Expression of STAT1 target genes and interferon gamma in human mammary carcinoma tissue

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Speaker abstracts
S.01
The challenges in translating present knowledge of
the molecular biology of breast cancer into clinical
use
N Davidson
Johns Hopkins University, USA
Breast Cancer Research 2005, 7(Suppl 2):S.01 (DOI 10.1186/bcr1044)
Abstract not submitted.
S.02
Stromal and epithelial TGF-β β signaling in mammary
tumorigenesis
HL Moses, N Cheng, A Chytil, AE Gorska, M Aakre, E Forrester,
EG Neilson, NA Bhowmick
Vanderbilt-Ingram Cancer Center, Department of Cancer Biology and
Department of Medicine, Vanderbilt University Medical Center,
Nashville, Tennessee, USA
Breast Cancer Research 2005, 7(Suppl 2):S.02 (DOI 10.1186/bcr1045)
There is compelling evidence from transgenic mouse studies and
analysis of mutations in human carcinomas indicating that the TGF-β
signal transduction pathway is tumor suppressive. We have shown that
overexpression of TGF-β1 in mammary epithelial cells suppresses the
development of carcinomas and that expression of a dominant negative
type II TGF-β receptor (DNIIR) in mammary epithelial cells under
control of the MMTV promoter/enhancer increases the incidence of
mammary carcinomas. Studies of human tumors have demonstrated
inactivating mutations in human tumors of genes encoding proteins
involved in TGF-β signal transduction, including DPC4/Smad4,
Smad2, and the type II TGF-β receptor (TβRII). There is also evidence
that TGF-β can enhance the progression of tumors. This hypothesis is
being tested in genetically modified mice. To attain complete loss of
TβRII, we have generated mice with loxP sites flanking exon 2 of Tgfbr2
and crossed them with mice expressing Cre recombinase under
control of the MMTV promoter/enhancer to obtain Tgfbr2mgKOmice.
These mice show lobuloalveolar hyperplasia. Mice are being followed
for mammary tumor development. Tgfbr2mgKOmice that also express
polyoma virus middle T antigen under control of the MMTV promoter
(MMTV-PyVmT) develop mammary tumors with a significantly shorter
latency than MMTV-PyVmT mice and show a marked increase in
pulmonary metastases. Our data do not support the hypothesis that
TGF-β signaling in mammary carcinoma cells is important for invasion
and metastasis, at least in this model system.
The importance of stromal–epithelial interactions in mammary gland
development and tumorigenesis is well established. These interactions
probably involve autocrine and paracrine action of multiple growth
factors, including members of the TGF-β family, which are expressed in
both stroma and epithelium. Again, to accomplish complete knockout
of the type II TGF-β receptor gene in mammary stromal cells, FSP1-Cre
and Tgfbr2flox/floxmice were crossed to attain Tgfbr2fspKOmice. The
loss of TGF-β responsiveness in fibroblasts resulted in intraepithelial
neoplasia in prostate and invasive squamous cell carcinoma of the
forestomach with high penetrance by 6 weeks of age. Both epithelial
lesions were associated with an increased abundance of stromal cells.
Activation of paracrine hepatocyte growth factor (HGF) signaling was
identified as one possible mechanism for stimulation of epithelial
proliferation. TGF-β signaling in fibroblasts thus modulates the growth
and oncogenic potential of adjacent epithelia in selected tissues.
More recently, we have examined the effects of Tgfbr2fspKOfibroblasts
on normal and transformed mammary epithelium. We analyzed the role
of TGF-β signaling by stromal cells in mammary tumor progression. To
avoid the possibility of endogenous wild-type fibroblasts masking
potential effects of Tgfbr2fspKOcells on tumor progression, we
implanted PyVmT mammary carcinoma cells with Tgfbr2fspKOor wild-
type fibroblasts in the subrenal capsule of nude mice. Mammary tumor
cells implanted with Tgfbr2fspKOcells exhibited an increase in tumor
growth and intravasation associated with an increase in tumor cell
survival, proliferation and an increase in tumor angiogenesis compared
with tumor cells implanted with control fibroblasts. We demonstrated
increased expression of several growth factors by Tgfbr2fspKOfibroblasts
compared with control fibroblasts in primary culture. These included
HGF, MSP and TGF-α. There was an increase in tumor cell activating
phosphorylation of the cognate receptors, c-Met, RON, erbB1, and
erbB2 in carcinomas accompanied by Tgfbr2fspKOfibroblasts.
The Tgfbr2fspKOmouse model illustrates that a signaling pathway
known to suppress cell-cycle progression when activated in epithelial
cells can also have an indirect inhibitory effect on epithelial proliferation
when activated in adjacent stromal fibroblasts in vivo. Loss of this
inhibitory effect can result in increased epithelial proliferation and may
even progress to invasive carcinoma in some tissues.
S.03
Genomic analysis of human breast cancer in families
and populations
M-C King
University of Washington, USA
Breast Cancer Research 2005, 7(Suppl 2):S.03 (DOI 10.1186/bcr1046)
Abstract not submitted.
S.04
Abstract withdrawn.
S.05
ATM mutations associated with breast cancer
RA Gatti1, P Concannon2
1UCLA School of Medicine, Department of Pathology and Laboratory
Medicine, Los Angeles, California, USA; 2Benaroya Research Institute
at Virginia Mason, Seattle, Washington, USA
Breast Cancer Research 2005, 7(Suppl 2):S.05 (DOI 10.1186/bcr1048)
Despite over a decade of scrutiny and over 20 published reports from
various countries, the degree to which ATM mutations lead to breast
Breast Cancer Research Volume 7 Supplement 2, June 2005
Meeting abstracts
The Third International Symposium on the
Molecular Biology of Breast Cancer
Molde, Norway
22–26 June 2005
Received: 15 April 2005 Published: 17 June 2005
© 2005 BioMed Central Ltd

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cancer in the general population remains unclear. Furthermore, the
methodology of ATM mutation detection is still laborious and costly.
Because the ATM protein kinase phosphorylates such a wide array of
downstream targets, many pathways to oncogenesis are possible and
largely unexplored. What seems clear is that: A-T heterozygotes are at
a fourfold to fivefold increased risk of breast cancer, although
confidence intervals are large; and the spectrum of ATM mutations is
distinct for A-T families versus breast cancer cohorts. Only a handful of
mutations have been identified in both A-T families and breast cancer
cohorts. Missense mutations represent <10% of mutations in A-T
patients and >80% in breast cancer cohorts. ATM missense mutations
are also more common in some leukemias and lymphomas.
Experimental data suggest that some missense mutations represent
dominant interfering mutations [1-5]; however, clinical support for a
dominant interfering model is minimal in family studies, suggesting
either that the model is flawed or that penetrance of these mutations is
very low. Histological classifications of breast cancer are largely
grouped as genetically homogeneous models, although expression
microarray data suggest otherwise. Other studies have associated
ATM-SNPs with increased breast cancer risk; however, just three SNP
haplotypes across the ATM locus include ~95% of a global population,
and this must be factored into such association models. Without the
benefit of mRNA analyses, of minigene experiments, of Maximum
Entropy Scores, of site-directed mutagenesis or of functional assays of
ATM activity, most ‘missense’ mutations cannot be reliably
distinguished from polymorphisms or from other types of mutations,
such as splicing variants that lead to secondary stop codons. Our
recent analyses have focused on two ATM missense mutations,
7271T>G and IVS10-6T>G. For each of these mutations, there are
published functional data suggesting that they act as dominant
interfering mutations, and epidemiological data suggesting a role in
breast cancer. Some family studies of the 7271T>G mutation suggest
that it is a highly penetrant breast cancer susceptibility allele. However,
its infrequency in the population means that its contribution to breast
cancer risk is slight and it is possible that 7271T>G represents only
one of a diverse array of uncommon ATM mutations leading to
increased cancer risk. We found that the frequency of the IVS10-6T>G
mutation was not increased in breast cancer cases as compared with
controls. Furthermore, the evidence that IVS10-6T>G is an A-T
mutation is called into question by our recent evidence that, in the one
known example of a homozygous IVS10-6T>G individual with A-T, a
homozygous mutation at 5644C>T was also present (Purayidom and
colleagues, submitted). Taken together, these studies suggest that
whereas no single ATM mutation impacts significantly upon breast
cancer risk, it may be possible to group mutations that do modulate risk
for breast cancer based on their phenotypic effects. This group of
patients might benefit substantially from a therapeutic approach to
correct missense mutations.
Acknowledgements These efforts were partially funded by NIH grant
NS35322 and the A-T Medical Research Foundation, Los Angeles,
California, USA.
References
1. Gatti RA, Tward A, Concannon P: Cancer risk in ATM heterozy-
gotes: a model of phenotypic and mechanistic differences
between missense and truncating mutations. Mol Biol Metab
1999, 68:419-423.
2. Spring K, Ahangari F, Scott SP, Waring P, Purdie DM, Chen PC,
Hourigan K, et al.: Mice heterozygous for mutation in Atm, the
gene involved in ataxia-telangiectasia, have heightened sus-
ceptibility to cancer. Nat Genet 2002, 32:185-190.
3. Scott SP, Bendix R, Chen P, Clark R, Dork T, Lavin MF: Missense
mutations but not allelic variants alter the function of ATM by
dominant interference in patients with breast cancer. Proc Natl
Acad Sci USA 2002, 99:925-930.
4. Concannon P: ATM heterozygosity and cancer risk. Nat Genet
2002, 32:89-90.
5.Chenevix-Trench G, Spurdle AB, Gatei M, Kelly H, Marsh A, Chen
X, Donn K, et al.: Dominant negative ATM mutations in breast
cancer families. J Natl Cancer Inst 2002, 94:205-215.
S.06
DNA damage response pathways in cancer causation
and treatment
MB Kastan, R Kitagawa, CJ Bakkenist
Department of Hematology–Oncology, St Jude Children’s Research
Hospital, Memphis, Tennessee, USA
Breast Cancer Research 2005, 7(Suppl 2):S.06 (DOI 10.1186/bcr1049)
Cellular responses to DNA damage impact many aspects of cancer
biology. First, damage to cellular DNA causes cancer. We know this
from epidemiologic studies, from animal models, and from the
observation that many human cancer susceptibility syndromes arise
from mutations in genes involved in DNA damage responses. For
example, the genes mutated in Fanconi’s anemia, ataxia-telangiectasia,
xeroderma pigmentosum, Li–Fraumeni syndrome, hereditary breast and
ovarian cancers, and hereditary non-polyposis colon cancer are all
involved in DNA damage responses. Second, DNA damage is used to
cure cancer. The majority of the therapeutic modalities that we
currently use to treat malignancies target the DNA, including radiation
therapy and many chemotherapeutic agents. Third, DNA damage is
responsible for the majority of the side effects of therapy. Bone marrow
suppression, GI toxicities, and hair loss are all attributable to DNA
damage-induced cellular apoptosis of proliferating progenitor cells in
these tissues. Thus, DNA damage causes the disease, is used to treat
the disease, and is responsible for the toxicity of therapies for the
disease. Significant progress has been made in recent years in
elucidating the molecular controls of cellular responses to DNA
damage in mammalian cells. These insights now provide us with
approaches to attempt to manipulate these responses for patient
benefit, such as enhanced tumor cell kill with therapy, protection of
normal tissues from toxic effects of therapy, and even prevention of
cancer development.
