a

valuable epigenetic marker for screening. We also demonstrate

that RASSF2 acts as a tumour suppressor gene and that it

contains a functional NLS that is important for its tumour

suppressor gene function.

Acknowledgement Supported by Breast Cancer Campaign.

P10

Role of poly(ADPribosyl)ation of CTCF in cancer and

normal breast cells

F Docquier, G Kita, D Farrar, I Chernukhin, E Klenova

Department of Biological Sciences, University of Essex, Colchester,

Breast Cancer Res 2008, 10(Suppl 2):P10 (doi: 10.1186/bcr 1894)

Background CTCF is a conserved, ubiquitous and multifunctional

transcription factor with features of a tumour suppressor. We have

previously reported that CTCF function is modulated by post-

translational poly(ADPribosyl)ation [1,2]. Poly(ADPribosyl)ation of

CTCF protein results in two isoforms: a highly poly(ADPribosyl)ated

form (called CTCF180) and a hypopoly(ADPribosyl)ated form

(called CTCF130). In this study we assessed the presence of both

CTCF isoforms in normal and cancer breast tissues and

investigated their function using immortalised cell lines.

Methods CTCF expression was analysed in breast tissues and

breast cancer cell lines by western blotting, immunohistochemistry

and immunofluorescence, using antibodies that specifically

recognise different CTCF isoforms. Functional investigations of

CTCF isoforms in cell culture included induction of apoptosis,

senescence and cell-cycle arrest using various chemical

treatments and analysis of cells by flow cytometry.

Results We discovered, using a large panel of breast tumours and

paired peripheral tissues, that only the CTCF180 isoform was

present in normal breast tissues, whereas CTCF130 was

exclusively detected in breast tumour tissues and immortalised cell

lines. Immunohistochemical staining revealed that 91% of the

breast tumours contained CTCF130. In addition, correlations were

found between the levels of CTCF130 and tumour grade, lymph

node metastases and neoadjuvant chemotherapy treatment. In

breast cancer cell lines, induction of cell death by apoptosis and

senescence resulted in a transition from the CTCF130 to the

CTCF180 isoform. This shift was not observed following cell-cycle

arrest.

Conclusion The present study demonstrates that CTCF180 is

characteristic for normal breast tissues, whereas CTCF130 is

specific for breast tumours and breast cancer cell lines. The

CTCF130 isoform may therefore be used as a specific biological

marker for breast tumourigenesis. Our data indicate that loss of

CTCF poly(ADPribosyl)ation may be involved in breast tumour

development. Poly(ADPribosyl)ation of CTCF, on the other hand,

correlates with induction of cell death in breast cancer cell lines.

Acknowledgements Supported by Breast Cancer Campaign, the

Medical Research Council, and the University of Essex.

References

1. Klenova E, Ohlsson R: Poly(ADPribosyl)ation and epigenet-

ics: is CTCF PARt of the plot? Cell Cycle 2005, 4:96-101.

2. Yu W, Ginjala V, Pant V, Chernukhin I, Whitehead J, Docquier

F, Farrar D, Tavoosidana R, Mukhopadhyay R, Kanduri C,

Oshimura M, Feinberg AP, Lobanenkov V, Klenova E, Ohlsson

R: Poly(ADPribosyl)ation regulates CTCF dependent chro-

matin insulation. Nat Genet 2004, 36:1105-1110.

P11

NRG1 is frequently silenced by methylation in breast

cancers and is a strong candidate for the 8p tumour

suppressor gene

Y-L Chua

, Y Ito

, JCM Pole

, S-F Chin

, IO Ellis

, C Caldas

MJ O’Hare

, AM Murrell

, PAW Edwards

Department of Pathology, University of Cambridge, Hutchison/

MRC Research Centre, Cambridge, UK;

CR-UK Cambridge

Research Institute, Cambridge, UK;

Department of Pathology,

University of Nottingham, UK;

Ludwig Institute for Cancer

Research Breast Cancer Laboratory, University College, London, UK

Breast Cancer Res 2008, 10(Suppl 2):P11 (doi: 10.1186/bcr 1895)

Background It has long been suspected that there is an important

breast cancer tumour suppressor gene on the short arm of

chromosome 8, 8p, and our array CGH data suggest that it may be

close to NRG1 [1]. NRG1 encodes growth factors that bind to

tyrosine kinases ErbB3 and ErbB4, and can both stimulate cell

proliferation and apoptosis. NRG1 is also quite frequently broken

by chromosome translocations [2].

Methods and results By quantitiative PCR, NRG1 expression was

repressed or abolished in many breast cancer cell lines and

tumours as compared with normal breast. Methylation analysis by

sequencing or pyrosequencing bisulphite-treated DNA showed

striking DNA methylation at a CpG island in NRG1, which is

correlated with an absence of NRG1 transcripts. Treatment of

Breast Cancer Research May 2008 Vol 10 Suppl 2 Breast Cancer Research 2008

cancer cell lines with 5-aza-2-deoxycytidine reactivated the

expression of NRG1 by 7 to 100 times. NRG1 was also methy-

lated in tumour tissue samples while it was not in uncultured

normal breast epithelium. Knocking down NRG1 expression by

siRNA led to an increase in net cell proliferation.

Conclusion NRG1 could be the 8p tumour suppressor gene. It is

located in the right place. It is silenced by methylation or other

mechanisms in many breast cancer cell lines and tumours.

Functionally, NRG1 expression is antiproliferative – shown both by

our siRNA experiments and older work that showed expression to

be proapoptotic to breast cancer cell line MCF7 [3].

Acknowledgements Supported by Breast Cancer Campaign and

also Cancer Research UK and the Ludwig Institute for Cancer

Research.

References

1. Pole JC, Courtay-Cahen C, Garcia MJ, Blood KA, Cooke SL,

Alsop AE, Tse DM, Caldas C, Edwards PA: High-resolution

analysis of chromosome rearrangements on 8p in breast,

colon and pancreatic cancer reveals a complex pattern of

loss, gain and translocation. Oncogene 2006, 25:5693-5706.

2. Huang HE, Chin SF, Ginestier C, Bardou VJ, Adelaide J, Iyer

NG, Garcia MJ, Pole JC, Callagy GM, Hewitt SM, Gullick WJ,

Jacquemier J, Caldas C, Chaffanet M, Birnbaum D, Edwards

PA: A recurrent chromosome breakpoint in breast cancer

at the NRG1/neuregulin 1/heregulin gene. Cancer Res

2004, 64:6840-6844.

3. Grimm S, Weinstein EJ, Krane IM, Leder P: Neu differentia-

tion factor (NDF), a dominant oncogene, causes apoptosis

in vitro and in vivo. J Exp Med 1998, 188:1535-1539.

P12

p53

ββ

isoform modulates differentially p53 transcriptional

activity in response to stress

K Fernandes, JC Bourdon

Department of Surgery & Molecular Oncology, Inserm European

Associated Laboratory, University of Dundee, Inserm U858,

Dundee, UK

Breast Cancer Res 2008, 10(Suppl 2):P12 (doi: 10.1186/bcr 1896)

We recently established that the p53 gene expresses nine

different p53 protein isoforms. The p53 isoforms bind preferentially

to some p53-responsive promoters and modulate differentially p53

transcriptional activity [1]. We characterized further p53β activity.

p53β is differentially recruited to p21 and bax promoters in the

absence or in the presence of DNA-damaging drugs.

p53β enhances p53 transcriptional activity on the p21 promoter in

a dose-dependent manner in the absence of cellular stress but

inhibits p53 transcriptional activity on the p21 promoter in the

presence of DNA-damaging agents. On the contrary, p53β has no

effect on p53 transcriptional activity on the bax promoter in the

absence of stress but enhances p53 transcriptional activity on the

bax promoter in response to stress without increasing the p53

protein level.

Our data indicate that p53β is involved in the choice of p53 target

gene expression in response to cellular signals, switching cell fate

outcome from G

arrest/DNA repair to cell death.

The present finding supports our hypothesis that differential

expression of the p53 isoforms in primary breast tumours may help

to link p53 status to biological properties and drug sensitivity.

Reference

1. Bourdon JC, Fernandes K, Murray-Zmijewski F, Liu G, Diot A,

Xirodimas DP, Saville MK, Lane DP: p53 isoforms can regu-

late p53 transcriptional activity. Genes Dev 2005, 19:2122-

2137.

P13

Inhibitor of apoptosis proteins as a therapeutic target in

breast cancer

FM Foster, NJ Bundred, CH Streuli

Faculty of Life Sciences, University of Manchester, UK

Breast Cancer Res 2008, 10(Suppl 2):P13 (doi: 10.1186/bcr 1897)

Background Apoptosis is the process of programmed cell death

by which damaged or unhealthy cells are normally destroyed.

Cancer cells are able to avoid apoptosis and thereby survive

inappropriately. Inhibitor of apoptosis proteins (IAPs) are a family of

proteins that block apoptosis in normal cells, by binding to active

caspases, the proteases that mediate cell death. There are eight

human IAPs, including NAIP, XIAP, cIAP1, cIAP2, livin, survivin and

apollon. An upregulation of IAPs could cause resistance to

apoptosis. Targeting IAPs in cancer therapy may therefore improve

the clinical effectiveness of apoptosis-inducing chemotherapeutics.

A number of studies have shown that XIAP and survivin are up-

regulated in cancer, and inhibiting these IAPs increased the

apoptotic response induced by some chemotherapeutics. We aim,

first, to examine the expression profile of all IAPs in breast cancer

and, second, to determine whether inhibiting IAPs will enhance the

apoptotic response to traditional chemotherapeutics and newly

developed targeted therapies, such as Herceptin.

Methods IAP levels were detected in patient and cell line samples

by immunoblotting with validated antibodies using the Li-Cor

Odyssey system (Li-Cor Biosciences, Lincoln, NE, USA). IAPs

were inhibited using siRNA or cell-permeable mimics of endo-

genous inhibitors. Control cells and cells with XIAP knocked down

or inhibited were exposed to TNF-related apoptosis inducing

ligand (10 ng/ml), Herceptin (100 μg/ml), Iressa (10 μM), or

Lapatinib (100 nM) for 48 hours. Apoptosis was scored by examin-

ing nuclear morphology (DAPI) or active caspase 3 staining.

Proliferation was examined by Ki67 staining.

Results We have found that IAPs are widely upregulated in breast

cancer. In particular cIAP2, XIAP and survivin were more prevalent

in breast cancer cells than normal breast epithelium. Knock down

of XIAP or inhibition with small molecule inhibitors resulted in an

increased apoptotic response to TNF-related apoptosis inducing

ligand, in both sensitive and resistant cell lines. Knocking down

XIAP also increased the apoptotic response to a number of growth

factor receptor-targeted therapies such as Herceptin, Iressa and

Lapatinib.

Conclusion Inhibiting IAPs in combination with both chemo-

therapeutic agents and targeted therapies, such as Herceptin and

Lapatinib, which act as receptor antagonists, will improve clinical

outcome.

P14

Cellular localization of the proto-oncogenic p53 inhibitor

AGR2 protein in breast cancer

A Fourtouna, T Hupp

University of Edinburgh, Western General Hospital, Cell Signalling

Unit, Edinburgh, UK

Breast Cancer Res 2008, 10(Suppl 2):P14 (doi: 10.1186/bcr 1898)

Background Proteomic technologies verified AGR-2 as a protein

family overexpressed in human cancers, including breast, prostate

and oesophagus cancers, with the ability to inhibit the tumour

suppressor protein p53 [1]. The AGR-2 gene is a hormone

responsive gene with an unexpected induction by the anticancer

drug tamoxifen highlighting the proto-oncogenic role of this

protein. The hAGR-2 gene was first described in the MCF-7 breast

Available online http://breast-cancer-research.com/supplements/10/S2

S10

carcinoma cell line, and was found to be coexpressed with the

estrogen receptor (ER), in ER-positive cell lines [2,3]. Moreover, it

was recently revealed that AGR-2 is secreted from androgen-

inducible cell lines in prostate cancer cell lines [4].

Methods Localization studies of AGR-2 were performed using

fluorescence microscopy in order to determine in which

compartment the protein functions. Yeast two-hybrid analysis has

identified potential nuclear and cytoplasmic binding proteins for

AGR-2, essential for the upstream or downstream regulation of the

AGR-2 pathway.

Results Anterior gradient 2 encodes one protein that gives rise to

two forms: the full-length and the mature. Full-length AGR2wt,

which bears the leader sequence, localizes to the ER and the

Golgi compartment whereas the mature protein requires the C-

terminal KTEL sequence for strong nuclear localization. Deletion of

the KTEL, putative ER retention, sequence does not alter the

localization of the wt full-length form to a large extent but has a

strong effect on the localization shift of the mature form.

Subcellular fractionation data verified the difference in the

localization patterns of the wt forms and their mutants. Moreover,

the localization of the protein and each of the mutants differs

significantly in various cell lines, suggesting a multipotent role of

the protein when it comes to activation pathways and localization

patterns within the cell. Furthermore, we present data showing

models of how the AGR-2 family might function as a drug-

resistance survival factor in cancer as well as a p53 inhibitor.

Conclusion All of the above suggest a multipotent role of AGR-2

when it comes to trafficking, cellular localization and activation or

inhibition pathways in cancer. The localization of the protein can

therefore determine the level of p53 inhibition.

Acknowledgement Breast Cancer Campaign funded this project.

References

1. Pohler E, Craig AL, Cotton J, Lawrie L, Dillon J, Ross P, Ker-

nohan N, Hupp T: The Barrett’s antigen anterior gradient-2

silences the p53 transcriptional response to DNA damage.

Mol Cell Proteomics 2004, 3:534-547.

2. Thompson DA, Weigel RJ: hAG-2, the human homologue of

the Xenopus laevis cement gland gene XAG-2, is coex-

pressed with estrogen receptor in breast cancer cell lines.

Biochem Biophys Res Commun 1998, 251:111-116.

