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S1
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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
© 2008 BioMed Central Ltd
KEYNOTE LECTURES
Abstracts not available at time of publication.
L1
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)
L2
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)
L3
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
O1
Use of BRCA1 protein:protein interactions to classify
cancer risk
JR Morris
1
, SV Tavtigian
2
1
Department of Medical & Molecular Genetics, King’s College
London, UK;
2
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.
S2
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.
O2
Dietary patterns across the life course, mammographic
density and implications for breast cancer: results from a
British prospective cohort
G Mishra
1
, I dos Santos Silva
2
, S McNaughton
3
, V McCormack
2
,
R Hardy
1
, A Stephen
4
, D Kuh
1
1
MRC National Survey of Health and Development, Department of
Epidemiology and Public Health, University College London,
London, UK;
2
Department of Epidemiology and Population Health,
London School of Hygiene and Tropical Medicine, London, UK;
3
Centre for Physical Activity & Nutrition Research, Deakin University,
Australia;
4
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
12
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.
O3
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
S3
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.
O4
Suppression of the NF-
κκ
B cofactor Bcl3 inhibits mammary
epithelial cell apoptosis and, in breast tumours, correlates
with poor prognosis
A Wakefield
1
, L Piggott
1
, D Croston
1
, WG Jiang
2
, R Clarkson
1
1
Life Sciences, School of Biosciences, University of Cardiff, UK;
2
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.
O5
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.
S4
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.
O6
Development of breast cancer immunotherapy using
MUC1-retargeted T lymphocytes
S Wilkie
1
, G Picco
1
, J Foster
2
, D Davies
1
, S Julien
1
, L Cooper
1
,
S Arif
3
, S Mather
2
, J Taylor-Papadimitriou
1
, J Burchell
1
, J Maher
1,4
1
Breast Cancer Biology Group, King’s College London, Guy’s
Hospital, London, UK;
2
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;
3
Department of Immunobiology, King’s College London, Guy’s
Hospital, London, UK;
4
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.
S5
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
P1
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
P2
Two functionally distinct epithelial progenitors exist within
the luminal cell compartment of the mouse mammary gland
J Stingl
1
, CJ Eaves
1,2
, CJ Watson
1
1
Department of Pathology, University of Cambridge, Cambridge,
UK;
2
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.
P3
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
S6
meiosis. These results lead us to suggest that BRCA2 is a
universal regulator of RAD51/DMC1 recombinase actions.
P4
Lineage commitment in mammary epithelium is regulated
by type 2 cytokines and Stat6
WT Khaled
1
, SE Nicholson
2
, FO Baxter
1
, N Sprigg
2
, JP Stingl
1
,
ANJ McKenzie
3
, CJ Watson
1
1
Department of Pathology, University of Cambridge, UK;
2
Division
of Cancer and Haematology, The Walter and Eliza Hall Institute of
Medical Research, Parkville, Victoria, Australia;
3
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).
P5
Topoisomerase II expression as a determinant of
chromosomal radiosensitivity and possible susceptibility in
breast cancer
PE Bryant
1
, AC Riches
1
, S Terry
1
, O Shovman
1
, D Adamson
2
1
Bute Medical School, University of St Andrews, UK;
2
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
2
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
2
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
2
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
G
2
-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.
P6
Chromosome translocations in breast cancer
K Howarth
1
, K Blood
1
, B Ng
2
, J Beavis
1
, Y Chua
1
, S Cooke
1
,
J Pole
1
, S Chin
3
, K Ichimura
4
, VP Collins
4
, I Ellis
5
, C Caldas
3
,
N Carter
2
, PAW Edwards
1
1
Department of Pathology, Hutchison/MRC Research Centre,
University of Cambridge, UK;
2
The Wellcome Trust Sanger
Institute, Cambridge, UK;
3
Cancer Research UK Cambridge
Research Institute, Cambridge, UK;
4
Division of Molecular
Histopathology, Department of Pathology, University of Cambridge,
UK;
5
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
S7
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.
P7
ZNF366 is a novel corepressor for estrogen receptor alpha
that mediates its effects through interaction with CtBP
S Ali
1
, M Periyasamy
1
, J Lopez-Garcia
1
, RS Thomas
1
,
M Christian
2
, MG Parker
2
, L Buluwela
1
1
Department of Oncology, Imperial College London, UK;
2
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.