Many of the insights that we have gained into the mechanisms involved
in cellular DNA damage response pathways have come from studies of
human cancer susceptibility syndromes that are altered in DNA
damage responses. One of these disorders, ataxia-telangiectasia (A-T),
is characterized by multiple physiologic abnormalities, including
neurodegeneration, immunologic abnormalities, cancer predisposition,
sterility, and metabolic abnormalities. The gene mutated in this
disorder, Atm, is a protein kinase that is activated by the introduction of
DNA double-strand breaks in cells. Atm activity is required for cell cycle
arrests induced by ionizing irradiation (IR) in G1, S, and G2 phases of
the cell cycle. Several targets of the Atm kinase have been identified
that participate in these IR-induced cell cycle arrests. For example,
phosphorylation of p53, mdm2, and Chk2 participate in the G1
checkpoint; Nbs1, Brca1, FancD2, and Smc1 participate in the
transient IR-induced S-phase arrest; and Brca1 and hRad17 have been
implicated in the G2/M checkpoint. Although Atm is critical for cellular
responses to IR, related kinases, such as Atr, appear to be important
for responses to other cellular stresses [1]. Some substrates appear to
be shared by the two kinases, with the major difference being which
stimulus is present and which kinase is used to initiate the signaling
pathway.
Characterization of these Atm substrates permitted us to manipulate
these proteins in cell lines and to selectively abrogate single or multiple
checkpoints. Using this approach, we demonstrated that abrogation of
checkpoints does not by itself result in radiosensitivity. Although this
has been known for several years in regards to the S-phase
checkpoint, it was a surprising finding that abrogation of the G2/M
checkpoint did not cause radiosensitivity. This observation suggested
that some other function of Atm, other than checkpoint control, was
important for cellular survival following ionizing irradiation. In
characterizing targets of the Atm kinase, the only substrate whose
phosphorylation seems to impact on radiosensitivity is Smc1 [2]. We
previously demonstrated that the phosphorylation of Smc1 by ATM
required the presence of both Nbs1 and Brca1 proteins. We recently
found that this dependence results from the role that these two
proteins play in recruiting both Smc1 protein and activated Atm to the
sites of DNA breaks. We generated mice in which the two Atm
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phosphorylation sites in the Smc1 protein are mutated; cells from these
mice demonstrate normal ATM activation, normal phosphorylation of
both Nbs1 and Brca1 after IR, and normal migration of these proteins
to DNA breaks [3]. Despite these normal activities of Atm, Nbs1 and
Brca1, these cells exhibit a defective S-phase checkpoint,
radiosensitivity, and increased chromosomal breakage after IR similar
to that seen in cells lacking Atm. These results suggest that the
phosphorylation of Smc1 is the critical target of this signaling pathway
for these endpoints, and that the reason why cells lacking Nbs1 and
Brca1 are radiosensitive and exhibit chromosomal breakage is due to a
failure to recruit Smc1 to the sites of DNA breaks where it gets
phosphorylated by previously activated Atm.
Recent studies also elucidated the mechanism by which DNA damage
activates the Atm kinase and initiates these critical cellular signaling
pathways [4]. Atm normally exists as an inactive homodimer bound to
nuclear chromatin in unperturbed cells, and introduction of DNA
damage induces intermolecular autophosphorylation on serine 1981 in
both Atm molecules. This phosphorylation causes a dissociation of the
Atm molecules and frees it up to now circulate around the cell and
phosphorylate the substrates that regulate cell cycle progression and
DNA repair processes. This regulation of Atm activity in the cell
represents a novel mechanism of protein kinase regulation and appears
to result from alterations in higher order chromatin structure rather than
direct binding of Atm to DNA strand breaks. Although Nbs1 and Brca1
are not required for the initial activation of Atm after IR, these two
proteins are required for the migration of activated Atm to the sites of
DNA breaks. It is this process of recruitment of activated Atm along with
Smc1 recruitment to the DNA breaks that leads to Smc1 phosphorylation
by Atm and presumably initiation of some repair process(es) that reduce
chromosomal breakage and enhance cell survival.
References
1.Bakkenist CJ, Kastan MB: Initiating cellular stress responses.
Cell 2004, 118:9-17.
2. Kim S-T, Xu B, Kastan MB: Involvement of the cohesin protein,
Smc1, in Atm-dependent and independent responses to DNA
damage. Genes Dev 2002, 16:560-570.
3. Kitagawa R, Bakkenist CJ, McKinnon PJ, Kastan MB: Phosphory-
lation of SMC1 is a critical downstream event in the ATM–
NBS1–BRCA1 pathway. Genes Dev 2004, 18:1423-1438.
4. Bakkenist CJ, Kastan MB: DNA damage activates ATM through
intermolecular autophosphorylation and dimer dissociation.
Nature 2003, 421:499-506.
S.07
SNPS in putative regulatory loci controlling gene
expression in cancer
VN Kristensen
Department of Genetics, Institute for Cancer Research, The Norwegian
Radium Hospital, Oslo, Norway
Breast Cancer Research 2005, 7(Suppl 2):S.07 (DOI 10.1186/bcr1050)
Given the increasing clinical importance of microarray expression
classification of breast tumours and the different biology it may reveal
[1], identifying an associated SNP profile may be of considerable value
for pharmacogenetics, early diagnostics and cancer prevention.
Studying the promoter composition of the genes that strongly predict
the patient subgroups, we observed clear separation of the gene
clusters based solely on their promoter composition, making feasible
the hypothesis that SNPs in the regulatory regions of genes that create
or abrogate transcription binding sites have the potential to influence
the expression profiles. Morley and colleagues [2] reported linkage
analysis of expression levels of 3554 genes and 2500 SNPs in 14
CEPH families (retrieved online [3]), and found significant evidence for
the existence of regulation hot spots, suggesting both cis and trans
regulatory effects. We report similar observations from a study with a
different design, performing actual genotyping of 49 unrelated breast
cancer patients, whose tumours have previously been analysed by
genome-wide expression microarrays leading to a robust tumour
classification with strong prognostic impact [4]. These patients were a
part of a pharmacogenetic study of 193 patients who had received
radiation therapy or chemotherapy. A high-throughput solid-phase,
array-based method using primer extension chemistry has been used to
perform the genotyping (GenomeLab™ SNPstream genotyping system;
Beckman Coulter, Fullerton, CA, USA). A total of 583 SNPs in 203
selected genes (1–19 SNPs/gene) were genotyped and tumour
genome-wide expression was studied in 49 patients. Association in
both cis and trans was detected for SNPs in 42 genes. SNP–
expression associations with the top 0.25% best P values (9.81 ×
10–6< P < 0.001) revealed regulatory SNPs in 115 genes in trans.
The subsets of transcripts that were observed to have significantly
many associations in common with a set of SNPs were further
analysed using the gene ontology (GO) annotations. The GO terms of
the unselected mRNA transcripts found associated to the SNPs in the
selected candidate genes were often similar, suggesting that the
observed associations are within the same functional pathway. Taken
together these data suggest that the observed SNP–expression
associations do exist and are observable even in a small set of
unrelated individuals. A given expression profile of the tumour may be
potentially associated and predicted by the genotype of the patient.
References
1. Perou CM, et al.: Nature 2000, 406:747-452.
2. Morley M, et al.: Nature 2004, 430:743-747.
3.
The SNP Consortium Ltd [http://snp.cshl.org/]
4. Sørlie T, et al.: Proc Natl Acad Sci USA 2001, 98:10869-10874.
S.08
Potential mechanisms whereby estrogens induce
breast cancer in women
RJ Santen, W Yue, J-P Wang
University of Virginia Health Sciences System, Charlottesville, Virginia,
USA
Breast Cancer Research 2005, 7(Suppl 2):S.08 (DOI 10.1186/bcr1051)
Long-term exposure to estradiol is associated with an increased risk of
breast cancer in women. The data supporting this conclusion include:
measurements of plasma total and free estradiol, estrone, and estrone
sulfate and the aromatase substrate testosterone in postmenopausal
women; the effect of oophorectomy before age 35; the effect of early
menarche and late menopause; the relationship between bone density
and breast cancer risk; and the role of menopausal hormone therapy on
risk. However, the mechanisms responsible for estradiol-induced
carcinogenesis are not firmly established. The prevailing theory
postulates that estrogens increase the rate of cell proliferation by
stimulating estrogen receptor (ER)-mediated transcription, thereby
increasing the number of errors occurring during DNA replication. An
alternative theory suggests that estradiol is metabolized to quinone
derivatives, which directly remove base pairs from DNA through a
process called depurination. Error-prone DNA repair then results in
point mutations. We postulate that both processes act in an additive or
synergistic fashion. If correct, aromatase inhibitors would block both
processes, whereas anti-estrogens would only inhibit receptor-
mediated effects. Our initial studies demonstrated that depurinating
catechol-estrogen metabolites are formed in MCF-7 human breast
cancer cells in culture. We then utilized an ERKO animal model that
allows dissociation of ER-mediated function from the effects of
estradiol metabolites, and demonstrated formation of genotoxic
estradiol metabolites. We also examined the incidence of tumors
formed in these ERα knockout mice bearing the Wnt-1 transgene. The
absence of estradiol induced by castration markedly reduced the
incidence of tumors and delayed their onset. Re-administration of
estradiol to castrate animals induced tumors in a dose-responsive
fashion. To ensure that all ER functionality was lacking, we
administered fulvestrant and demonstrated that estrogen still induced
breast tumors in these animals. On aggregate, our results support the
concept that metabolites of estradiol may act in concert with ER-
mediated mechanisms to induce breast cancer. These findings support
the possibility that aromatase inhibitors might be more effective than
anti-estrogens in preventing breast cancer. Data from four clinical
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studies have now suggested that fewer contralateral breast cancers
occur in women treated with aromatase inhibitors in the adjuvant setting
than with tamoxifen. Taken together, our data provide experimental
support for a genotoxic role for estradiol in hormonal carcinogenesis.
S.09
The future of breast cancer prevention
A Howell, A Sims, M Harvie, KR Ong, G Evans, R Clarke
CRUK Department of Medical Oncology, University of Manchester,
Christie Hospital, Manchester, UK
Breast Cancer Research 2005, 7(Suppl 2):S.09 (DOI 10.1186/bcr1052)
At present, large numbers of at-risk women are treated in order to
prevent relatively small numbers of breast cancers. There is a need to
define risk more precisely in order to target interventions and a need to
improve their efficacy. Risk estimations currently depend upon
integration of familial and endocrine risk factors. We have
demonstrated that the Tyrer–Cuzick model that takes both factors into
account more fully is superior to other risk prediction models in our
clinic [1]. However, prediction remains imprecise for the individual.