3. Kuang WW, Thompson DA, Hoch RV, Weigel RJ: Differential

screening and suppression subtractive hybridization iden-

tified genes differentially expressed in an estrogen recep-

tor-positive breast carcinoma cell line. Nucleic Acids Res

1998, 26:1116-1123.

4. Zhang J, Gong A, Cheville J, Smith D, Young C: AGR-2, an

androgen-inducible secretory protein overexpressed in

prostate cancer. Genes Chromosomes Cancer 2005, 43:

249-259.

P15

Investigating h-Prune activation of Wnt signalling in breast

cancer

J Freeman

, M Zollo

, T Dale

Cardiff School of Biosciences, Cardiff University, Cardiff, UK;

CEINGE, Biotecnologie Avanzate Scarl, Naples, Italy

Breast Cancer Res 2008, 10(Suppl 2):P15 (doi: 10.1186/bcr 1899)

We have been investigating a novel link between two independent

processes linked to breast cancer: Wnt signalling and h-Prune

overexpression. The canonical Wnt signalling pathway was

activated in 40% to 60% of human breast cancers through

mechanisms that are not understood. Similarly, the phospho-

diesterase h-Prune was overexpressed or amplified in 54% of

breast cancers and was linked to breast tumour progression

through unknown mechanisms.

We have shown that overexpression of xenopus Prune induced

formation of a secondary axis in a standard assay to identify

activators of the Wnt signalling pathway. In HEK293 cells, xenopus

Prune overexpression induced a 300-fold increase in Wnt/TCF-

dependent transcription. Whilst human prune does not appear to

be able to activate Wnt signalling as potently as its xenopus

homologue, it does synergise with other activators of the pathway

to increase TCF-dependent transcription.

Here we show whether there is a correlation between over-

expression of h-Prune and active Wnt signalling in breast cancer,

and whether the synergistic responses described are mediated

through the enzymatic activity of prune, or through binding to GSK-3.

P16

Coactivation of estrogen receptor alpha by the DEAD-box

RNA helicases p68 and p72 and its role in breast cancer

FV Fuller-Pace

, E Ahamed

, NC Wortham

, S Ali

Division of Pathology and Neuroscience, University of Dundee,

Ninewells Medical School, Dundee, UK;

Department of Oncology,

SORA, Faculty of Medicine, Imperial College London, UK

Breast Cancer Res 2008, 10(Suppl 2):P16 (doi: 10.1186/bcr 1900)

We have previously demonstrated that the DEAD-box RNA

helicase p68 is an important regulator of gene expression [1,2],

whilst other groups have shown that p68 interacts with and

coactivates estrogen receptor alpha (ERα) [3,4]. The main focus of

our project is to investigate the molecular mechanism of ERα

coactivation by p68 and to examine the potential consequences for

breast cancer development.

We have established that the interaction of p68 and ERα requires

the DNA binding domain of ERα and the C terminus of p68.

Importantly, this region of p68 lies outside the conserved helicase

core and was previously shown by us to be essential for

transcriptional regulation by p68. Additionally, coactivation of ERα

by p68 requires the ligand binding/AF2 region of ERα and is

consistent with the model that p68 is recruited to ERα-responsive

promoters in response to estrogen [4]. We have also shown that

p72, a helicase that is very highly related to p68 and that had

previously been suggested to act in an analogous fashion to p68

[3], poorly coactivates ERα in standard transcriptional coactivation

assays, using ER-responsive promoters. This is underscored by our

finding that overexpression of p68, but not of p72, in cell lines

results in stimulation of expression of physiological target genes of

ERα.

Interestingly, siRNA-mediated knockdown of endogenous p68 has

little effect on the expression of ERα target genes. This observation

is consistent with the idea that p68 has little effect on ERα function

physiologically, but that the elevated p68 levels found in tumours

may stimulate ERα-mediated gene expression in a pathological

context. Strikingly, however, in contrast to p68, knockdown of

endogenous p72 results in a marked inhibition of both baseline

and estrogen stimulated-expression of these genes. These findings

suggest, firstly, that p72 is important physiologically for ERα

activity in the cell and, secondly, that p68 and p72 may be acting

in an opposing rather than analogous fashion (as had been

previously suggested [3]). Moreover, our preliminary data suggest

that overexpression of p68 in cells may additionally coactivate ERα

in an estrogen-independent manner, a finding that may have implica-

tions in the development of resistance to endocrine therapies.

We are currently developing inducible p68/p72 overexpression

and siRNA cell lines with a view to examining the effect of

augmenting or suppressing p68/p72 expression in mouse

Breast Cancer Research May 2008 Vol 10 Suppl 2 Breast Cancer Research 2008

S11

xenograft models. Additionally we are screening a large panel of

breast cancers to examine p68 and p72 expression/localisation in

the context of ERα expression.

References

1. Bates GJ, Nicol SM, Wilson BJ, Jacobs AM, Bourdon JC,

Wardrop J, Gregory DJ, Lane DP, Perkins ND, Fuller-Pace FV:

The DEAD box protein p68: a novel transcriptional coacti-

vator of the p53 tumour suppressor. EMBO J 2005, 24:

543-553.

2. Wilson BJ, Bates GJ, Nicol SM, Gregory DJ, Perkins ND,

Fuller-Pace FV: The p68 and p72 DEAD box RNA helicases

interact with HDAC1 and repress transcription in a pro-

moter-specific manner. BMC Mol Biol 2004, 5:11.

3. Watanabe M, Yanagisawa J, Kitagawa H, Takeyama K, Ogawa

S, Arao Y, Suzawa M, Kobayashi Y, Yano T, Yoshikawa H,

Masuhiro Y, Kato S: A subfamily of RNA-binding DEAD-box

proteins acts as an estrogen receptor alpha coactivator

through the N-terminal activation domain (AF-1) with an

RNA coactivator, SRA. EMBO J 2001, 20:1341-1352.

4. Métivier R, Penot G, Hübner MR, Reid G, Brand H, Kos M,

Gannon F: Estrogen receptor-alpha directs ordered, cycli-

cal, and combinatorial recruitment of cofactors on a

natural target promoter. Cell 2003, 115:751-763.

P17

TSC22 in mammary gland development and breast cancer

C Huser

, V Heath

, M-A Pringle

, AK Bell

, D Crighton

K Ryan

, G Inman

, T Stein

, B Gusterson

Division of Cancer Sciences and Molecular Pathology, University

of Glasgow, UK;

Division of Cancer Sciences and Molecular

Pathology, CRUK Beatson Laboratories, Glasgow, UK

Breast Cancer Res 2008, 10(Suppl 2):P17 (doi: 10.1186/bcr 1901)

Mammary gland involution is characterised by a high degree of

apoptosis. By identifying genes that are upregulated at this

developmental stage, we aimed to discover key factors that are

involved in the induction of mammary epithelial cell death and

therefore present potential tumour suppressors for breast cancer.

Among 96 genes recently identified as specifically upregulated

early during involution were the transforming growth factor beta

(TGFβ)-stimulated clone 22 homologue (TSC-22/TGFβ

-induced

transcript 4) and TGFβ

[1]. TGFβ

has recently been shown to be

necessary for induction of apoptosis during mammary gland

involution, while TSC-22 overexpression can lead to cell death. We

have therefore tested whether TSC-22 mRNA expression can be

induced by TGFβ

and whether it is involved in or necessary for

TGFβ-induced apoptosis. We further show that TSC-22 can

enhance TGFβ

-induced Smad response and epithelial cell death.

In addition, overexpression of TSC-22 alone can induce a Smad

response and apoptosis in mammary epithelial cell cultures, which

is independent of p53. Further, we have performed tests to study

the necessity for Smad proteins during TSC-22-induced

apoptosis, and to establish the intracellular localisation of TSC-22.

A pilot study on a small cohort of archival breast cancer cases,

representing all stages of malignant progression, shows that TSC-

22 protein was reduced or undetectable in 60% of breast

carcinomas when compared with adjacent normal breast tissue,

suggesting that TSC-22 could indeed be a potential novel tumour

suppressor gene. We shall present data showing that methylation

of the TSC-22 promoter is not involved in the reduction of TSC-22

protein in breast cancer.

Acknowledgement Funded by a project grant from Breast Cancer

Campaign to TS.

Reference

1. Stein T, Morris JS, Davies CR, Weber-Hall SJ, Duffy MA,

Heath VJ, Bell AK, Ferrier RK, Sandilands GP, Gusterson BA:

Involution of the mouse mammary gland is associated

with an immune cascade and an acute-phase response,

involving LBP, CD14 and STAT3. Breast Cancer Res 2004,

6:R75-R91.

P18

Accurate prediction of BRCA1 and BRCA2 heterozygous

genotypes using expression profiling of lymphocytes after

irradiation-induced DNA damage

Z Kote-Jarai

, S Jugurnauth

, L Matthews

, I Giddings

E Bancroft

1,3

, R Williams

, M Girolami

, C Campbell

Carrier Clinic Collaborators

, RA Eeles

1,3

Translational Cancer Genetics, The Institute of Cancer Research,

Sutton, UK;

Molecular Carcinogenesis, The Institute of Cancer

Research, Sutton, UK;

The Royal Marsden NHS Foundation Trust,

London, UK;

Paediatric Oncology, The Institute of Cancer

Research, Sutton, UK;

Bioinformatics Research Centre, University

of Glasgow, UK;

Computational Intelligence Group, University of

Bristol, UK

Breast Cancer Res 2008, 10(Suppl 2):P18 (doi: 10.1186/bcr 1902)

Background Germline mutations in BRCA1 and BRCA2 genes

predispose women to an increased risk of breast/ovarian cancer.

Both genes have important roles in DNA damage repair and are

implicated in gene expression regulation. We have previously

shown that normal fibroblasts from mutation carriers can be distin-

guished from noncarriers following radiation-induced DNA damage.

In this new study we used lymphocytes to determine whether these

also show differential response to induced DNA damage and

whether expression profiling using microarray technology could be

used to accurately predict the BRCA genotype.

Methods Short-term lymphocyte cultures were established from

fresh blood samples from 20 BRCA1 and 20 BRCA2 mutation

carriers and from 10 negative controls (individuals tested negative

for the mutation present in the family). Lymphocytes were

subjected to 8 Gy ionizing irradiation to induce DNA damage and

RNA was extracted 1 hour post γ-irradiation. For expression pro-

filing, genome-wide (30 K) spotted cDNA microarrays manufac-

tured by the Cancer Research UK Microarray Facility were used.

We then applied the support vector machine (SVM) classifier with

statistical feature selection to determine the best feature set for

predicting BRCA1 and BRCA2 heterozygous genotypes. We also

investigated the prediction accuracy using a nonprobabilistic

classifier (SVM) and a probabilistic classifier (Gaussian process

classifier).

Results and conclusion We achieved high accuracy (92% to

96%) in predicting the mutation carrier status. We shall present

the detailed outcome of using the SVM classifier and the Gaussian

process classifier in the task of distinguishing between the three

classes, BRCA1 and BRCA2 mutation carriers and noncarriers,

and evaluate whether this microarray technology can be used to

facilitate the clinical detection and classification of mutations.

Available online http://breast-cancer-research.com/supplements/10/S2

S12

P19

Investigation into the molecular mechanism of the

antiapoptotic functions of CTCF in breast cancer cells

using a proteomics approach

CF Méndez-Catalá

, I Cherhukhin

, F Docquier

, D Farrar

E Pugacheva

, A Vostrov

, E Klenova

Department of Biological Sciences, University of Essex,

Colchester, UK;

Molecular Pathology Section, Laboratory of

Immunopathology, National Institute of Allergy and Infectious

Diseases, National Institutes of Health, Bethesda, MD, USA

Breast Cancer Res 2008, 10(Suppl 2):P19 (doi: 10.1186/bcr 1903)

Background CCCTC binding protein (CTCF) is a highly conserved

and ubiquitous transcription factor with versatile functions. It is

involved in transcriptional regulation, chromatin insulation and

epigenetic control [1]. Although CTCF has features of a tumour

suppressor gene, it is overexpressed in breast cancer cells; this

phenomenon is associated with the resistance of these cells to

apoptosis [2]. The aim of the present study is to investigate the

molecular mechanisms of the CTCF-dependent resistance of breast

cancer cells to apoptosis.

Methods A proteomics approach was used to generate protein

profiles of breast cancer cells, ZR75.1, with normal and reduced

levels of CTCF. In the latter cells CTCF was knocked-down using

siRNA and iRNA. Cell extracts were analysed using two-dimen-

sional PAGE, and differentially expressed proteins were identified

by matrix-assisted laser desorption/ionization time-of-flight or liquid

chromatography/mass spectrometry/mass spectrometry.

Results More than 20 putative candidates have so far been

obtained; they belong to various protein families involved in the

control of signalling, metabolic, apoptotic, stress response and

mammary gland specific regulatory pathways. One of the

candidates, the proapoptotic protein Bax, was further validated as

a target for negative regulation by CTCF. We demonstrated that

expression of Bax correlated inversely with CTCF levels.

Furthermore, Bax promoter was negatively regulated by CTCF in

reporter assays. Two putative CTCF binding sites were identified

within the promoter of Bax gene; contact nucleotides were

determined by footprinting and methylation interference assays.

Conclusion Our data suggest that high levels of CTCF may cause

repression of Bax and inhibition of apoptosis. Lower levels of

CTCF lead to activation of Bax, resulting in apoptosis. Selective

reduction of CTCF can therefore be an attractive option in the

development of antibreast cancer therapies.

Acknowledgements Supported by Breast Cancer Campaign and

CONACyT (National Council of Science and Technology, Mexico).

References

1. Klenova EM, Morse HC, 3rd, Ohlsson R, Lobanenkov VV: The

novel BORIS + CTCF gene family is uniquely involved in

the epigenetics of normal biology and cancer. Semin

Cancer Biol 2002, 12:399-414.