P8
A novel role for C-terminal binding proteins in the
regulation of mitotic fidelity in breast cancer cells
LM Bergman
1
, CN Birts
1
, M Darley
1
, B Gabrielli
2
, JP Blaydes
1
1
Somers Cancer Research Building, University of Southampton,
MP824 Southampton General Hospital, Southampton, UK;
2
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
S8
P9
RASSF2 can suppress the growth of breast cancer cell
lines and is epigenetically inactivated in breast tumours
WN Cooper
1
, RE Dickinson
1
, A Dallol
1
, LB Hesson
1
, I Bieche
2
,
GJ Clark
3
, ER Maher
1
, ER Zabarovsky
4
, F Latif
1
1
Department of Medical and Molecular Genetics, Institute of
Biomedical Research, University of Birmingham, Edgbaston,
Birmingham, UK;
2
Laboratoire d’Oncogenetique-INSERM E0017,
Centre Rene Huguenin, St-Cloud, France;
3
JG Brown Cancer
Center, Department of Medicine, Molecular Targets Group,
University of Louisville, KY, USA;
4
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,
UK
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
1
, Y Ito
2
, JCM Pole
1
, S-F Chin
2
, IO Ellis
3
, C Caldas
2
,
MJ O’Hare
4
, AM Murrell
2
, PAW Edwards
1
1
Department of Pathology, University of Cambridge, Hutchison/
MRC Research Centre, Cambridge, UK;
2
CR-UK Cambridge
Research Institute, Cambridge, UK;
3
Department of Pathology,
University of Nottingham, UK;
4
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
S9
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
1
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
1
, M Zollo
2
, T Dale
1
1
Cardiff School of Biosciences, Cardiff University, Cardiff, UK;
2
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
1
, E Ahamed
2
, NC Wortham
1
, S Ali
2
1
Division of Pathology and Neuroscience, University of Dundee,
Ninewells Medical School, Dundee, UK;
2
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
1
, V Heath
1
, M-A Pringle
1
, AK Bell
1
, D Crighton
2
,
K Ryan
2
, G Inman
2
, T Stein
1
, B Gusterson
1
1
Division of Cancer Sciences and Molecular Pathology, University
of Glasgow, UK;
2
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
1
-induced
transcript 4) and TGFβ
3
[1]. TGFβ
3
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
3
and whether it is involved in or necessary for
TGFβ-induced apoptosis. We further show that TSC-22 can
enhance TGFβ
3
-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
1
, S Jugurnauth
1
, L Matthews
2
, I Giddings
2
,
E Bancroft
1,3
, R Williams
4
, M Girolami
5
, C Campbell
6
,
Carrier Clinic Collaborators
3
, RA Eeles
1,3
1
Translational Cancer Genetics, The Institute of Cancer Research,
Sutton, UK;
2
Molecular Carcinogenesis, The Institute of Cancer
Research, Sutton, UK;
3
The Royal Marsden NHS Foundation Trust,
London, UK;
4
Paediatric Oncology, The Institute of Cancer
Research, Sutton, UK;
5
Bioinformatics Research Centre, University
of Glasgow, UK;
6
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
1
, I Cherhukhin
1
, F Docquier
1
, D Farrar
1
,
E Pugacheva
2
, A Vostrov
2
, E Klenova
1
1
Department of Biological Sciences, University of Essex,
Colchester, UK;
2
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
1
, ES Dornan
1
, MM Donaldson
1
, M MacFarlane
1
,
P Herzyk
2
, IM Morgan
1
1
Insitute of Comparative Medicine, University of Glasgow Faculty of
Veterinary Medicine, Glasgow, UK;
2
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
2
/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
1
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
1
are both essential for normal
breast development and mutation or aberration of their expression
is associated with breast cancer [1,2]. Cyclin D
1
is best known as
a G
1
cyclin where it regulates the G
1
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
1
levels in
G
2
/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
1
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
1
protein levels are inversely related to BRCA1 and
BARD1 levels in several model systems. Furthermore, regulation of
cyclin D
1
levels occurs through a post-transcriptional mechanism
and requires the ligase activity of BRCA1. Interestingly, this
phenomenon is cell-cycle regulated, occurring in G
2
/M.