Attempts are being made to take additional risk factors into account,
including mammographic density [2], serum estradiol concentration
and bone density. It seems probable that a better understanding of the
interactions between stromal and epithelial cells in the breast including
fibroblasts, adipocytes, macrophages and blood vessels will ultimately
lead to better prediction. We have shown that 5% loss of body weight
during mid life reduces postmenopausal breast cancer risk by 40% [3],
and overviews indicate that use of NSAIDs [4] and exercise [5] may
reduce risk by approximately 30%. The mechanisms of these risk
reductions are not clear but gene array studies indicate that calorie
restriction and exercise predominantly reduce the expression of genes
related to inflammation [6,7]. This raises the question of whether all
these interventions act by similar mechanisms. A better understanding
of the mechanisms of mammographic density and mammary cell
senescence is required. Both are associated with fibroblasts that
increase and stimulate proliferation of local epithelial cells [8,9]. Since
mammographic density is a major risk factor, its reversal is likely to be
beneficial. Another stromal target is aromatase. All adjuvant aromatase
inhibitor (AI) trials have shown an approximately 50% contralateral
breast cancer reduction compared with tamoxifen [10]. Since
tamoxifen reduces contralateral risk by about 50% compared with
placebo, AIs may reduce risk by 70–80%. Trials to test this hypothesis
are underway (IBIS II, MAP3). The aforementioned considerations
indicate that the stroma and stroma–epithelial interactions are already
targets for preventive measures, and this is likely to expand and lead to
new interventions such as NF-κB inhibition [11] and SIRT1 activation
[12].
References
1. Amir E, et al.: J Med Genet 2003, 40:807.
2. Warwick J, et al.: Breast 2003, 12:10.
3. Harvie M, et al.: Cancer Epidemiol Biomarkers Prev 2005,
14:656-661.
4.Khuder SA, Mutgi AB: Br J Cancer 2001, 84:1188-1192.
5. Berglund G: IARC Sci Publ 2002, 156:237-241.
6. Clement K, et al.: FASEB J 2004, 18:1658.
7. Bronikowski A, et al.: Physiol Genomics 2003, 12:129.
8. Tlsty T: Keystone Symposium, 5 February 2005.
9. Parinello S, et al.: J Cell Sci 2005, 118:485.
10. Howell A, et al.: Lancet 2005, 365:60.
11. Greten F, et al.: Cell 2004, 118:285.
12. Howitz K, et al.: Nature 2003, 425:191.
S.10
Targeting estrogen to kill ER-positive and
ER-negative breast cancer
VC Jordan
Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA
Breast Cancer Research 2005, 7(Suppl 2):S.10 (DOI 10.1186/bcr1053)
The current fashion of using long-term antihormonal therapies for the
treatment and prevention of breast cancer has been remarkably
successful over the past 20 years but this strategy has consequences
for the development of drug resistance in remaining tumor tissue.
Although estrogen is considered to be a survival signal that causes
increased breast cancer cell replication, the study of drug resistance to
antihormonal therapies has revealed an unanticipated new biology of
estrogen action. Long-term antihormonal therapy eventually results in
either tamoxifen or raloxifene (selective estrogen receptor modulators
[SERMs]) stimulated growth and tumors are also stimulated to grow
with estrogen. This is why aromatase inhibitors are effective treatments
after the development of SERM resistance once the SERM is stopped.
Long-term estrogen deprivation initially causes a cessation of breast
tumor cell growth but eventually cells grow out that remain ER-positive
but grow spontaneously. Estrogen deprivation with SERMs or
aromatase inhibitors for more than 5 years causes a remarkable
switching of the estrogen signaling pathway [1]. Instead of being a
survival signal, physiologic concentrations of estrogen now cause
apoptosis and tumor cell death. This knowledge provides an
opportunity to test the hypothesis that low-dose estrogen therapy
following exhaustive antihormonal therapy could be used as a
successful treatment for patients. Studies are in place to evaluate the
mechanism of action of estrogen-induced apoptosis so that a new
target can be discovered to develop a novel apoptotic drug group. The
ER-negative breast cancer cell is the ultimate hormone-resistant cell.
Reintroduction of an active ER gene re-sensitizes the cells to estrogen
that now causes blockade of the cell cycle [2] and apoptosis if cell
survival signaling is also blocked. These data suggest that a universal
target could be identified using the estrogen receptor mediated
mechanism that will permit the broad application of new anti-apoptotic
medicines.
References
1.Jordan VC: Selective estrogen receptor modulation: concept
and consequences in cancer. Cancer Cell 2004, 5:207-213.
2.Jiang SY, Jordan VC: Growth regulation of estrogen receptor-
negative breast cancer cells transfected with complementary
DNAs for estrogen receptor. J Natl Cancer Inst 1992, 84:580-
591.
S.11
ERβ β in normal and malignant breast
J-Å Gustafsson, G Cheng, M Warner
Department of BioSciences and Department of Medical Nutrition,
Novum, Karolinska Institute, Huddinge, Sweden
Breast Cancer Research 2005, 7(Suppl 2):S.11 (DOI 10.1186/bcr1054)
Both ERα and ERβ are expressed in not only normal breast of the
rodent, cow, monkey and human, but also in breast cancer. Cells that
express ERα are found within the luminal epithelium, but not in the
myoepithelium or stroma in the human breast. ERβ, on the other hand,
is expressed not only in the luminal epithelial cells, but also in
myoepithelial cells, stromal cells and in passenger lymphocytes. This
widespread distribution of ERβ suggests multiple roles for ERβ in the
mammary gland.
We have shown that in the rodent mammary gland ERβ is the dominant
ER, and that, in response to E2, ERα but not ERβ is downregulated in
the early G1 phase of the cell cycle. Cells that contain ERα receive the
signal to proliferate from E2, and within 4 hours of that signal ERα is
lost from the nucleus. The cells then go through a complete cycle and
ERα reappears in daughter cells. ERβ levels do not change in cell
nuclei during the cell cycle. This pattern of ER regulation holds true in
human breast cancer since ERα is never co-localized with proliferation
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markers in breast cancer samples. This means that under the
conditions of a constant high level of E2, ERα does not reappear in the
nucleus. A similar situation exists during pregnancy when there is a
constant high level of E2 and there is no ERα in the mammary
epithelium. This resistance to the proliferative response to E2 in the
presence of a constant high dose of E2 probably explains the very
successful use of high-dose E2 in the treatment of breast cancer. ERβ,
on the other hand, appears to have a differentiative role not a
proliferative role in the mammary gland, and the lactating rodent
mammary gland of ERβ–/–mice does not express gap junction and
adhesion proteins, typical indicators of fully differentiated cells.
In recent years there have been several publications showing that ERβ
is expressed in human breast cancer, and conclusions and
speculations about a causative role for ERβ in breast cancer
development and/or progression have been made. We have studied
500 frozen breast biopsies in collaboration with Prof. RC Coombes,
London, in order to clarify the role of ERβ in normal and malignant
breast. In this study we measured ERα and ERβ proteins by several
techniques (immunohistochemistry, western blotting, ligand binding in
sucrose gradients, and RT-PCR) in various human samples obtained
from both benign breast and malignant breast. We found that ERβ is
the predominant estrogen receptor in the normal mammary gland and
in benign breast disease. There is very little ERα in the normal
mammary gland. This low expression of ERα is one of the striking
differences between rodents and humans. This is in stark contrast to
ERβ, which is expressed in 80% of epithelial cells and is also present
in the stroma.
We found that ERα is abundantly expressed in invasive and in situ
ductal carcinoma but not in medullary cancer. ERβ is also expressed in
breast cancer, both ductal and medullary.
In this study we also found that, in the human breast, the major ER in
breast stroma is ERβ. This surprising finding has necessitated several
new lines of investigation about the function of ERβ in the breast. It has
long been thought that ERα in the stroma was responsible for
secretion of growth factors in response to E2 and that these growth
factors were responsible for epithelial cell proliferation. The discovery
that it is ERβ that is present in the stroma might suggest a role of ERβ
in growth factor secretion.
S.12
Molecular approaches to understanding pregnancy-
induced protection against breast cancer
CM Blakely, SE Moody, A Stoddard, E Tombler, C Liu,
LA Chodosh
Abramson Family Cancer Research Institute, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
Breast Cancer Research 2005, 7(Suppl 2):S.12 (DOI 10.1186/bcr1055)
The marked protection against breast cancer afforded women by an
early first full-term pregnancy has important clinical implications for
designing chemopreventive approaches to breast cancer and, more
generally, for understanding how cancer susceptibility can be
modulated by normal developmental events. Epidemiologic studies
have repeatedly demonstrated that women who undergo an early first
full-term pregnancy have a significantly reduced lifetime risk of breast
cancer. Similarly, rodents that have previously undergone a full-term
pregnancy are highly resistant to carcinogen-induced breast cancer
compared with age-matched nulliparous controls. Relatively little
progress has been made, however, towards understanding the
molecular basis of this phenomenon. We have used microarray
expression profiling to identify persistent changes in gene expression in
the mouse and rat mammary gland that are induced by an early first full-
term pregnancy. Using this approach, we have isolated a panel of
genes whose expression is persistently altered in multiple strains of
mice and rats by a reproductive event known to reduce breast cancer
risk. Additional studies are underway to compare gene expression
patterns in mammary tissues from parous and nulliparous mice, rats,
and women with parity-induced changes in gene expression that are
evolutionarily conserved. Similarly, gene expression patterns in rats that
have been treated with hormonal regimens that mimic parity-induced
protection are being compared with those induced by non-protective
control regimens in order to identify genes whose expression patterns
are most closely correlated with protection. Finally, gene expression
changes induced by parity in strains of rats that exhibit different levels
of susceptibility to carcinogen-induced tumorigenesis are being
compared. These gene expression changes suggest novel hypotheses
for the mechanisms by which parity may modulate breast cancer risk
and will be useful for probing the mechanisms by which the
developmental state of the mammary gland modulates the response to
an oncogenic stimulus.
S.13
Predicting response/resistance to endocrine therapy
for breast cancer
WR Miller1, TJ Anderson1, D Evans2, A Krause2, JM Dixon1
1Breast Unit, University of Edinburgh, Western General Hospital,
Edinburgh, UK; 2Novartis Pharma AG, Femara GBTR-Research, Basel,
Switzerland
Breast Cancer Research 2005, 7(Suppl 2):S.13 (DOI 10.1186/bcr1056)
Background Endocrine therapy for breast cancer is a major modality
for the treatment of breast cancer, producing response rates between
30% and 40% of unselected patients with the minimum of toxicity.
However, the majority of patients receive no benefits and, after
successful treatment, tumour regrowth may occur. Optimal manage-
ment therefore requires accurate predictors of response and early
identification of resistance. The present article reviews results from
neoadjuvant studies in which endocrine therapy was given to patients
whose primary breast cancer was still within the breast so that
changes in tumour volume could be used to assess clinical response
and so that sequential biopsies could be taken for molecular analyses
designed to identify predictive markers.
Methods All patients had histologically confirmed breast cancer and
were treated for 3–4 months with either tamoxifen or an aromatase
inhibitor (anastrozole, exemestane or letrozole). Core or excisional
tumour biopsies were taken before and at the end of treatment (and at
10–14 days in certain studies). Oestrogen receptors (ER), progestogen
receptors and c-erbB1 and c-erbB2 were measured by immuno-
histochemistry. Microarray analysis was performed on tumour RNA
extracted and amplified before hybridization on Affymetrix HG_U133A
GeneChips for microarray analysis.