2. Docquier F, Farrar D, D’Arcy V, Chernukhin I, Robinson AF,

Loukinov D, Vatolin S, Pack S, Mackay A, Harris RA, Dorricott

H, O’Hare MJ, Lobanenkov V, Klenova E: Heightened expres-

sion of CTCF in breast cancer cells is associated with

resistance to apoptosis. Cancer Res 2005, 65:5112-5122.

P20

Inhibition of apoptosis by Notch signalling in breast

epithelial cells

O Meurette, S Stylianou, GM Collu, AP Gilmore, K Brennan

Wellcome Trust Centre for Cell Matrix Research, Faculty of Life

Sciences, University of Manchester, UK

Breast Cancer Res 2008, 10(Suppl 2):P20 (doi: 10.1186/bcr 1904)

Background Aberrant Notch signalling has been shown to be

involved in many cancers. We have recently observed accumu-

lation of the Notch intracellular domain (NICD) in breast cancer cell

lines and tissue samples in comparison with normal cell lines and

tissues. Moreover, Notch activation has been shown to inhibit

apoptosis induced by chemotherapeutics that activate the p53

pathway. We are thus investigating the role of Notch signalling in

breast cancer by studying the molecular mechanisms that underlie

its suppression of apoptosis.

Methods Notch signalling was activated in the normal breast

epithelial cells MCF10A by expression of NICD or a fusion protein

CBF1-VP16. To inhibit Notch signalling in MCF7, BT474 and

Hs578t cancer cells we expressed NUMB, a natural inhibitor of the

pathway or a dominant-negative Mastermind protein. Signalling

through apoptotic pathways and extent of cell death were

monitored by western blot analysis and chromatin condensation by

Hoechst staining, respectively. Apoptosis was induced by

melphalan treatment (100 μM). Pretreatment with SH6 (10 μM) or

SP600125 (10 μM) was used to inhibit AKT pathways or JNK

activity, respectively. Nutlin-3 (10 μM) was used to inhibit p53–

MDM2 interaction.

Results Activation of Notch signalling in MCF10A cells caused

resistance to p53-dependent apoptosis induced by DNA damage.

Conversely, inhibiting Notch signalling in MCF7, BT474 or Hs578t

cancer cell lines led to a sensitization to apoptosis. We further

showed that AKT was phosphorylated on serine 473 and that the

AKT targets ASK1 and MDM2 were phosphorylated on serine 83

and 166, respectively, in Notch-activated MCF10A cells. Further-

more, AKT inhibition by treatment with SH6 restored sensitivity of

NICD-expressing or CBF1-VP16-expressing cells to DNA-damage-

induced apoptosis, showing that AKT activation is sufficient to

confer resistance to apoptosis. We thus investigated the role of

AKT-mediated MDM2 phosphorylation. Inhibition of p53–MDM2

interaction by treatment with Nultin-3 restored neither NOXA or

PUMA accumulation nor sensitivity of NICD-expressing or CBF-1-

VP16-expressing MCF10A cells following DNA-damaging agent

treatment. We next showed that, following DNA-damaging agent

treatment, Notch activation prevented JNK phosphorylation and

PUMA and NOXA accumulation. Furthermore, JNK activation in

NICD-expressing or CBF1-VP16-expressing cells is sufficient to

induce cell death, and inhibition of JNK signalling by treatment with

SP600125 is sufficient to prevent cell death in normal MCF10A

cells.

Conclusion Notch activation of the AKT pathway inhibits DNA

damage-induced apoptosis by inhibition of JNK via ASK1

phosphorylation.

Breast Cancer Research May 2008 Vol 10 Suppl 2 Breast Cancer Research 2008

S13

P21

TopBP1 contains transcriptional regulatory domains and

regulates gene pathways involved in breast cancer

RHG Wright

, ES Dornan

, MM Donaldson

, M MacFarlane

P Herzyk

, IM Morgan

Insitute of Comparative Medicine, University of Glasgow Faculty of

Veterinary Medicine, Glasgow, UK;

The Sir Henry Wellcome

Functional Genomics Facility, Institute of Biomedical and Life

Sciences, University of Glasgow, UK

Breast Cancer Res 2008, 10(Suppl 2):P21 (doi: 10.1186/bcr 1905)

TopBP1 is a nuclear protein with eight BRCT domains and is

involved in many aspects of nucleic acid metabolism: it is involved

in the initiation of DNA replication in the Xenopus in vitro

replication system by assisting loading of polymerase onto the

replication complex; it is a substrate for ATM/ATR and is essential

for the ATR DNA damage signalling pathway, and is also probably

involved in the actual DNA repair process; it acts as a

transcriptional cofactor for E2F1 where it regulates the apoptotic

function of this protein. In addition, the yeast homologues of

TopBP1, Dbp11 (Saccharomyces cerevisiae) and Cut5 (Schizo-

saccharomyces pombe), are also involved in replication and repair

processes. TopBP1 also shares functions with BRCA1; both are

involved in regulating an intact G

/M checkpoint, they colocalise to

sites of DNA damage, they share sequence homology (even

outside the BRCT domains), they are substrates for ATM/ATR, and

they can regulate expression of the c-myc gene. All of these

properties of TopBP1 led to us investigating whether TopBP1

plays a role in breast cancer.

There is a polymorphism in TopBP1 that gives an increased risk of

breast cancer [1], and work from our laboratory has demonstrated

that TopBP1 is aberrantly expressed in a significant number of

human breast cancers [2]. Clearly the role of TopBP1 in replication

and genome maintenance would mean that disturbance of expres-

sion could result in genomic instability contributing towards

cancer. Our studies have focused on an additional aspect of

TopBP1 that could contribute to the transformed phenotype; gene

regulation. We have identified several chromatin modification

domains on TopBP1 that could contribute not only to trans-

criptional regulation but also to the replication and repair functions

of this protein [3]. Using siRNA knockdown of TopBP1 in MCF7

cells, we identified genes that are regulated by TopBP1. Following

knockdown of TopBP1, the short-term growth of the MCF7 cells

was not affected. This was surprising as it has been predicted that

TopBP1 is essential for DNA replication and our results demon-

strate that this is not the case in all cell lines (we have tested other

lines in which TopBP1 is essential for S phase). However, even

though these cells cycled for several days, they did not survive long

term, presumably due to accumulated damage following replication

in the absence of TopBP1. Using this MCF7 system we carried out

microarray experiments that revealed the absence of TopBP1

alters the expression of genes involved in many cellular pathways

implicated in breast cancer, including the oestrogen signalling

pathway, and the mitogen-activated protein kinase signalling network.

Future work will focus on determining how TopBP1 regulates

these pathways and what cellular interacting partners TopBP1

requires for chromatin modification. Such studies will increase our

understanding of breast cancer and assist in developing diagnostic

and prognostic gene profiling for breast cancer management.

References

1. Karppinen SM, Erkko H, Reini K, Pospiech H, Heikkinen K,

Rapakko K, Syväoja JE, Winqvist R: Identification of a

common polymorphism in the TopBP1 gene associated

with hereditary susceptibility to breast and ovarian cancer.

Eur J Cancer 2006, 42:2647-2652.

2. Going JJ, Nixon C, Dornan ES, Boner W, Donaldson MM,

Morgan IM: Aberrant expression of TopBP1 in breast

cancer. Histopathology 2007, 50:418-424.

3. Wright RH, Dornan ES, Donaldson MM, Morgan IM: TopBP1

contains a transcriptional activation domain suppressed by

two adjacent BRCT domains. Biochem J 2006, 400:573-582.

P22

Regulation of cyclin D

by the BRCA1–BARD1 complex

MM Murray, DP Harkin

Centre for Cancer and Cell Biology, Queen’s University, Belfast, UK

Breast Cancer Res 2008, 10(Suppl 2):P22 (doi: 10.1186/bcr 1906)

Background BRCA1 and cyclin D

are both essential for normal

breast development and mutation or aberration of their expression

is associated with breast cancer [1,2]. Cyclin D

is best known as

a G

cyclin where it regulates the G

to S phase transition by

acting as a rate-limiting subunit of CDK4/6 kinase activity. More

recently, however, Stacey has demonstrated that cyclin D

levels in

/M determine whether a cell continues to proliferate or exits the

cell cycle [3]. The majority of BRCA1 in the cell is bound to

BARD1 through their N-terminal RING domains. Hetero-

dimerization is essential for the stability and correct localization of

the complex and confers ubiquitin ligase activity to BRCA1. The

importance of the ligase activity of BRCA1 to breast cancer

development is inferred from the fact that N-terminal disease-

associated mutations are proposed to reduce ligase activity [4].

Methods Protein–protein interactions were demonstrated using

yeast-two-hybrid and coimmunoprecipitation. Protein levels were

altered through overexpression, siRNA and antisense technology.

The effect of proteasome inhibitors and cycloheximide treatment

was also examined.

Results We initially identified cyclin D

as a binding partner of

BARD1 in a yeast-two-hybrid screen and defined the minimal

binding region as the N-terminus of BARD1. This interaction was

confirmed in vivo by coimmunoprecipitation. The N-terminus of

BARD1 also binds BRCA1 and imparts ubiquitin ligase activity to

the complex. Covalent modification of proteins with ubiquitin is a

common regulatory mechanism in eukaryotic cells. Traditionally,

polyubiquitin chains linked through lysine 48 target proteins for

degradation by the 26 S proteasome. We have demonstrated that

cyclin D

protein levels are inversely related to BRCA1 and

BARD1 levels in several model systems. Furthermore, regulation of

cyclin D

levels occurs through a post-transcriptional mechanism

and requires the ligase activity of BRCA1. Interestingly, this

phenomenon is cell-cycle regulated, occurring in G

/M.

Conclusion We propose that cyclin D

is a potential substrate for

BRCA1 ubiquitination and that this targets cyclin D

for

proteasomal-mediated degradation. Future work will focus on

ascertaining the functional consequence of cyclin D

regulation by

the BRCA1–BARD1 complex; in particular, the impact of BRCA1,

mediated through regulation of cyclin D

, on the proliferation

versus differentiation decision.

References

1. Sicinski P, Donaher JL, Parker SB, Li T, Fazeli A, Gardner H,

Haslam SZ, Bronson RT, Elledge SJ, Weinberg RA: Cyclin D

provides a link between development and oncogenesis in

the retina and breast. Cell 1995, 82:621-630.

2. Xu X, Wagner KU, Larson D, Weaver Z, Li C, Ried T, Hen-

nighausen L, Wynshaw-Boris A, Deng CX: Conditional muta-

tion of BRCA1 in mammary epithelial cells results in

blunted ductal morphogenesis and tumour formation. Nat

Genet 1999, 22:37-43.

Available online http://breast-cancer-research.com/supplements/10/S2

S14

3. Stacey, D: Cyclin D

serves as a cell cycle regulatory

switch in actively proliferating cells. Curr Opin Cell Biol

2003, 15:158-163.

4. Morris JR, Pangon L, Boutell C, Katagiri T, Keep NH, Solomen

ES: Genetic analysis of BRCA1 ubiquitin ligase activity

and its relationship to breast cancer susceptibility. Hum

Mol Genet 2006, 15:599-606.

P23

Modelling estrogen receptor alpha-positive breast cancer

by transformation of normal human mammary epithelial

cells

X Schmidt

, S Duss

, RD Iggo

1,2

Bute Medical School, University of St Andrews, UK;

NCCR

Molecular Oncology, Swiss Institute for Experimental Cancer

Research, Epalinges, Switzerland

Breast Cancer Res 2008, 10(Suppl 2):P23 (doi: 10.1186/bcr 1907)

Two-thirds of breast cancers express estrogen receptor alpha

(ERα) and are estrogen-dependent for growth, yet the unavailability

of accurate in vivo models has long impeded the characterisation

of critical events that lead to the development of these luminal

subtypes of the disease. Previously, our group successfully created

an ERα-positive breast cancer model by quantitative transfor-

mation of normal human mammary epithelial cells (HMECs) derived

from reduction mammoplasties. HMECs were grown as mammo-

spheres in suspension to enrich for progenitor cells, which were

then transformed using lentiviral vectors encoding ERα and TERT

as well as the polycomb gene BMI1 and MYC, both of which have

been implicated in ERα-positive breast cancer. Injection of

transformed HMECs into mammary fad pads of NOD/SCID mice

resulted in the formation of estrogen-dependent tumours that

metastasised to multiple organs [1], confirming the creation of a

model that mirrors the characteristics of human estrogen-

dependent breast cancer. Somewhat surprisingly, we observed

islands of squamous differentiation in the tumours that formed in

the NOD/SCID mice, whereas the large majority of human breast

tumours are adenocarcinomas. To address this discrepancy, we

are currently testing a combination of our established protocol with

new HMECs in vitro culture conditions that have recently been

shown to abrogate the squamous phenotype of the resulting

tumours in mice [2]. Our model system is a powerful tool for the in

vivo characterisation of candidate genes that have been implicated

in development of ERα-positive breast cancer. Genes of interest

include TNRC9, which has recently been identified in genome-

wide association studies as a potential novel breast cancer

susceptibility gene [3], as well as TBX3, which is known to play a

role in mammary gland development as well as breast tumouri-

genesis. We are currently testing these genes in our model using

overexpression and knockdown approaches.

References

1. Duss S, André S, Nicoulaz AL, Fiche M, Bonnefoi H, Brisken

C, Iggo RD: An oestrogen-dependent model of breast

cancer created by transformation of normal human

mammary epithelial cells. Breast Cancer Res 2007, 9:R38.

2. Ince TA, Richardson AL, Bell GW, Saitoh M, Godar S,

Karnoub AE, Iglehart JD, Weinberg RA: Transformation of

different human breast epithelial cell types leads to dis-

tinct tumor phenotypes. Cancer Cell 2007, 12:160-170.

3. Easton DF, Pooley KA, Dunning AM, Pharoah PD, Thompson

D, Ballinger DG, et al.: Genome-wide association study

identifies novel breast cancer susceptibility loci. Nature

2007, 447:1087-1093.