Conclusion We propose that cyclin D
1
is a potential substrate for
BRCA1 ubiquitination and that this targets cyclin D
1
for
proteasomal-mediated degradation. Future work will focus on
ascertaining the functional consequence of cyclin D
1
regulation by
the BRCA1–BARD1 complex; in particular, the impact of BRCA1,
mediated through regulation of cyclin D
1
, 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
1
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
1
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
1
, S Duss
2
, RD Iggo
1,2
1
Bute Medical School, University of St Andrews, UK;
2
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
1
, E Cosimo
1
, P Smith
2
, R Simon
3
, K Price
4
, L Baird
5
,
AK Bell
1
, G Sauter
3
, T Crook
2
, BA Gusterson
1
1
Division of Cancer Sciences and Molecular Pathology, Western
Infirmary, University of Glasgow, UK;
2
Breakthrough Breast Cancer
Centre, Institute of Cancer Research, London, UK;
3
Department of
Pathology, University Medical Center Hamburg–Eppendorf,
Hamburg, Germany;
4
Frontier Science and Technology Research
Foundation, Boston, MA, USA;
5
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)
5end 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 5UTR 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
v
ββ
6
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
6
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
6
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
β
6
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
β
6
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
6
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
4
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
v
β
6
(DCIS-1089 MEC) demonstrated enhanced
binding and migration to the β
6
ligand LAP, and activation of a
transforming growth factor beta reporter, indicating that the α
v
β
6
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
v
β
6
. 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
6
-
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
1
, MR Christiaens
3
,
S Ye
4
, R Paridaens
1,3
1
Laboratory for Experimental Oncology, KU Leuven, Belgium;
2
Biomedical Research Centre, School of Biological Sciences,
University of East Anglia, Norwich, UK;
3
Multidisciplinary Breast
Center, University Hospitals Leuven, Belgium;
4
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
3
, RC Laxton
4,5
, XO Shu
3
, C Hodgkinson
4
,
W Hendrickx
1
, EG Pearce
5
, YT Gao
6
, AC Pereira
7
, R Paridaens
1
,
W Zheng
3
, S Ye
4,5
1
Laboratory for Experimental Oncology, KU Leuven, Belgium;
2
Biomedical Research Centre, School of Biological Sciences,
University of East Anglia, Norwich, UK;
3
Department of Medicine,
Vanderbilt Epidemiology and Cancer Centre, Vanderbilt University
School of Medicine, Nashville, TN, USA;
4
William Harvey Research
Institute, Barts and The London School of Medicine, London, UK;
5
Human Genetics Division, University of Southampton, UK;
6
Department of Epidemiology, Shanghai Cancer Institute,
Shanghai, China;
7
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
1
, R Mohammed
1
, M Salmi
2
, M Pepper
3
, EC Paish
1
,
I Ellis
1
, P Patel
1
, SG Martin
1
1
University of Nottingham, UK;
2
Turku University, Finland;
3
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
1
-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
1
-integrin chain, FAK and paxillin;
and, furthermore, promoted a rapid increase in the activation status
of the β
1
-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
1
-integrin and α
4
β
1
-
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
1
, S Pilgrim
1
, A Gutiérrez-Fernández
2
,
XS Puente
2
, C López-Otín
2
, JL Jones
3
, D Holliday
3
, PN Span
4
,
F Sweep
4
, DR Edwards
1
1
University of East Anglia, Norwich, UK;
2
Universidad de Oviedo,
Spain;
3
University of London, UK;
4
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
2
,
MB Peter
2,3
, K Horgan
3
, V Speirs
2
, AM Hanby
1,2
, TA Hughes
2
1
Department of Pathology, St James University Hospital, Leeds,
UK;
2
Leeds Institute of Molecular Medicine, Wellcome Trust
Brenner Building, University of Leeds, UK;
3
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
G
1
/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
1
, SS Cross
2
, NJ Brown
1
, MW Reed
1
, CA Staton
1
1
Microcirculation Research Group, Academic Unit of Surgical
Oncology, School of Medicine and Biomedical Sciences, University
of Sheffield, UK;
2
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
1
, CM Rodrigues
1
, N Nyquist
1
, JA Shaw
1
, JL Jones
2
,
RA Walker
1
1
Department of Cancer Studies and Molecular Medicine, University
of Leicester, RKCSB, Leicester Royal Infirmary, Leicester, UK;
2
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
4
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
and differentiation of luminal epithelial cells. Our hypothesis is that
altered myoepithelial cell function could lead to reduced apoptosis
and therefore a lower ability of the breast to remove cells with DNA
damage, predisposing to cancer development.
To investigate the expression of myoepithelial proteins, immuno-
histochemistry was used to examine two series of NTCCB and
equal numbers of age-matched normal breast controls, with a total
of 180 tissues assessed. FGF2, IGF1, oestrogen receptor beta,
p63, 14-3-3σ, glucocorticoid receptor and maspin were
investigated. There was a significant difference in the expression of
FGF2 (P = 0.02), with NTCCB having greater staining in both
series of tissues. p63 was significantly different (P = 0.008) in one
series but not the other. None of the other proteins showed a
significant difference between the NTCCB and controls.
Myoepithelial cells are isolated from reduction mammoplasties and
noninvolved tissue from mastectomies using positive selection [4].
Purity and changes in expression