Results Steroid hormone receptor status highly influences the
response to all endocrine therapies, negative tumours failing to
respond and response being more likely with increasing levels of ER
and the concomitant presence of PgR. Conversely, tumour over-
expression of c-erbB2 (and c-erbB1) is associated with resistance to
tamoxifen but not aromatase inhibitors. While these receptors are
helpful in identifying groups of tumours with differing sensitivity to
endocrine therapy, they fail to predict accurately in individual cases. To
address this deficiency, in Edinburgh we have looked for early genetic
changes (at 10–14 days) that occur with treatment and might be
associated with subsequent response to the aromatase inhibitor
letrozole. Clinical response data were available for 43 cases, of which
33 (77%) were classified as responders (>50% reduction in tumour
volume) and 30 (70%) displayed evidence of pathological response.
No gene changed substantially with treatment in all cases; however,
there was consistent upregulation of three genes and downregulation
of 65 genes in 50 of the cases. Based on clustering techniques, it was
possible to identify highly consistent changes in gene expression with
treatment, which allowed tumours to be subdivided into groups
showing distinct patterns of molecular changes. While the change in
expression of any single gene failed to correlate with response,
significant differences in change of expression in 125 genes were
detected between non-responders and responders. A combination of
gene changes produced increased discrimination. The identity of the
genes and their relevance to the prediction of response and
mechanisms of resistance will be discussed.
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Conclusions Early changes in gene expression profiles may define
tumour groups with differing sensitivity to endocrine therapy and permit
early recognition of response and resistance. However, clinical utility at
the level of individual patients has yet to be validated and explored.
S.14
Genetic and epigenetic changes in early
carcinogenesis
TD Tlsty
Department of Pathology and UCSF Comprehensive Cancer Center,
University of California at San Francisco, California, USA
Breast Cancer Research 2005, 7(Suppl 2):S.14 (DOI 10.1186/bcr1057)
Studies of human epithelial cells and fibroblasts from healthy
individuals are providing novel insights into how early epigenetic and
genetic events affect genomic integrity and fuel carcinogenesis. Key
epigenetic changes, such as the hypermethylation of the p16 promoter
sequences, create a previously unappreciated pre-clonal phase of
tumorigenesis in which a subpopulation of epithelial cells is positioned
for progression to malignancy [1]. These key changes precede the
clonal outgrowth of premalignant lesions and occur frequently in
healthy, disease-free individuals [2]. Prior work from our laboratory has
shown that surrounding stroma can dramatically influence tumori-
genesis. Proper stromal–epithelial interactions can actually suppress
the expression of preneoplastic phenotypes in epithelial cells and,
conversely, altered stromal–epithelial interactions can promote the
probability that preneoplastic lesions progress to malignancy [3].
Understanding more about these early events should provide novel
molecular candidates for prevention and therapy of cancer.
References
1.
Nature 2001, 409:636.
2.
Cancer Cell 2004, 5:263.
3.
Cancer Res 1999, 61:5002.
S.15
A breast cancer progression model: the importance
of three-dimensional tissue architecture and
metalloproteinases
MJ Bissell, C Myers, G Lee, E Lee, A Rizki, S Mian, J Gray,
D Radisky
Life Sciences Division, Lawrence Berkeley National Laboratory,
Berkeley, California, USA
Breast Cancer Research 2005, 7(Suppl 2):S.15 (DOI 10.1186/bcr1058)
Previous studies from our laboratory have shown that non-malignant
and malignant cells can be distinguished easily and rapidly by their
morphology and growth rate when cultured in three-dimensional (3D)
laminin-rich basement membrane but not when cultured on traditional
tissue culture plastic (two-dimensional [2D]) [1,2]. In addition, we have
shown that cellular responses to signaling inhibitors and apoptotic
agents differ in cells cultured in 2D versus 3D [3,4]. This applies also
to our finding with reverted tumor cell lines [3-8]. In this presentation, I
will address two inter-related topics.
First, we asked how the 3D morphology and gene expression profiles
for a panel of 60 breast cancer cell lines for which the Gray laboratory
has obtained 2D expression as well as CGH profiles may differ, and
whether any of the surrogate genes or phenotypes could track with
response to therapy. The cell lines examined so far fell into four distinct
morphologies of ‘round’, ‘mass’, ‘grape-like’ and ‘stellate’. An ANOVA
analysis of Affymetrix gene expression profiles for each of these cell
lines was used to identify genes, the expression profiles of which could
distinguish the other known parameters of the cultured cells. Of the
22,283 genes on the Affymetrix 133A chip, ~5800 genes were
identified where expression patterns differed between different cell
lines both in 2D and 3D, and ~2000 genes were identified where
expression differed between the non-malignant and malignant cell lines.
About 700 genes differed between 2D and 3D, and ~800 correlated
with the morphological differences seen in 3D. These genes fall into a
number of functional classes, which we are currently analyzing to
identify common signaling themes and/or morphological regulators that
will be tested by manipulation of expression and correlated with
therapeutic response of these cell lines in 2D and 3D to Herceptin and
other chemotherapeutic drugs.
Second, we have also shown previously that loss of basement
membrane in both cultured mammary mouse cells [9] and in transgenic
animals led to epithelial to mesenchymal transition (EMT) and mammary
tumors [10]. We have now determined the molecular pathways
induced by MMP-3 to lead to EMT and genomic instability via
production of reactive oxygen species [11]. These mechanisms will be
discussed.
References
1. Petersen OW, Ronnov-Jessen L, Howlett AR, Bissell MJ: Proc
Natl Acad Sci USA 1992, 89:9064-9068.
2. Schmeichel KL, Bissell MJ: J Cell Sci 2003, 116:2377-2388.
3. Wang F, et al.: Proc Natl Acad Sci USA 1998, 95:14821-14826.
4. Weaver VM, et al.: Cancer Cell 2002, 2:205-216.
5. Weaver VM, et al.: [cover feature] J Cell Biol 1997, 137:231-246.
6.Wang F, et al.: J Natl Cancer Inst 2002, 94:1494-1503.
7.Liu H, et al.: J Cell Biol 2004, 164:603-612.
8. Bissell MJ, Rizki A, Mian IS: Curr Opin Cell Biol 2003, 6:753-762.
9. Lochter A, et al.: J Cell Biol 1997, 139:1861-1872.
10. Sternlicht MD, et al.: Cell 1999, 98:137-146.
11. Radisky DC, et al.: Nature 2005, in press.
S.16
Genomic and transcriptional events associated with
poor clinical responses to conventional therapies
K Chin1,2, S Devries1, J Fridlyand2, P Spellman1, W-L Kuo1,2,
A Lapuk1,2, R Neve1, T Tokuyasu2, C Kingsley2, S Dairkee3,
K Chew2, A Jain2, BM Ljung2, L Esserman2, F Waldman2,
JW Gray1,2
1Lawrence Berkeley National Laboratory, Berkeley, California, USA;
2University of California at San Francisco, California, USA; 3California
Pacific Medical Center, San Francisco, California, USA
Breast Cancer Research 2005, 7(Suppl 2):S.16 (DOI 10.1186/bcr1059)
Advances on several fronts have led to increases in survival duration
and to reduced mortality in patients with breast cancer. These include
improved procedures for earlier detection, optimization of combined
surgical and radiotherapy, and use of optimized selective estrogen
receptor modifiers (SERMS) and new chemotherapeutic strategies
including gene-targeted therapies. In addition, molecular stratification
strategies have been developed that stratify patients according to
outcome. Stratification based on measurement of expression
‘signatures’ have been particularly effective and seem likely to improve
treatment strategies. Patients at increased risk of progressive disease
can be offered standard of care chemotherapy. However, some of
these patients do not respond well to these treatments and current
stratification strategies provide little information to guide treatment of
these patients. This study of tumors from patients treated according to
standard of care identifies genomic and coordinated transcriptional
aberrations — especially amplification at 11q, and 20q in tumors with
the luminal A expression phenotype, and at 17q in tumors associated
with the ERBB2 expression phenotype — that are strongly associated
with poor response to such treatment. Our study identifies genes in
these regions of amplification that can be assessed to identify patients
that will respond poorly to the current standard of care and that are
targets for therapies that will be effective against these poorly
responding tumors. Interesting, this study also shows that patients with
basal-like tumors do not have substantially shorter survival durations
than patients with luminal-like tumors, suggesting that basal-like tumors
respond well to the adjuvant adriamycin and cyclophosphamide
therapies employed during their treatment.
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S.17
The role of the tumor microenvironment in breast
cancer progression
H Min, J Yao, M Allinen, L Cai, K Polyak
Dana-Farber Cancer Institute and Harvard Medical School, Boston,
Massachusetts, USA
Breast Cancer Research 2005, 7(Suppl 2):S.17 (DOI 10.1186/bcr1060)
We performed comprehensive molecular analysis of each cell type
composing normal breast tissue and in situ and invasive breast
carcinomas. Gene expression profiles were analyzed using serial
analysis of gene expression, genetic changes were analyzed by single
nucleotide polymorphism arrays, while epigenetic changes were
analyzed using methylation-specific digital karyotyping. Based on these
data we determined that gene expression and epigenetic changes
occur in all cell types during breast cancer progression, while genetic
alterations were only detected in tumor epithelial cells. Many of the
differentially expressed genes encode for secreted proteins and
receptors suggesting alterations in autocrine and paracrine interactions
in breast tumorigenesis. Two of these genes, the CXCL14 and
CXCL12 chemokines, overexpressed in tumor myoepithelial cells and
in myofibroblasts, respectively, bind to receptors on epithelial cells and
enhance their proliferation, migration, and invasion. Chemokines may
thus play a role in breast tumorigenesis by acting as paracrine factors.
The role of these chemokines, and myoepithelial and stromal cells in
the progression of in situ carcinomas to invasive carcinomas was
investigated using a xenograft model of human ductal carcinoma in
situ. Based on our studies we determined that changes in the tumor
microenvironment and epithelial–myoepithelial and epithelial–stromal
cell interactions play an important role in breast tumor progression.
Reference
1. Allinen M, Beroukhim R, Cai, L, Brennan C, Lahti-Domenici J,
Huang H, Porter D, Hu M, Chin L, Richardson A, et al.: Molecular
characterization of the tumor microenvironment in breast
cancer. Cancer Cell 2004, 6:17-32.
S.18
Biological features and xenograft models of a very
early human premalignant breast lesion
S Lee, Y Wu, SK Mohsin, D Medina, DC Allred
Breast Center, Baylor College of Medicine, Houston, Texas, USA
Breast Cancer Research 2005, 7(Suppl 2):S.18 (DOI 10.1186/bcr1061)
Background Most breast cancers appear to arise from certain
precursors over long periods of time. Enlargement (>50-fold) of normal
terminal duct lobular units (TDLUs) by hyperplastic epithelial cells is one
of the most common and earliest histologically recognizable alterations
with premalignant potential. Understanding how these hyperplastic
enlarged lobular units (HELUs) develop and progress could lead to new
and effective strategies for breast cancer prevention therapy.