P24

Regulation of estrogen receptor beta by 5

′′

untranslated

regions in breast carcinogenesis

L Smith, TA Hughes

Leeds Institute of Molecular Medicine, University of Leeds, St

James’s University Hospital, Leeds, UK

Breast Cancer Res 2008, 10(Suppl 2):P24 (doi: 10.1186/bcr 1908)

Background Estrogen receptor (ER) expression is a key

determinant of breast tumour behaviour. While the role of ERα in

carcinogenesis is relatively well understood, the role of ERβ, the

more recently identified receptor, remains uncertain. This is partly

because analyses have been confused by a consistent

discrepancy between ERβ expression at mRNA and protein levels

[1]. Recently, evidence has accumulated that deregulation of gene-

specific translation occurs during carcinogenesis in breast and

other tissues. Regulation of ERβ translation could therefore be

responsible for nonconcordance of its mRNA and protein levels,

and could provide an important level of modulation of ER activity

during breast cancer development.

Regulation of translation occurs mainly during initiation. Most

initiation occurs by cap-dependent scanning, which requires

binding of the initiation complex to the mRNA cap and recruitment

of other proteins. These scan along the 5′ untranslated region

(UTR) of the mRNA to the reading frame, where they recognise an

initiation codon, recruit more factors, and initiate protein synthesis.

5′ UTRs vary greatly in length and sequence with some containing

elements that allow regulation of factor recruitment or scanning,

and thereby allow regulation of translation of their specific mRNAs

[2]. It is thought that deregulation of translation, via 5′-UTR

sequences, is responsible for a significant proportion of the

expression changes in cancer cells and that this has a role in

carcinogenesis.

We identified three alternative 5′ UTRs for ERβ – UTRa (including

upstream exon 0K), UTRa long (UTRa containing an additional 5′

sequence) and UTRb (including upstream exon 0N) – from the

literature [3] and EST databases (Figure 1). Our hypothesis is that

these alternative 5′ UTRs allow differential post-transcriptional

regulation of ERβ expression, thereby providing critical regulation

of ER function.

Methods We investigated the properties of these three ERβ 5′

UTRs using established reporter assays [4]; each 5′ UTR was

cloned upstream of a GFP reporter. Breast cell lines (MCF7, MDA-

MB-453, MDA-MB-231, BT-20 and HB2) were transiently

transfected with either an unmodified GFP reporter as a control

(this is identical to experimental vectors except for its non-

specialised 5′ UTR), or with equal copy numbers of specific 5′-

UTR reporters. Effects of each 5′ UTR on translation were

assessed by measurement of relative GFP protein and mRNA

expression from each plasmid using flow cytometry and

quantitative PCR, respectively. Semi-quantitative PCR was also

used to analyse ERβ 5′ UTRa and UTRb expression in matched

normal/tumour breast tissues.

Results Our results are the first to show that these alternative 5′

UTRs do, in fact, allow the differential regulation of ERβ translation.

The UTRa and UTRa long 5′ UTRs strongly inhibited translation of

the GFP reporter whilst UTRb had little effect. In addition, our

preliminary data suggest that these alternative 5′ UTRs are

differentially expressed between breast normal and tumour tissue.

The expression of UTRa mRNA was found to be upregulated in a

panel of breast tumours compared with matched normal tissue. This

may have important implications in breast cancer development.

Work is currently ongoing to investigate the stability of these mRNA

messages and to identify important regulatory sequences.

Breast Cancer Research May 2008 Vol 10 Suppl 2 Breast Cancer Research 2008

S15

Conclusion Post-transcriptional regulation plays an important role

in determining the level of ERβ protein expression and may

therefore have an influence on overall estrogen receptor activity.

This may have important implications on our understanding of

breast cancer biology and treatment.

Acknowledgements LS is supported by Breast Cancer Campaign

while TAH is supported by the Breast Cancer Research Action

Group and Yorkshire Cancer Research.

References

1. Speirs V: Oestrogen receptor beta in breast cancer: good,

bad, or still too early to tell? J Pathol 2002, 197:143-147.

2. Hughes TA: 5

′′

untranslated regions: critical regulators of

cap-dependent translation. In Leading-Edge Research

Communications on Messenger RNA. Edited by Colubus F.

New York: Nova Science Publishers; 2007.

3. Hirata S, Shoda T, Kato J, Hoshi K: The multiple untranslated

first exons system of the human estrogen receptor beta

(ER

ββ

) gene. J Steroid Biochem Mol Biol 2001, 78:33-40.

4. Hughes TA, Brady HJ: Expression of axin2 is regulated by

the alternative 5

′′

-untranslated regions of its mRNA. J Biol

Chem 2005, 280:8581-8588.

P25

Reelin expression in breast tumours is associated with

increased survival and is controlled by promoter

methylation

T Stein

, E Cosimo

, P Smith

, R Simon

, K Price

, L Baird

AK Bell

, G Sauter

, T Crook

, BA Gusterson

Division of Cancer Sciences and Molecular Pathology, Western

Infirmary, University of Glasgow, UK;

Breakthrough Breast Cancer

Centre, Institute of Cancer Research, London, UK;

Department of

Pathology, University Medical Center Hamburg–Eppendorf,

Hamburg, Germany;

Frontier Science and Technology Research

Foundation, Boston, MA, USA;

Department of Pathology, Western

Infirmary, Glasgow, UK

Breast Cancer Res 2008, 10(Suppl 2):P25 (doi: 10.1186/bcr 1909)

Background Reelin is a secreted signalling protein whose major

function has so far been described in the developing brain, where

it is involved in cell positioning of neuronal progenitor cells.

Recently, the Reelin promoter has been found to be methylated in

pancreatic cancer and this was associated with increased

migratory ability [1], whereas in the prostate Reelin expression is

associated with high-grade cancers [2].

Methods We measured the Reelin expression and promoter

methylation status in breast cancer-derived cell lines and in a

cohort of 64 breast cancer cases. We further stained sections of

normal, benign, and cancerous human breast, as well as two tissue

arrays of 168 and 2,200 breast cancer patients, respectively.

Reelin staining was analysed with regards to other clinical

parameters and survival.

Results Promoter methylation status in breast cancer cell lines, as

well as in primary cancers, corresponds with reduced expression

of Reelin. In the normal breast, Reelin is expressed in the luminal

epithelium and myoepithelium, but is lost during breast cancer

progression. Reelin expression correlates with increased survival

(P = 0.01) and negative lymph node status. Treatment of breast

cancer cell lines with the demethylating agent decitabine leads to

re-expression of Reelin RNA.

Conclusion Reelin expression in the breast is associated with

increased survival and negative lymph node status, and is

controlled at least in part by promoter methylation. Reelin is

therefore a novel potential tumour suppressor gene in the breast.

Acknowledgement Funded by a project grant from Breakthrough

Breast Cancer to BAG and TS.

References

1. Sato N, Fukushima N, Chang R, et al.: Differential and epige-

netic gene expression profiling identifies frequent disrup-

tion of the RELN pathway in pancreatic cancers.

Gastroenterology 2006, 130:548-565.

2. Perrone G, Vincenzi B, Zagami M, et al.: Reelin expression in

human prostate cancer: a marker of tumor aggressive-

ness based on correlation with grade. Mod Pathol 2007,

20:344-351.

P26

Investigation of the roles of novel apoptosis-controlling

genes in breast cancer

GT Williams, M Mourtada-Maarabouni, MR Pickard, VL Hedge,

A Sutherland

Institute for Science and Technology in Medicine, Keele University,

Keele, UK

Breast Cancer Res 2008, 10(Suppl 2):P26 (doi: 10.1186/bcr 1910)

Background Normal mammary epithelial cells, like all other

nucleated cells in the body, have the innate capacity to undergo

programmed cell death by apoptosis. This process is controlled by

external factors such as hormones and growth factors, as well as

by cell–cell contacts and recognition of damaged DNA, and plays

an essential role in maintaining stable cell numbers. In breast

cancer cells, in common with most other cancer cells, the control

of apoptosis is defective, so that the rate of cell death falls below

that required to maintain a stable cell population size. The analysis

of the molecular control of apoptosis is therefore very important in

understanding breast cancer development and in producing novel

therapies.

We have successfully used functional expression cloning [1] to

identify novel genes playing controlling roles in the apoptosis

process. We have used the effects of the genes on cell survival

itself to isolate those genes that act at rate-limiting steps in the

control of this process, and whose level of activity therefore

Available online http://breast-cancer-research.com/supplements/10/S2

Figure 1 (abstract P24)

5′ end of the human ERβ gene (14q23) aligned with mRNAs

containing different ERβ 5′ UTRs. UTR exons (filled boxes),

transcriptional (black arrows) and translational (ATG) start sites, intron

sizes, and primers used for PCR analysis (grey arrows, specific UTRs;

open arrows, exon 1) are shown. Sequences strongly suggestive of

translational regulation are described: uORFs and stable RNA

structure, quantified as change in free energy, ΔG; for comparison, ΔG

of the nonregulatory β-actin 5′ UTR is only –24 kcal/mol.

S16

determines whether the cell lives or dies. The genes we have

identified include protein phosphatase 4 [2], Fau [3], vATPase E,

PLAC8 and GAS5 [4]. Subsequently we have studied the effects

of upregulation and downregulation of the activity of these genes

on breast cancer cells. We have also analysed the levels of

expression of these genes in normal breast epithelium and in

breast cancer tissue in order to determine which of the genes are

involved in the development of these cancers.

Methods Gene expression in breast cancer cell lines was

upregulated by transfection of full-length cDNAs in pcDNA3 or

pCMVSPORT expression vectors. Downregulation was achieved

by transfection of siRNAs (Ambion; Applied Biosystems, Warring-

ton, UK). Real-time quantitative RT-PCR was used to monitor gene

expression levels in the breast cancer cell lines, and also in breast

cancer and matched normal breast tissue.

Results Modulation of expression of several of the candidate

genes, particularly Fau, altered the sensitivity of breast cancer cell

lines to apoptosis. While expression of PLAC8 was not signifi-

cantly altered in the breast tumour samples as a whole, several of

the other genes, including Fau and vATPase E, did show

significant changes in their levels of expression in breast tumour

tissue, when compared with normal matched breast epithelial

tissue from the same patients.

Conclusion Several of the apoptosis-controlling genes identified

by functional expression cloning affect the sensitivity of breast

cancer cells to apoptosis, including that caused by DNA-damaging

agents. Those genes that show differential expression may play

particularly important roles in the development of breast cancer

and in determining breast cancer resistance or sensitivity to

cytotoxic therapy.

Acknowledgement Breast Cancer Campaign funded this project.

References

1. Williams GT, Farzaneh F: The use of gene function to iden-

tify the rate-limiting steps controlling cell fate. Cancer

Immunol Immunother 2004, 53:160-165.

2. Mourtada-Maarabouni M, et al.: Functional expression

cloning reveals pro-apoptotic role for protein phosphatase

4. Cell Death Differ 2003, 10:1016-1024.

3. Mourtada-Maarabouni M, et al.: Regulation of apoptosis by

Fau revealed by functional expression cloning and anti-

sense expression. Oncogene 2004, 23:9419-9426.

4. Williams GT, et al.: Isolation of genes controlling apoptosis

through their effects on cell survival. Gene Ther Mol Biol

2006, 10:255-262.

P27

D133p53 isoform is a direct p53 target gene that

modulates p53 tumour suppressor activity

M Aoubala

Breast Cancer Res 2008, 10(Suppl 2):P27 (doi: 10.1186/bcr 1911)

Abstract not available at time of publication.

P28

De novo expression of

αα

ββ

integrin by myoepithelial cells

in ductal carcinoma in situ may be an important marker of

disease progression

M Allen, K Mulligan, S Clark, I Hart, JF Marshall, JL Jones

Queen Mary’s University of London, John Vane Science Centre,

Institute of Cancer, Department of Tumour Biology, London, UK

Breast Cancer Res 2008, 10(Suppl 2):P28 (doi: 10.1186/bcr 1912)

Myoepithelial cells (MEC) are essential to the maintenance of

normal breast function, and loss of normal MEC function is

commonly associated with breast cancer. Most established

invasive breast carcinomas develop through an in situ phase

known as ductal carcinoma in situ (DCIS). We have identified up-

regulation of β

integrin on MEC in a subset of DCIS. Normal MEC

exhibit potent tumour suppressor function, but it is not clear

whether this is compromised in DCIS. The aim of the present study

is to investigate the effect of β

expression on myoepithelial tumour

suppressor function.

Immunohistochemical analysis of DCIS of different grades with and

without an invasive breast cancer was carried out to determine the

status. For in vitro studies magnetic bead sorting was used to

isolate a pure normal-like myoepithelial cell line from the immorta-

lised 1089 cell line (N-1089 MEC). These were used to generate

overexpressing myoepithelial cells (DCIS-modified MEC) by

retroviral transduction. The lines were characterised by immuno-

fluorescence and flow cytometry, and the tumour suppressor

function of both lines was compared with primary normal and DCIS

MEC in coculture with breast cancer cell lines.

Analysis of a series of primary DCIS tissues (n > 400) demon-

strated induction of β

in MEC in a subset of cases, predominantly

high grade. Upregulation was almost universal in DCIS associated

with invasive disease. Initial characterisation of the 1089 line

revealed a mixed phenotype from which pure MEC were selected

on the basis of β

integrin. This population exhibited all charac-

teristic myoepithelial markers. Coculture assays demonstrated that

N-1089 MEC could produce the same tumour suppressor effect

as primary MEC, leading to significant reduction in tumour invasion

(P < 0.001) and proliferation (P < 0.001). N-1089 MEC trans-

duced with α

(DCIS-1089 MEC) demonstrated enhanced

binding and migration to the β

ligand LAP, and activation of a

transforming growth factor beta reporter, indicating that the α

was functional. Whilst primary DCIS MEC showed loss of

suppressor function (P < 0.05), DCIS-1089 MEC exhibit altered

behaviour with a more migratory phenotype then the normal

counterpart, but at least some of the tumour-suppressor function

was maintained.

We have shown that MEC exhibit an altered phenotype in DCIS

with de novo expression of α

. We have generated normal-like

cell lines that exhibit all the characteristics of primary MEC and

recapitulate primary MEC tumour suppressor function. Primary

DCIS MEC show loss of suppressor function whereas β

overexpressing MEC (which resemble DCIS-like MEC) promote or

suppress breast cancer cell invasion in a cell-type-specific manner.

These findings suggest that changes in MEC during DCIS may

influence disease progression, and these cell lines provide a

powerful model to study further the mechanisms involved.

P29

Understanding and exploiting changes in O-linked

glycosylation in breast cancer

S Julien, J Coleman, G Picco, R Beatson, J Taylor-Papadimitriou,

J Burchell

Breast Cancer Biology, King’s College London School of Medicine,

Guy’s Hospital, London, UK

Breast Cancer Res 2008, 10(Suppl 2):P29 (doi: 10.1186/bcr 1913)

The differences in glycosylation patterns seen in breast malignancy

strongly influence the final structure of membrane and secreted

glycoproteins, and these novel tumour-associated glycoforms can

modify the behaviour of the malignant cell and its interaction with

immune effector cells. Changes in mucin type O-glycosylation

occur in breast carcinomas and are the result, at least in part, of

Breast Cancer Research May 2008 Vol 10 Suppl 2 Breast Cancer Research 2008

S17

changes in the expression of specific glycosyltransferases [1]. A

similar change in the expression of glycosyltransferases resulting in

the change of glycans attached to O-linked glycoproteins is seen

when normal dendritic cells mature and migrate to the lymph nodes

[2]. As 70% to 80% of metastatic breast cancers metastasize via

the lymphatics, a particular pattern of O-linked glycans may be

required for cells to migrate and/or settle in the lymph nodes.

Changes in O-linked glycosylation have a considerable influence

on the structure of mucin glycoproteins that carry hundreds of O-

linked glycans. The MUC1 membrane mucin is expressed by over

90% of breast carcinomas and in the change to malignancy

truncated O-glycans are added to this mucin. In vitro synthesis of

MUC1-based glycoproteins and glycopeptides carrying specific

tumour-associated glycans has allowed an investigation of how

individual glycoforms affect the immune response and interact with

immune effector cells [3,4]. It is becoming clear that some glyco-

forms of MUC1 can induce an immune response while others are

immunosuppressive. Understanding how the different tumour-

associated glycoforms induce or inhibit the immune response is

important for the design of clinical studies using MUC1-based

antigens.

Acknowledgements Supported by Cancer Research UK,

European Commission and Breast Cancer Campaign. The authors

would like to thanks all members of the European Prime Boost

Consortium, contract number QLK3-CT-2002-02010.

References

1. Burchell JM, Mungul A, Taylor-Papapdimitriou J: O-linked gly-

cosylation in the mammary gland: changes that occur during

malignancy. J Mammary Gland Biol Neoplasia 2001, 6:355-

364.

2. Julien S, Grimshaw M, Sutton-Smith M, Coleman J, Dell A,

Taylor-Papadimitriou J, Burchell J: O-linked glycosylation is

regulated during maturation of dendritic cells and has an

impact on their migration. J Immunol 2007, 179:5701-

5710.

3. Napoletano C, Rughetti A, Tarp MPA, Coleman J, Bennett EP,

Picco G, Sale P, Denda-Nagai K, Irimura T, Mandel U,

Clausen H, Frati L, Taylor-Papadimitriou J, Burchell J, Nuti M:

Tumor associated Tn-MUC1 glycoform is internalised

through the macrophage galactose-type C-type lectin and

delivered to the HLA class I and II compartments in den-

dritic cells. Cancer Res 2007, 67:8358-8367.

4. Tarp MA, Sorensen AL, Mandel U, Paulsen H, Burchell J,

Taylor-Papadimitriou J, Clausen H: Identification of a novel

cancer-specific immunodominant glycopeptide epitope in

the MUC1 tandem repeat. Glycobiology 2007, 117:197-

209.

P30

Downregulation of 15-hydroxyprostaglandin

dehydrogenase in hormone-resistant breast cancer

M Cummings, L Maraqa, MB Peter, AM Shaaban, AM Hanby,

MA Hull, V Speirs

Leeds Institute of Molecular Medicine, St James’s University

Hospital, Leeds, UK

Breast Cancer Res 2008, 10(Suppl 2):P30 (doi: 10.1186/bcr 1914)

Background Tamoxifen has been the principal endocrine therapy

for estrogen receptor alpha (ERα)-positive breast cancer patients

and still remains the therapy of choice in the premenopausal

setting. However, resistance and recurrence remain a serious

problem. Our previous work has indicated that 15-hydroxyprosta-

glandin dehydrogenase (15-PGDH) was significantly down-

regulated in two, independently derived, tamoxifen-resistant (TAMr)

MCF-7 derivatives compared with sensitive controls [1]. 15-PGDH

is the key enzyme for the biological inactivation of prostaglandins,

and has been shown to be a tumour suppressor in breast cancer.

However, a role for 15-PGDH downregulation in endocrine resis-

tance has not previously been identified.

Methods and results Downregulation of 15-PGDH mRNA and

protein in TAMr MCF-7 was confirmed by quantitative RT-PCR and

western blotting. To determine the role of 15-PGDH in TAMr, we

stably transfected TAMr MCF-7 cells with human 15-PGDH cDNA.

Overexpression of 15-PGDH partially restored sensitivity of TAMr

cells to 4-hydroxytamoxifen by the MTT assay, demonstrating that

15-PGDH downregulation plays a functional role in the acquisition

of TAMr. Treatment of TAMr MCF-7 cells with a DNA methyl-

transferase inhibitor (5-azacytidine), and a histone deacetylase

inhibitor (trichostatin A), led to re-expression of 15-PGDH mRNA

(by quantitative RT-PCR), suggesting that 15-PGDH is silenced via

epigenetic mechanisms during the acquisition of TAMr. To address

whether 15-PGDH downregulation is involved in clinical TAMr, we

assembled a tissue microarray comprising 89 relapsed primary

human breast cancers and 234 tamoxifen-sensitive controls. We

are currently optimizing 15-PGDH immunohistochemistry on our

tissue microarrays, and results will be presented.

Conclusion Our data suggest that the acquisition of TAMr in vitro

involves epigenetic silencing of 15-PGDH. Moreover, our data

show that 15-PGDH downregulation has a novel, functional role in

endocrine resistance.

Reference

1. Scott DJ, Parkes AT, Ponchel F, Cummings M, Poola I, Speirs

V: Changes in expression of steroid receptors, their down-

stream target genes and their associated co-regulators

during the sequential acquisition of tamoxifen resistance

in vitro. Int J Oncol 2007, 31:557-565.

P31

Plasma MMP1, MMP8 and MMP13 expression in breast

cancer: protective role of MMP8 against lymph node

metastasis

J Decock

1,2

, W Hendrickx

1,3

, U Vanleeuw

, MR Christiaens

S Ye

, R Paridaens

1,3

Laboratory for Experimental Oncology, KU Leuven, Belgium;

Biomedical Research Centre, School of Biological Sciences,

University of East Anglia, Norwich, UK;

Multidisciplinary Breast

Center, University Hospitals Leuven, Belgium;

William Harvey

Research Institute, Barts and The London School of Medicine,

London, UK

Breast Cancer Res 2008, 10(Suppl 2):P31 (doi: 10.1186/bcr 1915)

Elevated levels of matrix metalloproteinases (MMPs) have been

found to associate with poor prognosis in various carcinomas. This

study aimed at evaluating plasma levels of the collagenases

MMP1, MMP8 and MMP13 as diagnostic and prognostic markers

of breast cancer. Using ELISA, plasma levels of MMP1, MMP8 and

MMP13 were measured in 42 control individuals and in 208

patients – of which 21 were inflammatory breast cancer patients –

and were correlated with standard clinicopathological data. Plasma

MMP1 levels were higher in breast cancer patients than in control

individuals, while the opposite was true for MMP8. Plasma MMP13

levels could not be detected. We found a negative correlation of

plasma MMP1 with tumour size (P = 0.07); and a positive associa-

tion of MMP8 with the premenopausal status (P = 0.06),

Nottingham Prognostic Index (P = 0.06) and Her2 expression

(P = 0.07). Further, a twofold decrease in MMP1 (P = 0.025) and

MMP8 (P = 0.007) levels was observed in inflammatory breast

cancer patients, a very rare and not well understood aggressive

Available online http://breast-cancer-research.com/supplements/10/S2

S18

disease. Most interestingly, we observed a peculiar relation

between plasma MMP8 levels and lymph node metastasis. We

found that both control individuals and patients without lymph node

involvement (pN0) have lower plasma MMP8 levels than patients

with moderate lymph node involvement (pN1, pN2) (P = 0.001);

and that they show a trend for higher MMP8 levels as compared

with patients with extensive lymph node metastasis (pN3) and a

strong predisposition to distant metastasis. In summary, we

observed differences in MMP1 and MMP8 plasma levels between

distinct breast cancer patient groups. As it is not clear to date

whether MMPs in blood and body fluids have a physiological

function per se, we hypothesize that altered levels in blood reflect

local changes in the extracellular microenvironment. As such, a

positive association of blood MMP levels with clinical

characteristics and tumour features reflects a negative association

with tissue MMP levels and vice versa. Therefore, our results

suggest that both MMP1 and MMP8 in the tumour may contribute

to the aggressive phenotype of inflammatory breast carcinomas.

Interestingly, our results suggest that tumour MMP8 expression

may affect the metastatic behaviour of breast cancer cells with a

greater protective effect against lymph node metastasis than

against distant metastasis.

Acknowledgement Supported by the EU Cancerdegradome

Project Grant LSHC-CT-2003-503297.

P32

Association of MMP8 gene variation with breast cancer

prognosis

J Decock

1,2

, JR Long

, RC Laxton

4,5

, XO Shu

, C Hodgkinson

W Hendrickx

, EG Pearce

, YT Gao

, AC Pereira

, R Paridaens

W Zheng

, S Ye

4,5

Laboratory for Experimental Oncology, KU Leuven, Belgium;

Biomedical Research Centre, School of Biological Sciences,

University of East Anglia, Norwich, UK;

Department of Medicine,

Vanderbilt Epidemiology and Cancer Centre, Vanderbilt University

School of Medicine, Nashville, TN, USA;

William Harvey Research

Institute, Barts and The London School of Medicine, London, UK;

Human Genetics Division, University of Southampton, UK;

Department of Epidemiology, Shanghai Cancer Institute,

Shanghai, China;

Departamento de Biociencias e Diagnostico

Bucal, Faculdade de Odontologia de São José dos Campos –

UNESP, São José dos Campos, Brazil

Breast Cancer Res 2008, 10(Suppl 2):P32 (doi: 10.1186/bcr 1916)

Animal and cell line studies indicate an inhibitory effect of matrix

metalloproteinase 8 (MMP8) on tumorigenesis and metastasis [1-

3]. We investigated whether MMP8 gene variation was associated

with breast cancer metastasis and prognosis in humans. We first

studied nine tagging single nucleotide polymorphisms (SNPs) in

the MMP8 gene in 140 clinically and pathologically well-charac-

terized breast cancer patients. Four of the SNPs were found to be

associated with lymph node metastasis, the most pronounced

being a promoter SNP (rs11225395) with its minor allele (T)

associating with reduced susceptibility to lymph node metastasis

(P = 0.02). This SNP was further evaluated for association with

cancer relapse and survival among a cohort of approximately 1,100

breast cancer patients who had been followed for cancer

recurrence and mortality for a median of 7.1 years. The T allele was

associated with reduced cancer relapse and greater survival,

particularly among patients with earlier stage cancer. Among

patients of tumour-node-metastasis stage 0-II, the adjusted hazard

ratio of disease-free survival was 0.7 (95% CI, 0.5 to 0.9) for

patients carrying T allele compared with those homozygous for the

C allele (P = 0.02). In vitro experiments showed that the T allele

had higher promoter activity than the C allele in breast cancer

cells. Electrophoretic mobility shift assays showed binding of

nuclear proteins to the DNA sequence at the SNP site of the T

allele but not that of the C allele. The data suggest that MMP8 gene

variation may influence breast cancer prognosis and support the

notion that MMP8 has an inhibitory effect on cancer metastasis.

Acknowledgements Supported by the EU Cancerdegradome

Project (LSHC-CT-2003-503297), and research grants

(R01CA64227 and R01CA090899) from the National Cancer

Institute and CAPES/BRAZIL (PDEE 2730/05-7). The data in this

abstract are presented in a manuscript accepted for publication in

Cancer Research.

References

1. Montel V, Kleeman J, Agarwal D, et al.: Altered metastatic

behavior of human breast cancer cells after experimental

manipulation of matrix metalloproteinase 8 gene expres-

sion. Cancer Res 2004, 64:1687-1694.

2. Balbin M, Fueyo A, Tester AM, et al.: Loss of collagenase-2

confers increased skin tumor susceptibility to male mice.

Nat Genet 2003, 35:252-257.

3. Agarwal D, Goodison S, Nicholson B, et al.: Expression of

matrix metalloproteinase 8 (MMP-8) and tyrosinase-

related protein-1 (TYRP-1) correlates with the absence of

metastasis in an isogenic human breast cancer model.

Differentiation 2003, 71:114-125.