Methods The estrogen receptor (ER) and proliferation (Ki67) were
evaluated and compared in TDLUs and HELUs in the same breasts
(n = 250) by immunohistochemistry. Apoptosis was also assessed by
the TUNEL assay. The rate of ER expression in proliferating cells was
assessed by dual-labeled immunofluorescence. Comprehensive gene
expression profiling was performed in a subset of samples (currently six
matched pairs of TDLUs and HELUs) using RNA isolated from
microdissected formalin-fixed paraffin-embedded breast tissue samples
and Affymetrix U133-X3P microarrays analyzed by dCHIP software.
Xenografts of human TDLUs and HELUs were prepared by implanting
isolated epithelial cells into cleared mammary fat pads of estrogen-
stimulated immune-compromised mice ‘humanized’ by prior local
injection of immortalized (h-tert transfected) human mammary
fibroblasts.
Results The average ER expression was significantly elevated in
HELUs compared with adjacent TDLUs (85% vs 30% positive cells,
respectively; P < 0.0001). Proliferation was significantly higher (6% vs
2%; P < 0.0001) and apoptosis was significantly lower (0.6% vs
0.2%; P < 0.001) in HELUs than TDLUs. There was a large increase in
the proportion of ER-positive proliferating cells in HELUs compared
with TDLUs (35% vs 4%; P < 0.0001). In preliminary analysis of the
microarray results, HELUs and TDLUs segregated perfectly in
unsupervised hierarchical comparisons. In supervised comparisons,
many (n = 74) genes showed >3-fold (P < 0.05) differences in
expression, with 45 relatively up (from 3.5-fold to 9.5-fold) and 29
relatively down (from 3.5-fold to 12.5-fold) in HELUs versus TDLUs.
Especially prominent elevations in HELUs included several genes
involved in G-protein signaling, the retinoic acid pathway, and
detoxification. Prominent decreases included genes involved in cell
cycle inhibition, apoptosis, differentiation, and water transport.
Differences were also noted in the expression of genes for ligands of
the epidermal growth factor receptor between HELUs and TDLUs.
Several fresh human samples of TDLUs and HELUs are in various
stages (currently up to generation four) of implantation in mice in
attempts to establish stable xenografts.
Conclusions HELUs are one of the earliest histologically recognizable
lesions in the human breast with premalignant potential. They show
striking elevations of ER, which may partially explain the hyperplasia
leading to their development from TDLUs through increased
proliferation and decreased apoptosis, which are both regulated by
estrogen. DNA microarrays reveal many additional differences in the
expression of genes involved in growth and differentiation. Human
xenograft models are under development to support mechanistic
studies of these genes to understand their roles in the development
and progression of HELUs and how to prevent it.
Acknowledgments This work was supported by funds from the Astra
Zeneca/Baylor College of Medicine Research Alliance and NIH/NCI
grant U01-CA84243.
S.19
Regulation of epithelial cell polarity during
carcinogenesis
SK Muthuswamy
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA
Breast Cancer Research 2005, 7(Suppl 2):S.19 (DOI 10.1186/bcr1062)
Pathogenesis of cancer begins as hyperplastic lesions; some lesions
remain benign, while others progress to malignancy. An increase in cell
proliferation rates and changes in tissue architecture are two properties
commonly observed in hyperplastic lesions. A great deal is known
about the molecular events that regulate cell proliferation and the
knowledge gained is widely used for development of diagnostic and
treatment tools. Our understanding of the mechanisms that deregulate
tissue architecture is poor, and hence it is understandable that the use
of architectural features to determine prognosis of early lesions has
varying success. We used polarized epithelial cells and an inducible
method of ErbB2 activation to investigate whether the cell architecture
influences ErbB2-induced gene expression and to investigate how
activation of ErbB2 disrupts epithelial cell architecture. Activation of
ErbB2 in three-dimensional epithelial acini-like structures leads to
expression of a unique set of genes that was not observed when
ErbB2 was activated in cells grown on plastic dishes, suggesting that
the cell architecture can have significant influence on ErbB2-induced
gene expression. To investigate the effect of ErbB2 activation on
epithelial architecture, we activated ErbB2 in polarized epithelial cells.
ErbB2 induced a loss in apical–basal polarity, re-initiated proliferation
and induced multilayering of epithelial sheets. These changes correlate
with the ability of ErbB2 to regulate the Par complex, a protein complex
known to regulate establishment of epithelial cell polarity. Inactivation
of atypical protein kinase C, a component of the Par complex,
cooperates with ErbB2 to disrupt polarized epithelial cells, suggesting
that the Par complex is a mediator of ErbB2-induced effects on
polarized epithelial cells. In addition, we identify tricellular junctions,
and not bicellular junctions, as a novel site for ErbB2 action in cultured
epithelial cells and in primary breast cancer. We are thus beginning to
gain novel insights into the molecular mechanisms that regulate early
lesions.
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S.20
Expression profiling of peripheral blood cells for
early detection
A-L Børresen-Dale1, P Sharma2
1Department of Genetics, Institute for Cancer Research, The Norwegian
Radium Hospital, Oslo, Norway; 2DiaGenic ASA, Oslo, Norway
Breast Cancer Research 2005, 7(Suppl 2):S.20 (DOI 10.1186/bcr1063)
Existing methods to detect breast cancer in asymptomatic patients
have limitations, and there is a need to develop more accurate and
convenient methods. Especially, an accurate method for breast cancer
detection based on peripheral blood as a clinical sample will be highly
desirable because of the easy accessibility and less-invasive nature by
which samples can be obtained.
Results demonstrating that peripheral blood can be used to develop a
gene expression based test for early detection of breast cancer will be
presented. The rationale for using blood cells as monitors for a
malignant disease elsewhere in the body is based on the hypothesis
that a malignant growth will cause characteristic changes in the
biochemical environment of blood. These changes will affect the
expression pattern of certain genes in blood cells.
We initially conducted a pilot study where the expression pattern of
1368 genes in peripheral blood cells of 24 females with breast cancer
and 32 females with no signs of this disease were analyzed using
macroarrays and the expression data analyzed by PAM. The results
were validated using a standard leave-one-out cross-validation
approach. We were able to identify a set of genes that correctly
predicted the diagnostic class in at least 82% of the samples. The
majority of the identified genes had a decreased expression in samples
from breast cancer patients, and predominantly encoded proteins
implicated in ribosome production and translation control. In contrast,
the expression of some defence-related genes was increased in
samples from breast cancer patients.
In order to revalidate these findings and to increase the repertoire of
informative genes, we have now extended the study with a larger
number of breast cancer and non-breast cancer samples and used
Agilent WG oligo arrays for large-scale gene expression analysis. The
preliminary analysis of the data supports our previous finding that a
blood-based gene expression test can potentially be developed to
detect breast cancer in asymptomatic patients.
Reference
1. Sharma P, Sahni NS, Tibshirani R, Skaane P, Urdal P, Berghagen
H, Jensen M, Kristiansen L, Moen C, Sharma P, et al.: Early detec-
tion of breast cancer based on gene expression patterns in
peripheral blood cells. Breast Cancer Res 2005, in press.
S.21
Stem cells in human breast development and cancer
M Wicha, G Dontu, S Liu, I Mantle
University of Michigan Comprehensive Cancer Center, Ann Arbor,
Michigan, USA
Breast Cancer Research 2005, 7(Suppl 2):S.21 (DOI 10.1186/bcr1064)
The epithelial components of the breast are thought to arise from a
stem cell population that is capable of both self-renewal and lineage-
specific differentiation. We and others have hypothesized that
mammary stem cells or their immediate progeny are targets for trans-
formation during carcinogenesis. Normal stem cells and carcinoma
cells share many characteristics including self-renewal capacity,
telomerase expression, ability to differentiate, resistance to apoptosis,
and ability to home to specific sites. Mammary transformation may
require dysregulation of pathways that control normal stem cell self-
renewal such as Notch, Wnt, Hedgehog, and Bmi-1. In order to study
these pathways in normal mammary development, we have developed
an in vitro culture system in which primary human epithelial cells
isolated from reduction mammoplasties are cultured as ‘mammo-
spheres’ on non-adherent surfaces. Cells within mammospheres are
able to self-renew, as well as to differentiate into all the lineages found
in the mammary gland. Utilizing this system, we demonstrate bi-
directional interaction between Notch and Hedgehog signaling and
Bmi-1 in the regulation of stem cell self-renewal. When mammospheres
are admixed with irradiated human mammary fibroblasts and implanted
into the cleared fatpads of NOD/SCID mice, they are able to
reconstitute the ductal alveolar structures found in the human
mammary gland.
The stem cell model of carcinogenesis may also provide a partial
explanation for the generation of cellular heterogeneity seen within
mammary tumors. Using flow cytometry, we have identified a small
population of cells within primary or metastatic breast cancers that
bear the cell surface phenotype ESA+CD44+CD24–/lowLineage–that
have the properties of human tumor stem cells. As few as 200 of these
cells are able to reproducibly generate tumors in NOD/SCID mice,
while the vast majority of cells in these tumors that lack this phenotype
are incapable of tumor formation even when tens of thousands of cells
are injected. Consistent with a stem cell model, tumorigenic cells
generate tumors that recapitulate the phenotypic heterogeneity found
in the original tumors. We have demonstrated that pathways that
control normal stem cell self-renewal, such as Hedgehog, are activated
in mammary tumor stem cells, compared with their differentiated
progeny. Despite progress in breast cancer therapeutics, metastatic
breast cancer remains an incurable disease. Current therapies that
have been developed by virtue of their ability to induce tumor
regression may selectively target more differentiated cells in tumors,
while leaving the tumor stem cell population intact, accounting for
treatment resistance and relapse. Multiple mechanisms may account
for this resistance to apoptosis, including increased expression of anti-
apoptotic genes, increased DNA repair mechanisms, and transporter
proteins such as BCRP found in the tumor stem cell population. The
targeting of stem cell self-renewal pathways such as Hedgehog or
Notch may thus provide a novel and more effective approach for the
treatment of advanced breast cancer.
S.22
Molecular distinctions among ERBB2-overexpressing
breast cancers
SS Jeffrey
Stanford University, Stanford, California, USA
Breast Cancer Research 2005, 7(Suppl 2):S.22 (DOI 10.1186/bcr1065)
HER2 or c-ERBB2/neu is a member of the epidermal growth factor
receptor (EGFR) family and encodes a tyrosine kinase receptor. Over-
expression of HER2 protein is generally attributable to gene amplification.
HER2 is overexpressed in 20–30% of primary invasive breast
carcinomas and in a greater proportion of in situ breast cancers. Invasive
breast cancers that overexpress HER2 are generally higher stage, show
lymph node positivity, and have higher S-phase. Moreover, they are often
associated with poor prognosis, particularly in node-positive patients.
Microarray studies have subdivided breast cancers into several sub-
types. HER2-overexpressing ER-negative tumors are generally classified
within a single subtype denoted ERBB2-overexpressing. However, ER-
positive HER2-overexpressing tumors are usually intermixed with other
ER-positive tumors that do not show HER2 overexpression.
Our recent population-based study evaluating HER2 overexpression
and hormone receptor status has unexpectedly found that the majority
of HER2-overexpressing tumors are hormone receptor-positive and are
more common than HER2-overexpressing ER-negative breast cancers.