P33

CD44 signalling increases cathepsin K and MT1MMP

expression to potentiate breast cancer cell invasion

through collagen I

A Hill, S McFarlane, PG Johnston, DJJ Waugh

Centre for Cancer Research and Cell Biology, Queen’s University

Belfast, UK

Breast Cancer Res 2008, 10(Suppl 2):P33 (doi: 10.1186/bcr 1917)

Background Bone metastasis is a frequent and often incurable

complication of breast cancer causing severe bone pain, patho-

logical fractures, spinal cord compression and hypercalcaemia. We

have focused on establishing the significance of the cell surface

hyaluronan receptor CD44 in underpinning the preferential meta-

stasis of breast cancer cells to bone. In prior in vitro studies we

have demonstrated that depletion of CD44 expression in breast

cancer cells attenuates their adhesion to bone marrow endothelial

cells (BMECs). Our recent experiments have also determined that

the expression of CD44 is elevated in a bone-homing breast

cancer subline MDAMB231BO relative to that detected in the

parental MDAMB231 breast cancer cell line. Together these

experiments suggest a physiological role for this receptor in

promoting the entry of breast cancer cells into the bone micro-

environment.

Methods To further understand the potential significance of CD44

signalling to breast cancer metastasis, we established a tetra-

cycline-regulated CD44 expression system in the minimally

invasive, CD44-negative 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

Breast Cancer Research May 2008 Vol 10 Suppl 2 Breast Cancer Research 2008

S19

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 meta-

stasis of breast cancer. Quantitative real-time PCR, immuno-

blotting and ELISA-based experiments have demonstrated that the

transcript and protein expression of cathepsin K and MT1MMP

increase in response to CD44/HA signalling in a panel of CD44-

expressing breast cancer cell lines (MDA231, MDA157 and

MCF7F). Further experiments conducted using a parental and bone-

homing subclone of the MDAMB231 cell line (MDAMB213BO)

have shown that the expression of CD44, cathepsin K and

MT1MMP is elevated in the MDAMB231BO cells relative to their

parental counterparts. Furthermore, CD44/HA signalling was

shown to increase cathepsin K and MT1MMP mRNA and protein

expression in the MDAMB231BO cells. Consistent with their

increased metastatic phenotype, MDAMB231BO cells displayed

enhanced invasion on HA-supplemented Matrigel and collagen I

and demonstrated enhanced collagenolytic activity as demon-

strated using an in vitro fluorescence-based assay. RNAi mediated

depletion of CD44 and MT1-MMP expression and pharmacological

inhibition of cathepsin K attenuated CD44 promoted invasion

through a collagen I matrix. We are currently investigating the

mechanistic basis underpinning the transcription of these

proteases in breast cancer cells, and using the MDA231BO cell

line to determine the in vivo significance of CD44 expression to

osteolytic metastasis of breast cancer.

Conclusion Our studies demonstrate that CD44 signalling

regulates collagenase activity in breast cancer cells underpinning

their invasion through matrix substrates that are enriched within

breast tissues and organs to which this disease preferentially

metastasises. The long-term objective of our research will be to

determine whether CD44 expression and that of its transcriptional

targets may be predictive for those breast cancer patients at higher

risk of developing skeletal disease and/or may potentially lead to

the development of novel and more effective therapeutic strategies

to attenuate bone metastasis.

Acknowledgement Research funded by Breast Cancer Campaign.

P34

Overexpression of CD44 in acquired tamoxifen-resistant

breast cancer cells augments their migratory response to

heregulin beta 1

S Hiscox, L Goddard, N Jordan, C Smith, M Harper,

RI Nicholson, J Gee

Tenovus Centre for Cancer Research, Welsh School of Pharmacy,

Cardiff University, Cardiff, UK

Breast Cancer Res 2008, 10(Suppl 2):P34 (doi: 10.1186/bcr 1918)

Background Acquired endocrine resistance in breast cancer cells

is accompanied by altered growth factor receptor signalling [1]

and a highly migratory cell phenotype [2]. Interestingly, in

tamoxifen-resistant (TamR) MCF7 cells, our microarray analysis has

demonstrated elevated levels of CD44, a transmembrane

glycoprotein known to interact with, and modulate the function of,

growth factor receptors [3]. Here we have explored the role of

CD44 as a modulator of heregulin beta-1-induced migratory

signalling in TamR cells.

Methods Expression of CD44 (standard and v3 isoforms) were

confirmed by RT-PCR and western blotting and their association

with erbB family members determined by both immuno-

fluorescence microscopy and immunoprecipitation. Activation of

intracellular signalling following heregulin beta 1 treatment

(10 ng/ml) in the presence or absence of CD44 (using siRNA-

mediated inhibition) was determined by western blotting using

phosphospecific antibodies. Cellular migration was determined by

seeding cells (control and CD44 siRNA-treated) into fibronectin-

coated transwell chambers (8.0 μm pore size) in the presence or

absence of heregulin beta 1. After 24 hours, migratory cells were

fixed, stained with crystal violet and counted.

Results Both standard and v3 isoforms of CD44 were

overexpressed in TamR cells at both gene and protein levels (mean

fold increase in CD44s protein (TamR versus MCF7): 4.26 ± 1.2,

P < 0.05). Moreover, CD44s and v3 colocalised with Her2 and

Her3 receptors at the cell surface and were also detectable in

Her2/Her3 cellular immunoprecipitates. Treatment of TamR cells

with heregulin resulted in phosphorylation of erbB receptors

together with a number of downstream signalling intermediates,

including Akt, Src and FAK, and resulted in enhanced cellular

migration. Significantly, heregulin-induced intracellular signalling

was dramatically reduced in cells in which the expression of CD44

was suppressed (via siRNA), with a corresponding loss of

heregulin-induced migratory behaviour (mean fold change in cell

migration versus untreated control: 6.7 ± 1.1, P < 0.05 (heregulin

beta 1); 1.8 ± 0.9 (CD44 siRNA); 1.47 ± 0.6, P < 0.05 (heregulin

beta 1 + CD44 siRNA)).

Conclusion These data demonstrate a role for CD44 as a

modulator of erbB receptor function in endocrine-resistant breast

cancer cells, where it augments heregulin beta 1 migratory signalling.

Acknowledgements The authors acknowledge the support of

Breast Cancer Campaign and the Tenovus charity in these studies

References

1. Jones HE, Gee JM, Hutcheson IR, Knowlden JM, Barrow D,

Nicholson RI: Growth factor receptor interplay and resis-

tance in cancer. Endocr Relat Cancer 2006, 13:45-51.

2. Hiscox S, Morgan L, Green TP, Barrow D, Gee J, Nicholson

RI: Elevated Src activity promotes cellular invasion and

motility in tamoxifen resistant breast cancer cells. Breast

Cancer Res Treat 2006, 97:263-274.

3. Bertotti A, Comoglio PM: Tyrosine kinase signal specificity:

lessons from the HGF receptor. Trends Biochem Sci 2003,

28:527-533.

P35

Role of CLEVER-1 in breast cancer metastasis

A Ammar

, R Mohammed

, M Salmi

, M Pepper

, EC Paish

I Ellis

, P Patel

, SG Martin

University of Nottingham, UK;

Turku University, Finland;

University of the Witwatersrand, South Africa

Breast Cancer Res 2008, 10(Suppl 2):P35 (doi: 10.1186/bcr 1919)

The role that the novel lymphatic-associated adhesion molecule

CLEVER-1 plays in breast cancer metastasis has been examined

by assessing its expression in human breast tumour specimens

and by conducting in vitro experiments to monitor its involvement in

regulating cell adhesion to human umbilical vein endothelial cells

(HUVEC) and hTERT immortalised lymphatic endothelial cells

(LEC).

CLEVER-1 expression was examined in tonsil, lymph node and

148 formalin-fixed paraffin-embedded archival breast carcinoma

specimens using standard immunohistochemistry protocols. In

vitro CLEVER-1 expression was studied, in HUVEC and LEC, via

fluorescence-activated cell sorting. Tumour cell (breast MCF-7 and

MDA-MB-231, and melanoma SKMEL-30) adhesion and leukocyte

adhesion to parental and CLEVER-1 siRNA knockdown endothelial

cells was also examined.

The results show that, in tissue specimens, CLEVER-1 is present

in blood and lymphatic vessels and in certain leukocyte sub-

Available online http://breast-cancer-research.com/supplements/10/S2

S20

populations (macrophage or dendritic cells). Although expression,

in tumours, is higher in blood vessels than in lymphatic vessels

(62.4% versus 18.2%), only lymphatic expression is associated

with lymph node metastasis (P = 0.027). CLEVER-1 expression in

blood vessels and lymphatic vessels correlates with the density of

inflammatory infiltrate (P < 0.001 and P = 0.004, respectively) and

expression in macrophages (P < 0.001). In vitro results show that

although CLEVER-1 is expressed intracellularly in both HUVEC

and LEC, only LEC exhibit surface expression. Interestingly,

adhesion assays show that tumour cells adhere preferentially to

LEC with maximal adhesion exhibited at 30 to 40 minutes. Tumour

cells adhere less to CLEVER-1 knockdown LEC than to control

LEC. The role of CLEVER-1 in cellular adhesion is being further

investigated, using tumour cells and different leukocyte

populations, to determine its involvement in adhesion and migration

of different cell types across lymphatics.

P36

Characterization of a cytoskeletal signaling pathway

underpinning CD44-initiated, integrin-mediated adhesion

of breast cancer cells to bone marrow endothelium

S McFarlane, A Hill, PG Johnston, DJJ Waugh

Centre for Cancer Research and Cell Biology, Queen’s University

Belfast, UK

Breast Cancer Res 2008, 10(Suppl 2):P36 (doi: 10.1186/bcr 1920)

Background Bone metastasis is a frequent complication of breast

cancer. It is estimated that up to 85% of breast cancers will

metastasize to the bone. The selective metastasis of breast cancer

to the bone is thought to result from the preferential adhesion of

breast cancer cells to the bone marrow endothelial cells (BMECs)

lining the bone marrow sinusoids. Our studies have shown that

CD44 promotes the primary adhesion of breast cancer cells to

bone marrow endothelium in vitro. The aim of the present study

was to further explore the cascade of events underpinning CD44-

initiated adhesion.

Methods Experiments using parental and bone-homing (BO)

clones of the MDA-MB-231 breast cancer cell line established the

importance of CD44 to integrin-mediated adhesion to BMECs.

Results MDA-MB-231BO cells displayed increased CD44

expression and adhesion to both BMECs and fibronectin, relative

to parental cells. MDA-MB-231BO cells also displayed increased

expression and activation of the β

-integrin subunit. In addition, the

bone-homing cells exhibited elevated constitutive phosphorylation

of the kinases Src and FAK and the cytoskeletal proteins cortactin

and paxillin relative to the parental cells. Stimulation of MDA-MB-

231BO cells with the CD44 ligand hyaluronan (HA) induced an

increase in the expression of the β

-integrin chain, FAK and paxillin;

and, furthermore, promoted a rapid increase in the activation status

of the β

-integrin subunits, Src, cortactin and paxillin in these cells.

The HA-induced phosphorylation of paxillin was attenuated by

depletion of CD44 and cortactin expression using selective RNAi

strategies, suggesting that it is a downstream target of HA-CD44-

cortactin signaling. MDA-MB-231BO cell adhesion to fibronectin

or to hBMECs was attenuated by RNAi-mediated suppression of

CD44, cortactin and paxillin expression or following administration

of two neutralizing antibodies that inhibit β

-integrin and α

integrin receptor signaling. Antibody-based inhibition of integrin

signaling also attenuated the HA-induced phosphorylation of

cortactin and paxillin, suggesting that these proteins constitute a

signaling cascade activated downstream of a CD44-initiated,

integrin-dependent process.

Conclusion Our results describe a molecular pathway promoting

cytoskeletal reorganization that is activated downstream of a

CD44-induced, integrin-dependent event and that is critical to

efficient breast cancer cell adhesion to hBMECs.

P37

Identification of genes involved in the formation of lymph

node metastasis from human tumour xenograft models of

breast cancer

L Paon, SA Eccles

The Institute of Cancer Research, Sutton, UK

Breast Cancer Res 2008, 10(Suppl 2):P37 (doi: 10.1186/bcr 1921)

Background Lymph node metastasis is associated with

considerable morbidity and is linked to poor prognosis in breast

cancer. We have developed experimental models of lymphatic

metastasis from the human breast carcinoma cell lines GI 101a

and MDA-MB-435. Several sublines of cells derived from lymph

node metastases in vivo have been developed. When injected into

mammary fat pads (MFP) of athymic mice, all cell lines produced

spontaneous lymph node metastases. These cell lines also

generated lymph node metastases (in addition to the expected

lung metastases) when injected intravenously. In the latter, the

tumour cells need to traverse the pulmonary capillary bed and

either show tropism for, or adaptation to, the lymph node

environment. These distinct patterns of spread – due respectively

to direct (intralymphatic) and indirect (haematogenous) colonisa-

tion of nodes – will enable us to explore determinants of both putative

passive and active (nodal tropism) mechanisms independently.

Methods RNA was extracted from frozen primary tumours and

lymph node metastases derived from the different cell lines, after

MFP or intravenous injection, and was used to generate gene

expression profiles. A supervised learning method from the BRB

ArrayTools 3.5.0 software was used to identify the genes that were

differentially expressed between the lymph node metastases

obtained from the two routes of dissemination, as well as between

matched primary tumours and their lymph node metastases.

Results Microarray results indicate that it is possible to distinguish

between the lymph node metastases and matched primary

tumours. Additionally, the nodal metastases derived from the MFP

primary site segregate from those derived from the peripheral

circulation. These samples cluster together irrespective of the cell

line of origin. We have now identified genes upregulated and

downregulated in each cluster, and are validating their expression

at the protein level.

Conclusion The presented results will provide more information

about the molecules involved in the generation of lymph node

metastases. Furthermore, the identification of genes differentially

expressed between metastases originating from MFP and

intravenously suggests that at some level distinct molecular

mechanisms may be in operation in active and passive modes of

dissemination.

Acknowledgement Funded by the European Framework 6

Program (MetaBre – LSHC-CT-2004-50304).