This implies that the ERBB2-overexpressing molecular subtype, which
is associated with ER-negative status, only includes a minority of
HER2-overexpressing tumors. We therefore studied gene expression
patterns of HER2-overexpressing breast cancers and found several
tumor subtypes with distinctive molecular signatures. These ERBB2-
overexpressing subtypes spanned the range of hormone receptor
status and highlighted different biological characteristics. Since the
clinical course varies among patients with HER2-positive tumors, as
does their response to targeted therapy, differences in global gene
expression among HER2-overexpressing tumors could be important in
distinguishing patients for the design and delivery of individualized
targeted therapies.
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S.23
Insulin-like growth factor regulation of mammary
gland development and tumorigenesis
AV Lee
Breast Center, Baylor College of Medicine and the Methodist Hospital,
Houston, Texas, USA
Breast Cancer Research 2005, 7(Suppl 2):S.23 (DOI 10.1186/bcr1066)
Insulin-like growth factors (IGFs) are potent mitogens and survival
factors. In the mammary gland, IGFs stimulate proliferation,
differentiation, and survival during numerous developmental stages;
IGF signaling is required for puberty-dependent ductal outgrowth,
stimulates lobuloalveolar development during pregnancy, and is
reduced or absent during apoptosis-driven involution. Much of our
knowledge of IGF action in the mammary gland in vivo comes from
knockout or transgenic models. However, very few of these studies
have examined the consequence of these gene alterations on IGF
signaling in vivo. We have recently shown that intravenous injection of
IGF-I stimulates IGF-IR and IRS phosphorylation in the mammary gland,
and we are currently assessing the effect of targeted gene deletion of
overexpression of IGF signaling components on downstream signaling
in the mammary gland in vivo.
Many years of research have shown that the proliferative and survival
functions of the IGFs are not only important in mammary gland
development, but are also strongly involved in mammary cancer. Early
work using breast cancer cell lines in vitro showed that IGFs could
increase cell growth and survival; in particular, that IGFs could block
the effects of chemotherapy. We have recently shown that breast
cancer cell lines grown as xenografts in vivo are also sensitive to
intravenous IGF stimulation, and several IGF-IR inhibitors have been
shown in the past year to block MCF-7 xenograft growth.
IGF-IR and its downstream signaling intermediate IRS-1 can transform
fibroblasts in vitro. To date there is no evidence for their transforming
ability in vivo using transgenic mice. We have recently characterized
mice that overexpress IGF-IR, IRS-1, or IRS-2 in the mammary gland,
using mouse mammary tumor virus directed overexpression. We have
found that overexpression of a constitutively active IGF-IR in the
mammary gland disrupts normal development, such that female mice
are unable to lactate, and that mice rapidly develop mammary tumors.
Interestingly, overexpression of IRS-1 or IRS-2 also causes mammary
tumorigenesis, albeit with a longer time to tumor formation than
dominant active IGF-IR. These are the first mouse models showing that
IGF-IR or IRS overexpression leads to tumorigenesis in vivo. We are
currently examining the pathways required for IGF-IR and IRS-mediated
tumorigenesis in the mammary gland.
S.24
Targeting the cell cycle for prognosis and therapy of
breast cancer
K Keyomarsi, S Akli
University of Texas, MD Anderson Cancer Center, Houston, Texas, USA
Breast Cancer Research 2005, 7(Suppl 2):S.24 (DOI 10.1186/bcr1067)
Cyclin E is a G1-cyclin that plays a key role in the G1to S transition of
the cell cycle. Cyclin E is processed in tumor cells by an elastase-like
protease into low-molecular-weight (LMW) isoforms that are bio-
chemically hyperactive. The LMW isoforms of cyclin E are unique to
cancer cells. In breast cancer, such alteration of cyclin E is a very
strong predictor of poor patient outcome.
Alterations in the binding properties of these LMW isoforms to CDK2
and the CDK inhibitors (CKIs), p21 and p27, result in their functional
hyperactivity. The LMW forms of cyclin E are several-fold more effective
at binding to CDK2. Additionally, compared with the full-length cyclin
E–CDK2 complexes, the LMW cyclin E–CDK2 complexes are
significantly more resistant to inhibition by p21 and p27, despite equal
binding of the CKIs to the LMW complexes. When both the full-length
and the LMW cyclin E are co-expressed, p27 preferentially binds to the
LMW forms yet is unable to inhibit the CDK2 activity. When
overexpressed in breast cancer cells, the LMW forms of cyclin E, but
not the full-length form, result in their hyperactivity due to increased
affinity for cdk2 and resistance to inhibition by the CDK inhibitors p21
and p27, result in resistance to the growth inhibiting effects of anti-
estrogens, and result in chromosomal instability. Finally, tumors from
breast cancer patients overexpressing the LMW forms of cyclin E are
polyploid in nature and are resistant to endocrine therapy.
To assess the oncogenic role of cyclin E-LMW as compared with full-
length cyclin E, we examined the consequences of overexpressing
these isoforms in the mammary glands of transgenic mice using the
MMTV promoter. Four constructs were generated: MMTV-M46A
coding for the full-length cyclin E (EL1), MMTV-EL1/EL4 coding for
EL1 and the isoform translated at methionine 46 (EL4), and MMTV-T1
and MMTV-T2 coding for the isoforms generated by elastase cleavage
at the first site (EL2 + EL3) and at the second site (EL5 and EL6),
respectively. For each construct at least two transgenic lines were
established. Transgene expression was demonstrated by RT-PCR,
northern blotting and western blotting. Overexpression of cyclin E was
seen in more than 90% of ductal and lobular cells of the mammary
glands for each independent line. Mammary-specific LMW cyclin E
overexpression induced extensive abnormalities at 2 months, including
perturbed architecture, polyploidy, anysocytosis and apoptosis. Whole-
mount preparations of mammary glands at different development
stages showed that overexpression of EL1/EL4 and cyclin E-T1
induced growth delay, while at 6 months of age an increased
proportion of cells in the S phase was found (25.6 ± 5.6% for
EL1/EL4, 9.0 ± 2.7% for T1 compared with 3.9 ± 1.9% for non-
transgenic animals). We observed a 34% (13/38) incidence of
mammary adenocarcinomas in the EL1/EL4 transgenic lines with a
mean latency of 18.3 months, and observed a 20% (5/25) incidence in
the T1 transgenic lines with a mean latency of 17.1 months. The tumor
incidence rate of the other transgenic lines, M46A and T2, are still
unknown due to the young age of the mice (all under 7 months of age)
and the long latency of cyclin E-mediated tumor generation. Thirty
percent (4/13) of the EL1/EL4 and 40% (2/5) of the T1 tumor-bearing
animals developed lung metastasis. The tumors induced by the
EL1/EL4 and T1 transgenes were mainly solid adenocarcinomas with
very little differential to glandular for EL1/EL4 and mostly glandular for
T1. Since p53 alterations are common in human breast carcinomas, we
bred a T1 line with p53+/–mice. The T1 × p53+/–cross generated
tumors that are much more malignant than the T1 tumors; the
incidence increased to 100%, with a much shorter latency of
11 months. Biochemical analysis of the tumors revealed that 64%
(9/14) retained cyclin E expression and that, on average, the cyclin
E-overexpressing tumors had threefold higher cyclin E kinase activity
than the non-cyclin E-expressing tumors. Taken together, these data
indicate that tumor progression in cyclin E transgenic mice follow
sequential steps of dysplasia, mammary intraepithelial neoplasia and
invasive/metastatic tumors.
Collectively, the biochemical and biological differences between the
full-length and the LMW isoforms of cyclin E provide a molecular
mechanism for the poor clinical outcome observed in breast cancer
patients harboring tumors expressing high levels of the LMW forms of
cyclin E. The transgenic mouse model system can serve as a useful
system in which to study the mechanisms responsible for LMW cyclin
E-induced genetic instability and may help identify those factors that
promote tumor progression and metastasis. The properties of the LMW
forms of cyclin E suggest that they are not just surrogate markers of
poor outcome, but that they are bona fide mediators of aggressive
disease and potential therapeutic targets for patients whose tumors
overexpress these forms.
S.25
Apoptotic chemotherapies
AB Pardee, PV Reddy, DK Biswas, CJ Li
Dana-Farber Cancer Institute, Boston, Massachusetts, USA
Breast Cancer Research 2005, 7(Suppl 2):S.25 (DOI 10.1186/bcr1068)
Mutations derange growth regulations of cancer cells. They can also
make the cells more subject to apoptosis. We have investigated two
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drugs that produce specific apoptotic chemotherapeutic mechanisms
that cause tumor shrinkage in mice without deleterious side effects.
The small natural product b-lapachone is specifically apoptotic to a
variety of cancer cells. It synergizes strongly with taxol. It seems to have
several mechanisms of lethality depending on the tumor type and the
drug concentration. One mechanism is to elevate the major S-phase
transcription factor E2F-1, to an apoptotic concentration [1]. It is now
in clinical trial.
Tumor cells often mutate to apoptosis resistance; for example, by
inactivating the p53 protein. We have reported that Go6976, a kinase
inhibitory small molecule, can decrease activation of the anti-apoptotic
transcription factor NF-κB [2].
These two novel therapies thus specifically cause cancer cell
apoptosis; one by increasing an apoptotic factor, and the other
restoring apoptosis by decreasing an anti-apoptotic factor.
References
1. Li Y, Sun X, LaMont T, Pardee AB, Li C: Selective killing of
cancer cells by b-lapachone: direct checkpoint activation as a
strategy against cancer. Proc Natl Acad Sci USA 2003, 100:
2674-2678.
2. Biswas DK, Martin KJ, McAllister C, Cruz AP, Graner E, Dai S,
Pardee AB: Apoptosis caused by chemotherapeutical inhibition
of nuclear factor-kB activation. Cancer Res 2003, 63:290-295.
S.26
High-resolution representational oligonucleotide
microarray analysis and fluorescence in situ
hybridization analysis of aneuploid and diploid
breast tumors
J Hicks1, V Grubor1, N Navin1, P Lundin2, S Månér2,
T Hägerström2, L Skoog2, M Wigler1, A Zetterberg2
1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA;
2Cancer Center Karolinska, Karolinska Institute, Stockholm, Sweden
Breast Cancer Research 2005, 7(Suppl 2):S.26 (DOI 10.1186/bcr1069)
Background Combining representational oligonucleotide microarray
analysis (ROMA) of tumor DNA with quantitative multigene fluores-
cence in situ hybridization (QM-FISH) of individual tumor cells provides
the opportunity to detect and validate a wide range of gene amplifica-
tions, deletions, duplications and rearrangements directly in frozen
tumor samples.
Methods We have used these combined techniques to examine 101
aneuploid and diploid breast tumors (highly aneuploid A-tumors and
pseudo-diploid D-tumors), for which long-term follow-up and detailed
clinical information were available.
Results We have determined that ROMA provides accurate and
sensitive detection of duplications, amplifications and deletions, and it
yields defined boundaries for these events with a resolution of less than
50 kbp in most cases.
Conclusion Diploid tumors are particularly useful subjects for this
approach, revealing complex rearrangements and repeated sequential
amplification events on certain chromosomes that provide unique
insights into the genomic progression of the disease. First, the fine
structure of these amplification clusters, as detected by ROMA and
quantitatively validated by FISH, provides extremely high-resolution
‘pointers’ to potential novel oncogenes, since many of the detected
amplicons contain only one or two known or prospective genes.