P38

Matrix metalloproteinase-8 is a regulator of the clinical

aggressiveness of mammary tumours

CJ Pennington

, S Pilgrim

, A Gutiérrez-Fernández

XS Puente

, C López-Otín

, JL Jones

, D Holliday

, PN Span

F Sweep

, DR Edwards

University of East Anglia, Norwich, UK;

Universidad de Oviedo,

Spain;

University of London, UK;

University Medical Center

Nijmegen, The Netherlands

Breast Cancer Res 2008, 10(Suppl 2):P38 (doi: 10.1186/bcr 1922)

Breast Cancer Research May 2008 Vol 10 Suppl 2 Breast Cancer Research 2008

S21

Protease genes are involved in multiple steps of cancer

progression, including cell growth, migration and angiogenesis.

These genes are valuable as prognostic and/or diagnostic markers

of disease and are potential therapeutic targets.

We used TaqMan

real-time quantitative PCR to conduct the first

detailed quantitative expression profiling of the entire family of

metalloprotease and serine protease genes and their inhibitors in

breast cancer (over 380 genes). Using a bank of 60 samples (50

cancer and 10 normal mammary tissue) collected at the Norfolk

and Norwich University Hospital [1] we have identified a number of

genes that show significant disregulation in tumour samples

compared with normal breast tissue. Expression correlates either

positively or negatively with tumour grade in many genes.

A further cohort of 229 Dutch patients with more extensive clinical

history [2] was profiled in a subset of the metalloproteinase genes.

Among the genes that showed significant aberrant expression,

Matrix metalloproteinase-8 (MMP8) emerged as a candidate to

play a protective role during tumour progression. MMP8 was found

to have significant prognostic value and was strongly correlated

with prolonged survival. MMP8 is prognostic as a continuous

variable for relapse-free survival (hazard ratio = 0.76, P = 0.045)

and for overall survival (hazard ratio = 0.69, P = 0.025). Expression

of MMP8 also correlated with lymph node involvement, reduced

expression equating to greater nodal spread (P = 0.001).

Expression of MMP8 was independent of tumour grade. These

data show that MMP8 is prognostic in breast cancer, and suggest

that the function of MMP8 antagonizes metastasis.

Acknowledgement Supported by Breast Cancer Campaign.

References

1. Porter S, Scott SD, Sassoon EM, Williams MR, Jones JL,

Girling AC, Ball RY, Edwards DR: Dysregulated expression

of adamalysin-thrombospondin genes in human breast

carcinoma. Clin Cancer Res 2004, 10:2429-2440.

2. Porter S, Span PN, Sweep FC, Tjan-Heijnen VC, Pennington

CJ, Pedersen TX, Johnsen M, Lund LR, Rømer J, Edwards DR:

ADAMTS8 and ADAMTS15 expression predicts survival in

human breast carcinoma. Int J Cancer 2006, 118:1241-

1247.

P39

Actions of IGF-I are differentially regulated by fatty acids in

normal and breast cancer epithelial cells

CM Perks, AA Morrison, L Zeng, C Jarrett, J Shield, ZE Winters,

JMP Holly

Department of Clinical Sciences at North Bristol, IGFs and

Metabolic Endocrinology Group, The Medical School, Southmead

Hospital, University of Bristol, UK

Breast Cancer Res 2008, 10(Suppl 2):P39 (doi: 10.1186/bcr 1923)

Introduction Obesity will soon be the leading preventable risk

factor for many cancers. The insulin-like growth factors (IGFs) have

been strongly implicated as important risk factors for many

epithelial cancers, including breast cancer, and for mediating the

link between nutrition and these cancers. Obesity-related

increases in circulating fatty acids cause insulin resistance with

consequent morbidity but, despite the considerable overlap

between insulin and IGFs, there have been no studies of the

effects of fatty acids on IGF activity.

Objective To examine the effects of the most abundant circulating

fatty acids (oleate – unsaturated; palmitate – saturated) alone and

in combination with IGF-I on MCF-10A nonmalignant breast and

MCF-7 breast cancer epithelial cells.

Methods Following 24 hours in serum-free media, cells were

exposed to albumin-bound fatty acids (100 to 400 μM) for 48

hours with or without IGF-I (20 to 25 ng/ml). Cell growth and death

were assessed by cell counting and the trypan blue dye exclusion

assay, respectively. Data were analysed by ANOVA.

Results For MCF10-A and MCF-7 cells, IGF-I increased cell

growth (P < 0.01 and P < 0.001) whereas oleate (100 to 400 μM)

alone had no effect. However, IGF-induced growth was

differentially affected in combination with oleate: being enhanced

in the MCF-10A cells (by 57% at 400 μM; P < 0.001) but inhibited

in the cancer cells (by 28% at 400 μM; P < 0.05).

For both cell lines, palmitate alone only inhibited growth at the

highest dose (400 μM), which was coincident with the induction of

apoptosis. Palmitate did not affect IGF-induced proliferation in

either cell line. The cells were differentially sensitive to palmitate-

induced death (at 400 μM a 1.5-fold increase of MCF-7 cells; an

eightfold increase of MCF-10A cells). Palmitate-induced death in

MCF10-A cells was inhibited by a ceramide synthase inhibitor,

fumonisin B1 (0.1 μM) (61%), and by oleate (96% at 400 μM) but

was unaffected in the presence of IGF-I.

We are currently investigating the signalling pathways underlying

the differential effects of oleate on IGF-induced growth of MCF-

10A and MCF-7 cells.

Conclusion Palmitate had no effect on IGF-induced cell growth,

whereas oleate enhanced that of normal cells but inhibited that of

cancer cells. Unlike oleate, palmitate induced apoptosis although

cancer cells were relatively resistant to this. This apoptosis was via

ceramide production and was inhibited by oleate but not IGF-I.

Saturated and unsaturated fatty acids have differential effects on

IGF-induced growth and the survival of human breast epithelial

cells, supporting the notion that nutrition is a major environmental

influence on breast cancer progression.

Acknowledgement Funded by American Institute for Cancer

Research.

P40

Insulin-like growth factor binding protein 3 modulates

epidermal growth factor (EGF)-induced growth of breast

epithelial cells by altering EGF receptor internalization

GJ Dennison, JMP Holly, J McIntosh, ZE Winters, CM Perks

Department of Clinical Sciences North Bristol, IGFs and Metabolic

Endocrinology Group, The Medical School, Southmead Hospital,

Bristol, UK

Breast Cancer Res 2008, 10(Suppl 2):P40 (doi: 10.1186/bcr 1924)

Introduction Insulin-like growth factor binding protein 3 (IGFBP-3)

is the most abundant insulin-like growth factor binding protein in

human serum and is able to modulate cell proliferation inde-

pendently of its ability to bind insulin-like growth factor. Tumour-

associated increases in IGFBP-3 levels relate to upregulation of

epidermal growth factor receptor (EGFR) and HER-2 with

increasing oestrogen independence. Remodelling of the extra-

cellular matrix with increased fibronectin expression in poor

prognostic tumours further enhances EGFR levels and signalling.

Objective To explore the potential interactions of IGFBP-3 with the

EGFR/HER-2 pathways.

Methods Normal breast epithelial cells (MCF-10A) and breast

cancer cells (T47D) were dosed with EGF (5 ng/ml and 10 ng/ml),

IGFBP-3 (100 ng/ml), an EGFR/HER-2 tyrosine kinase inhibitor,

(Iressa, 0.25 μm) and a ROCK inhibitor (Y-27632, 5 μM) either

alone or in combinations on either plastic, laminin or fibronectin

(0.25 μg/ml). Cell growth was evaluated by cell counting and

tritiated thymidine incorporation. Internalisation of the EGFR and

Available online http://breast-cancer-research.com/supplements/10/S2

S22

HER-2 was assessed by biotinylation and affinity purification using

a Pin Point Cell Surface Isolation Kit (Pierce, Northumberland, UK)

on whole cell lysates followed by western immunoblotting for the

EGFR and HER-2. Statistical significance was determined using

ANOVA.

Results On plastic and laminin with MCF10A cells, EGF and

IGFBP-3 each increased cell proliferation alone (by 55.2%,

P < 0.001 and 31.7%, P < 0.01, respectively), and together there

was a synergistic increase of 278% (P < 0.001). In addition, the

proliferative effect of IGFBP-3 alone, like that of EGF, was

completely abrogated in the presence of Iressa. With T47D cells,

EGF increased cell proliferation (by 33.9%, P < 0.001), IGFBP-3

alone had no effect, but in combination, in contrast to the normal

cells, IGFBP-3 completely blocked EGF-induced growth

(P > 0.01). These actions of IGFBP-3 on EGF-induced growth

were reversed when the cells were cultured on fibronectin. Further-

more, we found that the modulation of EGF-induced proliferation

by IGFBP-3 was not mediated by changes in the phosphorylation

status of EGFR or HER-2. It was, however, associated with modu-

lation of the internalisation of the EGFR and activation of Rho.

Conclusion We found that IGFBP-3 had differential, matrix-

dependent effects on EGF-mediated proliferation in normal and

breast cancer cells, which was achieved through modulation of

EGFR internalisation and the activation of Rho. Breast tumour

levels of IGFBP-3 may determine their dependence on EGFR/

HER-2 activity and their response to therapies targeting these

receptors.

P41

Anti-oestrogen therapy switches off tumour suppressors

and proapoptotic genes in breast cancer and reveals a new

therapeutic opportunity

A Stone, H Jones, M Giles, J Gee, R Nicholson

Tenovus Centre for Cancer Research, Welsh School of Pharmacy,

Cardiff, UK

Breast Cancer Res 2008, 10(Suppl 2):P41 (doi: 10.1186/bcr 1925)

Background Previous studies in the Tenovus Centre have

demonstrated that the development of antioestrogen resistance in

vitro is accompanied by unfavourable changes in the breast cancer

phenotype leading to increase tumour cell growth rate. Here

evidence is presented to suggest that this is in part due to

antihormones causing the epigenetic silencing of oestrogen-

induced genes involved in the negative regulation of cell growth.

Importantly, we show that reversal of this process using the

demethylation agent 5-azacytidine (5AZA) allows oestrogen-

induced cell kill by a previously unrecognised mechanism.

Methods The breast cancer cell lines used in this study were

MCF7, MCF7-derived tamoxifen-resistant variant (TamR) and

TamR sublines that had been withdrawn from tamoxifen (TamRwd)

for up to 6 months. Cells were challenged by oestradiol (E2),

antihormones and 5AZA. Cell growth responses were assessed by

anchorage-dependent growth assays and alterations in

expression/activity of oestrogen receptor (ER) and ER-regulated

genes were analysed by real-time PCR, western blotting and/or

immunocytochemistry.

Results Compared with the parental MCF7 cells, TamR cells

showed a significant upregulated basal rate of growth that was

maintained on tamoxifen withdrawal for 6 months. Following the

tamoxifen withdrawal, the cells remained ER-positive and showed a

slight growth response to E2. In contrast, they showed no growth

inhibitory response to tamoxifen. Examination of the methylation

status of the promoters of two classically ER-regulated genes

switched off in TamR and TamRwd cells, pS2 and progesterone

receptor (PR), confirmed their increased methylation and that

5AZA was able to reverse this process, allowing the re-expression

of pS2 and PR on E2 treatment. Although pS2 and PR are not

thought to play a role in the regulation of cell growth, these data

provide proof of principal that gene silencing occurs in TamR cells

and that it can be reinstated by 5AZA plus E2. To determine

whether tamoxifen was capable of inducing the methylation of ER-

regulated genes involved in cell growth, TamRwd cells pretreated

with 5AZA were subject to an E2 dose–response challenge. In

contrast to TamRwd cells treated with E2, which promoted a

growth response, E2 in combination with 5AZA was strongly

inhibitory at physiological doses of the steroid (10

–9

M), with this

action being reversed by tamoxifen. An Affymetrix analysis of the

TamR cells has revealed multiple E2-regulated genes that are

switched off in the resistant cells whose ontology indicates tumour

suppressor/proapoptotic functions.

Conclusion Our data suggest that antihormone resistance may be

associated with the epigenetic silencing of growth inhibitory genes

leading to enhanced growth rates. We propose that reinstatement

of the expression of such genes using demethylation agents in

combination with E2 may provide a previously unrecognised

therapeutic opportunity in breast cancer.

P42

Zinc transporter HKE4 as a new target in antihormone

resistance of breast cancer

KM Taylor, N Jordan, S Hiscox, JM Gee, RI Nicholson

Tenovus Centre for Cancer Research, Welsh School of Pharmacy,

Cardiff University, Cardiff, UK

Breast Cancer Res 2008, 10(Suppl 2):P42 (doi: 10.1186/bcr 1926)

Background Oestrogen receptor-positive breast cancers develop

resistance to anti-oestrogens by utilising alternative growth factor

pathways as observed in our tamoxifen-resistant cell line (TAMR).

These include EGFR, IGF1-R and Src signalling as well as

increased growth and invasion. Zinc is elevated in breast cancer

tissue and has been demonstrated to activate certain growth factor

signalling pathways. We have tested the expression level of

members of the LIV-1 family of zinc influx transporters and

discovered that HKE4 (SLC39A7, ZIP7), previously shown by us

capable of increasing the intracellular zinc levels, has increased

expression in TAMR. We have therefore investigated whether the

development of the more aggressive phenotype observed in our

TAMR cells, including activation of these signalling pathways as

well as increased growth and invasion, is due to an increase of

intracellular zinc and as a direct result of increased expression of

HKE4.

Methods All nine members of the LIV-1 subfamily of ZIP

transporters were measured in our model of tamoxifen-resistant

breast cancer using Affymetrix arrays. Zinc-induced activation of

growth factor signalling pathway components was investigated by

western blot and/or fluorescent microscopy. Short-term (15-min)

treatments with 20 μM zinc included ionophore, whereas long-term

(hours/days) did not. Recombinant LIV-1 family members with a V5

tag were expressed using pcDNA3.1/V5-His-TOPO vector, and

siRNA (Dharmacon smartpools with relevant controls) was used to

reduce endogenous expression.

Results HKE4 (SLC39A7), a ZIP transporter from the LIV-1

subfamily, was discovered to be elevated in TAMR cells by

Affymetrix analysis and confirmed by PCR and western blot. We

have observed that our TAMR cells have a twofold increase in

intracellular zinc compared with wild-type cells, using the zinc-

Breast Cancer Research May 2008 Vol 10 Suppl 2 Breast Cancer Research 2008

S23

specific fluorescent dye Newport Green. Short-term zinc treatment

of TAMR cells activates the signalling pathways implicated in

antihormone-resistant proliferation and is reduced by both the zinc

chelator TPEN and the Src kinase inhibitor SU6556. The same

effects are observed after longer term (6 days) zinc treatment with

additional increases in cell growth and invasion through Matrigel.

Since we have previously demonstrated that HKE4 is capable of

increasing intracellular zinc in cells and, more recently, that these

TAMR have elevated intracellular zinc levels, we have tested the

hypothesis that elevated HKE4 expression is directly responsible

for the aggressive phenotype observed in our TAMR cells. Reduc-

ing HKE4 levels by siRNA demonstrated a role for this molecule in

driving the zinc-induced activation of multiple signalling pathways.

In the presence of siRNA for HKE4, the previously observed zinc-

induced activation of EGFR, Src, and IGF1-R was eradicated and

the EGF-stimulated activation was also decreased. Additionally, we

have demonstrated the converse by transfecting recombinant

HKE4 into wild-type cells and/or treating them with zinc to observe

the activation of these signalling pathways and increases in

invasive capability. Interestingly, we have observed a similar role of

HKE4 in our model of faslodex-resistant breast cancer.

Conclusion The presented results propose that HKE4, a member

of the LIV-1 subfamily of ZIP transporters, is directly involved in the

activation of the aggressive phenotype observed with the

development of antihormone resistance, and as such is a potential

new target for the prevention of resistance to antihormones in

breast cancer progression.

P43

Stromal fibroblasts with nuclear

ββ

-catenin are present

within breast tumours and increase proliferation and

invasion of epithelial breast cancer cells

E Verghese

1,2

, HG Shenoy

2,3

, AM Shaaban

1,2

, A Waterworth

MB Peter

2,3

, K Horgan

, V Speirs

, AM Hanby

1,2

, TA Hughes

Department of Pathology, St James University Hospital, Leeds,

UK;

Leeds Institute of Molecular Medicine, Wellcome Trust

Brenner Building, University of Leeds, UK;

Department of Breast

Surgery, Leeds General Infirmary, Leeds, UK

Breast Cancer Res 2008, 10(Suppl 2):P43 (doi: 10.1186/bcr 1927)

Background β-catenin, when located within the nucleus, acts as

an oncoprotein by activating TCF/LEF transcription factors, which

in turn regulate transcription of a wide range of growth and

proliferation-associated genes. Nuclear β-catenin is frequently

seen in epithelial cancer cells as a result of either inappropriate

Wnt signalling or inactivating mutations in genes for key β-catenin

regulators or for β-catenin itself. Breast tumours are unusual,

however, in that nuclear β-catenin is relatively rare in epithelial

breast cancer cells. On the other hand, nuclear β-catenin expres-

sion has been documented in fibroblasts within breast fibro-

adenomas and benign phyllodes tumours [1]. Preliminary

observations within our laboratory indicated that stromal fibroblasts

in and around breast carcinomas also frequently express nuclear β-

catenin. Our aim in the present work was to validate this

observation, and to determine how fibroblasts with nuclear β-

catenin might influence cancer behaviour.

Methods We performed immunohistochemistry for β-catenin on

whole sections of breast cancers from 200 individual cases. A

scoring system based on the number of fibroblasts expressing

nuclear β-catenin was devised and fibroblasts around tumour and

normal breast tissue were scored. To examine the potential

influence of nuclear β-catenin-positive fibroblasts on breast tumour

behaviour, we have developed a tissue culture model. With

appropriate controls, fibroblasts (MRC5/immortalised primary

breast fibroblasts) were transfected to overexpress β-catenin and

the influence of these cells on breast cancer cells (MCF7/MDA-

MB-231) in vitro was determined. First, proliferation rates of breast

cancer cells treated with conditioned media from transfected

fibroblasts were determined (MTT assays). Secondly, invasion

assays were carried out in transwell plates; fibroblasts were β-

catenin or control transfected in lower chambers and breast

cancer cells were seeded into upper chambers onto membranes

coated with extracellular matrix (Matrigel/ECMatrix). Epithelial cells

invading through membranes were quantified (cell counting/

fluorometric assay using CyQuant GR dye).

Results and conclusion We found that fibroblasts expressing

nuclear β-catenin are frequent in and around breast tumours, while

they are very rare around normal breast. In our tissue culture

model, β-catenin-transfected fibroblasts stimulated both prolifera-

tion and invasion of breast cancer cells. In conclusion, nuclear β-

catenin within stromal fibroblasts may have a potent influence on

breast cancer behaviour. This influence further highlights the

importance of stromal–epithelial interactions in breast carcino-

genesis.

Reference

1. Sawyer EJ, Hanby AM, Rowan AJ, Gillett CE, Thomas RE,

Poulsom R, Lakhani SR, Ellis IO, Ellis P, Tomlinson IP: The

Wnt pathway, epithelial–stromal interactions, and malig-

nant progression in phyllodes tumours. J Pathol 2002,

196:437-444.

P44

Proapoptotic protein Bid is regulated by phosphorylation

during anoikis and the cell cycle

J Lindsay, AP Gilmore

Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life

Sciences, University of Manchester, UK

Breast Cancer Res 2008, 10(Suppl 2):P44 (doi: 10.1186/bcr 1928)

Adhesion to the extracellular matrix is fundamental in cell survival,

proliferation and differentiation. In the absence of correct signals

from the extracellular matrix, normal cells die by a form of apoptosis

termed anoikis. These mechanisms are abrogated in invasive

breast cancer. The BH3-only protein Bid is involved in anoikis. The

full-length protein translocates to the mitochondria following cell

detachment from the matrix and this is not dependent on Bid

cleavage by caspase 8. Understanding the regulation of full-length

Bid during apoptosis will help identify ways to manipulate the

apoptotic machinery to prevent cell survival during metastasis. Bid

can be phosphorylated following a number of stimuli. We have

shown that Bid becomes dephosphorylated in epithelial cells

undergoing anoikis. We have also identified Bid as being

phosphorylated during normal cell cycle. Arresting the cell cycle at

/S results in accumulation of nonphosphorylated Bid.

Conversely, arresting cells during mitosis results in an increase in

the phosphorylated form of Bid. Mutant forms of the protein in

which potential phosphorylation sites were removed were used to

identify serine 66 as a critical site of phosphorylation. Inhibition of

the cell cycle kinase cdk1 in fibroblasts blocks Bid phosphorylation

and is therefore a potential regulatory kinase acting to control Bid

during the cell cycle. Our results indicate a novel site of regulation

in Bid at serine 66. This residue is phosphorylated during mitosis

and may act to control the sensitivity to apoptosis during this part

of the cell cycle.

Available online http://breast-cancer-research.com/supplements/10/S2

S24

P45

Breast cancer and environmental risk factors: an appraisal

of the scientific evidence

A Kortenkamp

The School of Pharmacy, University of London, UK

Breast Cancer Res 2008, 10(Suppl 2):P45 (doi: 10.1186/bcr 1929)

With a few exceptions, the number of new breast cancer cases

among women is increasing in almost all western countries.

Although lifestyle, life choices, genetics and the diet are shown to

contribute to the increase in breast cancer, the sheer number of

newly diagnosed cases cannot solely be explained by these

factors. The present review aims to evaluate evidence that environ-

mental factors, including chemical exposure, also play a role.

Studies among identical twins have shown that the most important

contributor to the causation of breast cancer is the environment

not shared by the pair, even under circumstances where the

genetic predisposition is very similar. Similarly, in families with a

heritable predisposition to breast cancer, time of birth, physical

activity and obesity can profoundly influence risk.

There is overwhelming evidence that oestrogens are strong

determinants of breast cancer risks. This is not limited to natural

oestrogens formed in a woman’s body, but extends to synthetic

hormones used as pharmaceuticals, such as those used for the

alleviation of menopausal symptoms. The demonstration of breast

cancer risks from oestrogen-only and, more pronounced, from

combined oestrogen–progesterone regimens is a case in point.

Very recent decreases in breast cancer incidence in the USA and

in parts of Germany could even be linked to a dropping off of

hormone therapy use.

To date, studies carried out to examine whether certain environ-

mental chemicals are implicated in breast cancer could neither

prove nor rule out a possible link. But to avoid wrongly dismissing a

role for chemicals in breast cancer, two issues must be addressed.

First, the available studies have largely focused on single

chemicals and have ignored the possibility that large numbers of

agents may act in concert. Recent evidence from Spain strongly

suggests that cumulative exposure to oestrogenic chemicals is

associated with breast cancer risks. Second, instead of looking at

exposures later in a woman’s life, when the breast tissue is less

vulnerable, critical periods of vulnerability during puberty and

development in the womb must be considered. Very recent studies

demonstrating breast cancer risks from exposure to the pesticide

DDT during puberty and from exposure to the oestrogenic anti-

miscarriage drug DES further underline the importance of chemical

exposure in breast cancer.

Taken together, there is a case for abandoning the view of breast

cancer as solely a lifestyle and genetic disease. It is necessary to

take account of the role of environmental factors, especially

chemical exposures. With UK breast cancer incidence at an all

time high, risk reduction will not be achievable without considering

preventable causes, such as exposure to chemicals.

P46

Assessment of angiogenesis in the hyperplasia

preinvasive, invasive breast carcinoma sequence

JE Bluff

, SS Cross

, NJ Brown

, MW Reed

, CA Staton

Microcirculation Research Group, Academic Unit of Surgical

Oncology, School of Medicine and Biomedical Sciences, University

of Sheffield, UK;

Department of Pathology, School of Medicine

and Biomedical Sciences, University of Sheffield, UK

Breast Cancer Res 2008, 10(Suppl 2):P46 (doi: 10.1186/bcr 1930)

Background Tissue factor (TF), the primary initiator of coagulation,

has been shown to stimulate angiogenesis, which is crucial for the

development and metastasis of solid tumours, in part by

upregulating vascular endothelial growth factor A (VEGF).

Angiogenesis in invasive breast cancer is well documented, but

little is known about the role of angiogenesis in premalignant

breast disease, or when the angiogenic switch occurs during the

development of breast malignancy. This study therefore quantifies

angiogenesis, VEGF and TF in the hyperplasia, preinvasive,

invasive breast carcinoma sequence.

Method One hundred and eighty-seven serial sections of normal

human breast (n = 12), benign hyperplastic breast (usual ductal

hyperplasia; n = 35), premalignant hyperplastic breast (atypical

ductal hyperplasia; n = 31), preinvasive cancer (ductal carcinoma

in situ; low/intermediate grade, n = 23; high grade, n = 43) and

invasive breast cancer specimens (n = 43) were immunohisto-

chemically stained for CD31 (pan endothelial cell (EC) marker),

endoglin (proliferating EC marker), VEGF and TF. The microvessel

density (MVD), a surrogate marker for angiogenesis, was quantified

using Chalkley grid analysis. VEGF staining was assessed semi-

quantitatively and TF expression was graded as present or absent.

Results CD31 staining was observed in ECs in all of the breast

specimens observed. There was a significant increase in MVD

between normal and hyperplastic/preinvasive breast cancer tissue

(P < 0.005) and between preinvasive and invasive carcinomas

(P < 0.0005), which was associated with a significant increase in

VEGF expression in breast epithelial (P < 0.0005) and tumour

cells, respectively (P < 0.0005). The significant increase in MVD

observed between preinvasive and invasive cancers was also

associated with a significant increase in TF expression in invasive

tumour cells (P < 0.0005). In contrast to CD31 staining, endoglin

was not expressed in normal breast, but was expressed by ECs in

11% of usual ductal hyperplasia cases, 13% of atypical ductal

hyperplasia cases, 17% and 26% of ductal carcinoma in situ

cases (low/intermediate grade and high grade, respectively) and

81% of invasive breast cancer specimens. A significant increase in

the number of proliferating ECs was seen in invasive cancers

compared with all the classes of breast tissue examined

(P < 0.0005). Moreover, the significant increase in proliferating

ECs seen between preinvasive and invasive carcinomas was

associated with a significant increase in VEGF and TF expression

in invasive tumour cells (P < 0.0005). There was evidence for a

close association between VEGF and TF in tumour cells of invasive

cancers (P = 0.007) and between VEGF and TF in ECs

(P < 0.0005), suggesting a role for both in angiogenesis.

Conclusion These data indicate that angiogenesis is initiated at

the earliest stages of dysplasia and increases rapidly between

preinvasive and invasive cancer. VEGF and TF expression patterns

suggest these factors play a role in this process.

Breast Cancer Research May 2008 Vol 10 Suppl 2 Breast Cancer Research 2008

S25

P47

Altered myoepithelial cell expression and function in

cancer-containing breasts

V Modes

, CM Rodrigues

, N Nyquist

, JA Shaw

, JL Jones

RA Walker

Department of Cancer Studies and Molecular Medicine, University

of Leicester, RKCSB, Leicester Royal Infirmary, Leicester, UK;

Centre for Tumour Biology, Institute of Cancer and CR-UK

Clinical Centre, Barts and The London, UK

Breast Cancer Res 2008, 10(Suppl 2):P47 (doi: 10.1186/bcr 1931)

We have previously identified that the noninvolved tissue from

breasts that contains a cancer (NTCCB) differs from age-matched

normal breast from women without cancer, having lower apoptotic

indices [1] and altered expression of epidermal growth factor

receptor [2] and β

integrin [3]. Both of the latter are proteins

expressed by myoepithelial cells. The myoepithelial cell is now

recognised as being important in the regulation of growth, apoptosis