Second, FISH patterns provide a means for interpretation of the
mechanism of these events. Third, the reproducibility and frequency of
these events, especially in very early stage tumors, provides insight into
the earliest chromosomal events in breast cancer. Finally, we have
identified correlations between certain sets of rearrangement events
and clinically relevant parameters such as long-term survival. These
correlations may enable novel and powerful prognostic indicators for
breast cancer and other cancers when more samples can be examined.
S.27
Tailored therapies based upon tumor subtype biology
CM Perou
Department of Genetics and Department of Pathology, Lineberger
Comprehensive Cancer Center, The University of North Carolina at
Chapel Hill, North Carolina, USA
Breast Cancer Research 2005, 7(Suppl 2):S.27 (DOI 10.1186/bcr1070)
Breast cancer is a spectrum of diseases comprised of different tumor
subtypes, each with a distinct biology and clinical behavior. To capture
this diversify, we characterized the variation in gene expression across
human breast tumors using DNA microarrays and identified at least five
distinct tumor subtypes that are statistically significant predictors of
patient overall survival [1]. Recently, we further validated these findings
using a training set of 102 tumors, which was used to derive a new
‘intrinsic gene set’. This gene set was then validated using a true test
set of 311 tumors compiled from three different microarray studies.
Our analyses demonstrate that common patterns of gene expression
can be identified across different microarray platforms, that the breast
tumor ‘intrinsic’ subtypes are reproducible across different datasets,
and that this classification was a significant predictor of outcomes after
correcting for standard clinical parameters such as estrogen receptor
(ER), grade and node status [2].
The biology of the ‘intrinsic’ subtypes is rich and extensive, and many of
these expression features suggest distinct therapies. The ‘intrinsic’
subtypes include at least two types of ER-negative tumors (Basal-like
and HER2+/ER–) and at least two types of ER-positive tumors (Luminal
A and Luminal B). Basal-like tumors typically show low expression of
HER2 and ER, and these tumors exhibit high expression of genes
characteristic of the basal epithelial cell layer, including expression of
keratin 5, keratin 6, keratin 17 and four Kallikrein genes (KLK5–KLK8).
The Basal-like tumors pose a challenge from the treatment perspective
because they lack ER and HER2. However, we have recently shown
that most are HER1-positive and/or c-KIT-positive [3], and we have
initiated a clinical trial to evaluate the efficacy of HER1-inhibitors in pre-
selected Basal-like tumor patients.
HER2-positive (i.e. gene amplified) tumors fall into at least two distinct
expression groups: those that are ER-negative and typically cluster
near the Basal-like tumors (HER2+/ER–), and those that are ER-positive
and cluster with tumors of luminal cell origin. These findings suggest
that both types of HER2+patients should receive transtuzumab, but
that the ER+/HER2+may gain a benefit from hormone therapy.
Finally, the Luminal subtype A and Luminal subtype B tumors express
ER, GATA3, and genes regulated by both ER and GATA3. Compared
with Luminal B tumors, Luminal A tumors express higher levels of ER,
BCL2 and GATA3, and they show more favorable patient outcomes.
Luminal B tumors more often express HER1, HER2 and/or cyclin E1,
and they show worse outcomes. Our data, when coupled with data from
others [4], suggests that Luminal A patients are likely to benefit from
hormone therapy and are not likely to benefit from chemotherapy, while
the opposite may be true of Luminal B patients. Experiments to answer
these questions in Luminal patients are underway and will be discussed.
References
1. Sørlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A,
Deng S, Johnsen H, Pesich R, Geisler S, et al.: Repeated obser-
vation of breast tumor subtypes in independent gene expres-
sion data sets. Proc Natl Acad Sci USA 2003, 100:8418-8423.
2.Hu Z, Fan C, Marron JS, He X, Qaqish BF, Karaca G, Livasy C,
Carey L, Reynolds E, Dressler L, et al.: The molecular portraits
of breast tumors are conserved across microarray platforms.
2005, submitted.
3. Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z,
Hernandez-Boussard T, Livasy C, Cowan D, Dressler L, et al.:
Immunohistochemical and clinical characterization of the
basal-like subtype of invasive breast carcinoma. Clin Cancer
Res 2004, 10:5367-5374.
4.Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, Baehner FL,
Walker MG, Watson D, Park T, et al.: A multigene assay to
predict recurrence of tamoxifen-treated, node-negative breast
cancer. N Engl J Med 2004, 351:2817-2826.
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S.28
Expression profiling as a prognostic and predictive
factor in breast cancer
LJ van ‘t Veer
The Netherlands Cancer Institute, Amsterdam, The Netherlands
Breast Cancer Research 2005, 7(Suppl 2):S.28 (DOI 10.1186/bcr1071)
Microarray gene expression profiling combined with advanced bio-
informatics is beginning to show its power in delineating disease
entities that are otherwise indistinguishable. This refinement in tumor
classification allows a more accurate prediction of outcome of disease
for patients that present with the same stage of disease based on
conventional clinical and histopathological criteria. Gene activities
determining the biological behaviour of the tumor may indeed be more
likely to reflect the aggressiveness of the tumor than general
parameters such as tumor size, age of the patient, or even tumor grade.
The immediate clinical consequences are therefore that treatment
schemes can be tailored based on the gene activity patterns of the
primary tumor.
Using gene expression profiling with cDNA microarrays, Perou and
colleagues showed that there are several subgroups of breast cancer
patients based on unsupervised cluster analysis: those of ‘basal type’
and those of ‘luminal type’. These subgroups differ with respect to
outcome of disease in patients with locally advanced breast cancer. In
addition, microarray analysis has been used to identify diagnostic
categories (e.g. BRCA1 and BRCA2, estrogen receptor status).
We used gene expression profiling with DNA microarrays harboring
25,000 genes on 78 primary breast cancers of young lymph-node-
negative patients to establish a signature, predictive for a short interval
to distant metastases. This ‘poor prognosis’ signature consists of
genes involved in the cell cycle, invasion and angiogenesis. The
prognosis signature is superior to currently available clinical and
histopathological prognostic factors in predicting a short interval to
distant metastases (odds ratio = 18 [95% confidence interval =
3.3–94], P < 0.001, multivariate analysis). We have validated our
findings of this poor prognosis profile on a large unselected
consecutive series of LN0 as well as lymph-node-positive (LN+) young
breast cancer patients (n = 295). The analyses confirm that the profile
is a strong independent factor in predicting outcome of disease for
LN0 patients in general (10-year overall survival for the good prognosis
profile 96% vs 50% for the poor prognosis profile). Furthermore, the
profile is also powerful for LN+ patients. At present, the prognostic
significance of the 70 genes is tested in older breast cancer patients.
Nowadays, consensus guidelines in the management of breast cancer
select up to 95% of lymph-node-negative young breast cancer patients
for adjuvant systemic therapy (e.g. NIH and St Gallen consensus
criteria). As 70–80% of these patients would have remained disease-
free without this adjuvant treatment, these patients are ‘overtreated’.
The ‘poor prognosis’ signature provides a novel strategy to accurately
select patients who would benefit from adjuvant systemic therapy and
can greatly reduce the number of patients that receive unnecessary
treatment.
Our data revealed that already small tumors display the metastatic
signature, and recent results show that the molecular program
established in a primary breast carcinoma is highly preserved in its
distant metastasis. These findings suggest that metastatic capability in
breast cancer is an inherent feature, and is not based on clonal
selections. The results further imply that neo-adjuvant treatment given
to patients based on (yet to be established) response expression
profiles of their primary breast tumor might indeed prevent the
outgrowth of micrometastases.
Currently, the EORTC breast group is preparing a 5000-patient
randomized trial to compare the efficacy of guidance of breast cancer
patients for adjuvant chemotherapy based on either ‘conventional’ St
Gallen consensus criteria or the microarray prognosis test (MINDACT
trial within the EU-TRANSBIG program). The aim of the study is to
confirm that the microarray test will save up to 30% of the patients
from unnecessary chemotherapy and to identify 5% of them who are
nowadays ‘undertreated’.
S.29
Genomic profiling of breast cancer
Å Borg
Lund University, Sweden
Breast Cancer Research 2005, 7(Suppl 2):S.29 (DOI 10.1186/bcr1072)
Cancer and other genetic diseases are characterized by genome
alterations, including DNA copy number changes. Comparative
genomic hybridization (CGH) represents a powerful technique to
detect and map these aberrations, and recent improvements in
resolution and sensitivity have been possible through implementation of
microarray-based platforms. Germline mutations in the two major
breast cancer susceptibility genes, BRCA1 and BRCA2, account for a
significant proportion of all hereditary breast cancers. Earlier studies
have shown that inherited and sporadic tumors progress along
different somatic genetic pathways and that global gene expression
profiles distinguish between these groups. Using 1 Mbp resolution
BAC-array CGH analysis, we now show that genomic copy number
profiles similarly discriminate between BRCA1/BRCA2-related tumors
and sporadic tumors. Overall, BRCA1 tumors had a higher frequency
of copy number alterations than sporadic breast cancers. In particular,
frequent losses on 4p, 4q and 5q in BRCA1 tumors and frequent gains
on 7p and 17q24 in BRCA2 tumors distinguish these from sporadic
breast cancer. Distinct amplicons at 3q27.1-q27.3 were identified in
BRCA1 tumors, and amplicons at 17q23.3-q24.2 in BRCA2 tumors.
Moreover, evidence of a homozygous deletion in a BRCA1 tumor on
5q12.1 was obtained. Using a set of 169 BAC clones that detect
significantly different frequencies of copy number changes in inherited
and sporadic tumors, these subsets could be discriminated into
separate groups using hierarchical clustering. Further validation may
prove this tumor classifier to be useful for selecting familial breast
cancer cases, likely to carry BRCA1 or BRCA2 germline mutations, for
further mutation screening, particularly as these data can be obtained
using DNA prepared from archival tumor tissue.
Further improved genomic profiling was obtained by construction of
microarrays comprising 32,433 BAC clones, offering complete
genome coverage at single gene resolution, on average <50 kbp.
These new tiling 32k-arrays were evaluated on breast cancer cell lines
(BT-474, MCF7, HCC1937, SK-BR-3, L56Br, ZR-75-1), validated by
FISH and gene expression analysis. Known amplicons were resolved
and found to include complex patterns of narrow peaks, occasionally
including a few or even single genes. Several amplified regions and
genes on 17q and 20q were depicted and confirmed by demonstrating
strong correlations between gene copy numbers and expression.
Previously described as well as novel homozygous deletions, ranging
from a few BAC clones (<300 kb) to several Mbp, were observed,
including PTEN and other regions on 10q, CDH1/CDH3 on 16q22,
and new regions on 4q34 and 19p12, emphasizing the power of array
CGH in pinpointing genes of importance in tumor development. Array
CGH is a promising diagnostic tool in profiling of somatic and
constitutional genomic alterations.
S.30
A single nucleotide polymorphism in the HDM-2 gene
regulates the p53 apoptotic response and influences
the age of onset of cancers in humans: the SNP 309
HDM-2 polymorphism
GL Bond1, AJ Levine1,2
1Cancer Institute of New Jersey, New Brunswick, New Jersey, USA;
2Institute for Advanced Study, School of Natural Sciences, Princeton,
New Jersey, USA
Breast Cancer Research 2005, 7(Suppl 2):S.30 (DOI 10.1186/bcr1073)
The HDM-2 gene in humans has two promoters for transcription. 5′ to
the first exon is a maintenance promoter providing low levels of HDM-2
in the cell. In the first intron are the P53 DNA binding sites and the p53
inducible promoter that yields threefold to 10-fold more HDM-2 mRNA
after a p53 activation and response. When this intronic promoter is
employed, transcriptional initiation starts at the second exon and this
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mRNA is translated more efficiently than mRNA that starts at the first
exon. The coding region of the HDM-2 protein starts in the third exon.
At residue 309 in this first intron is a single nucleotide polymorphism,
with 12% of people being a G/G homozygote, 40% being a G/T
heterozygote and 48% of people being T/T wild-type homozygotes (the
G/G genotype is lower in black Americans and the sample size is now
over 300 people). We have found that the G/G genotype creates a
better SP-1 transcription factor binding site, raises the level of m-RNA
in unstressed cells and produces threefold to sixfold more HDM-2
protein in cells (cancer cells in culture) with the G/G genotype. This
mRNA starts at the second exon, and is probably translated better in
unstressed cells. After DNA damage or other stresses, P53 activity in
cells with the G/G genotype is lower and the percentage of cells
undergoing apoptosis is lower when compared with cells in culture
with T/T genotypes. We have reproduced these observations with
lymphocytes taken from human volunteers and placed in culture, with
EBV-immortalized B cells in culture, with primary fibroblasts in cell
culture and with cancer cell lines in culture. In 92 individuals that have
donated lymphocytes we see individuals forming a distribution of
apoptotic responses between 20% and 60% after gamma radiation,
with individuals being quite reproducible in repeated experiments. The
lower half of the distribution is heavily weighted with the G/G
genotype, while the upper half of the distribution has mainly the T/T
genotype. The higher HDM-2 levels in cells thus result in a lower
apoptotic index in cells from these volunteers. It has become clear in
recent studies that SNP 309 has a clinical impact. We have genotyped
two cancer cohorts, one at MD Anderson and one in Germany,
containing patients with sarcomas and breast cancers. The results
have been statistically significant (P = 0.01–0.02) and clear in both
cohorts, and the average age of onset of these cancers is 10–15 years
earlier in people with the G/G genotype than in people with the same
cancer with the T/T genotype. The interpretation is then that the
probability of eliminating pre-cancerous clones of cells via a p53
mechanism is lower in people with a G/G genotype (high HDM-2
levels) and the probability of developing a cancer at an earlier time in
life is higher. In addition, in patients that have a germline mutation in the
p53 gene (this yields one-half of the p53 protein level in a cell) those
individuals that have a G/G genotype or a G/T genotype develop
multiple cancers (three, four or five cancers) over their lifetimes, while
no T/T homozygotes develop that many independent cancers.
S.31
Evading p53 action during tumor development and
therapy
SW Lowe
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA
Breast Cancer Research 2005, 7(Suppl 2):S.31 (DOI 10.1186/bcr1074)
Apoptosis is a regulated form of cell death that is important for normal
development and tissue homeostasis. Senescence produces ‘genetic
death’, in that the senescent cell is incapable of further propagation.
Both processes are frequently disrupted in cancer cells, and each act
as potent barriers to tumorigenesis. Since radiation and many
chemotherapeutic agents induce apoptosis or senescence, the
integrity of these programs can influence the outcome of cancer
therapy. Our laboratory strives to understand how cancer genes
control apoptosis and senescence in normal cells, and how mutations
that disrupt these processes impact tumor development and therapy.
The goal of these efforts is develop therapeutic strategies based on an
understanding of drug action and cancer genotype. We currently are
using genetically engineered mouse models to understand how
apoptosis and senescence are controlled in tumor cells, as well as the
response of tumors to conventional and targeted therapeutics. Recent
work exploring the action of tumor-derived myc mutants in oncogenesis
and the role of the p53 tumor suppressor network in the action of
targeted therapeutics will be discussed.
S.32
TP53 and additional pathways in therapy resistance
PE Lønning
Haukeland University Hospital, Bergen, Norway
Breast Cancer Research 2005, 7(Suppl 2):S.32 (DOI 10.1186/bcr1075)
Resistance to chemotherapy is the main obstacle to cancer cure.
Despite encouraging results from preclinical studies, we have limited
knowledge regarding mechanisms causing therapy resistance in vivo.
We previously identified mutations affecting the L2 and/or L3 domains
of the TP53 gene to predict resistance to anthracycline as well as
mitomycin therapy [1,2]. However, while TP53 mutations were
significantly associated with therapy failure, we observed tumours
resistant to therapy despite harbouring wild-type p53. We also saw
responding tumours among those harbouring TP53 mutations affecting
the L2 or L3 domains.
Based on these assumptions, we postulated that chemoresistance
could be due to failure of the ‘p53 pathway’ acting in concert with one,
or more, redundant pathways [3]. In a recent paper we thus reported a
mutation of the CHEK2 gene among one of the tumours resistant to
therapy despite harbouring wild-type TP53 [4]. In addition, we are
searching for redundant pathways that may compensate for the p53
mechanism. Strikingly, looking at genetic alterations associated with
resistance to other drugs with respect to other malignancies, this
seems to concentrate on drugs known to be involved in so-called
‘family cancer syndromes’, meaning genes involved either in growth
arrest, apoptosis or DNA damage repair [3]. This may seem logical, as
much of the damage created by chemotherapeutic drugs resemble
genetic events involved in carcinogenesis. Thus, at this stage, our
interest is focused on genetic pathways involving genes involved in
‘family cancer syndromes’. An update of our current results will be
presented.
References
1. Aas T, et al.: Specific P53 mutations are associated with de
novo resistance to doxorubicin in breast cancer patients. Nat
Med 1996, 2:811-814.
2.Geisler S, et al.: TP53 gene mutations predict the response to
neoadjuvant treatment with FUMI in locally advanced breast
cancer. Clin Cancer Res 2003, 9:5582-5588.
3.Lønning PE: Genes causing inherited cancer as beacons iden-
tifying the mechanisms of chemoresistance. Trends Mol Med
2004, 10:113-118.
4. Staalesen V, et al.: Alternative splicing and mutation status of
CHEK2 in stage III breast cancer. Oncogene 2004, 23:8535-
8544.
S.33
Dynamic imaging of plasticity and escape in tumor
cell invasion
P Friedl
Rudolf Virchow Center for Experimental Biomedicine and Department
of Dermatology, University of Würzburg, Germany
Breast Cancer Research 2005, 7(Suppl 2):S.33 (DOI 10.1186/bcr1076)
Cancer cell interactions with the extracellular matrix and the migration
therein require adhesion and traction provided by integrins, together
with pericellular proteolysis executed by extracellular matrix degrading
proteases. We have used experimental interference strategies and
identified plasticity of migration modes resulting in new ways of
dissemination. As imaged by three-dimensional matrix-based models
and intravital microscopy, quantitative reconstruction from movies has
shown how tumor cells depend on adhesion mechanisms but continue
to migrate after adhesion receptors are blocked, has shown how
proteases generate proteolytic tracks but are dispensable if ‘physical’
strategies allow cells to bypass tissue barriers, and has shown why
individual and collective invasion patters predispose to a different
outcome after pharmacotherapeutic intervention.
These findings have implications with reference to invasion as a
therapeutic target in progressive cancer disease.
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S.34
Role of HER2 in local relapse and metastasis
S Ménard
Molecular Targeting Unit, National Cancer Institute, Milan, Italy
Breast Cancer Research 2005, 7(Suppl 2):S.34 (DOI 10.1186/bcr1077)
HER2-positive breast carcinomas have been shown to display an early
peak of relapses in the first 4 years after surgery, especially in the
node-positive subgroup. To explain this observation, growth factors
released at the time of surgery were investigated. The level of growth
factors of the EGF family, detected in postsurgical sera from breast
carcinoma patients, was found to correlate with surgical invasiveness.
Indeed, following radical mastectomy, higher levels of serum EGF-like
factors were found than after conservative surgery. This implicates that
the growth of tumors overexpressing HER2, activated by these growth
factors, should be stimulated after invasive surgery. Two retrospective
analyses of the HER2 status of primary tumors included in a
randomized clinical trial addressing the issue of conservative versus
invasive surgery and of radiotherapy were performed by immuno-
histochemistry using the standardized herceptest. Survival analysis
according to surgery indicated no differences in HER2-negative cases
but indicated a poorer survival for HER2-positive node-positive patients
who had mastectomy in comparison with those who had conservative
surgery. Furthermore, local relapses in patients who had conservative
surgery without radiotherapy were found to be anticipated in the
HER2-positive subset. This is a ‘proof of principle’ that surgery by
inducing growth factor release may be detrimental for patients with
HER2-positive tumors. To verify these findings, a prospective analysis
of the follow-up of more than 2000 patients who have had mastectomy
or conservative surgery is ongoing. Preliminary data indicate a
significantly worst prognosis of HER2-positive tumors after invasive
surgery, above all for tumors scoring 2+ by immunohistochemistry.
Acknowledgement Partially supported by the AIRC.
S.35
Molecular profiling of early breast cancer in relation
to detection of micrometastases and outcome
B Naume1, T Sørlie2
1Department of Oncology and 2Department of Genetics,
The Norwegian Radium Hospital, Oslo, Norway
Breast Cancer Research 2005, 7(Suppl 2):S.35 (DOI 10.1186/bcr1078)
Background Molecular profiling of breast cancer by DNA microarrays
has been used to classify tumors into five distinct subclasses that show
significant differences in clinical outcomes. Of these subclasses, the
luminal subtype A is associated with a relatively good prognosis [1-3].
Detection of disseminated tumor cells (DTC) in bone marrow (BM) can
independently predict future metastasis, which was confirmed in our
study of 817 early BrCa patients [4].
Materials and methods Fresh tumor samples were prospectively
collected during primary surgery from 123 of these patients, for
evaluation of the clinical significance of gene expression profiling and
for comparison of tumor subtypes with DTC detection in BM. The BM
samples were collected from iliac crests at primary surgery, followed by
immunocytochemical staining (anti-cytokeratin mAbs) and morphology-
guided screening for DTC. Gene expression patterns of the primary
tumors were examined using 42,000 spot cDNA microarrays (Stanford
Functional Genomics Facility). Data were analyzed by hierarchical
clustering and were compared with our previously published breast
tumor subclassifications. Data were further analyzed by supervised
analysis methods (SAM, PAM).
Results The tumors were classified by gene expression analysis into
luminal A (41%), luminal B (13%), ERBB2+(17%), basal-like (14%)
and normal-like (12%). The luminal A subtype showed high ER/PgR-
positivity (98%), low ERBB2-positivity (4%) (assessed by IHC) and low
frequency of TP53 mutations (6%). Luminal B, ERBB2+and basal-like
subtypes showed high frequencies of TP53 mutations (43%, 65%, and
82%, respectively), whereas the ER/PgR-positivity was 94%, 24% and
6%, respectively. Expression of the ERBB2 protein differed between
these groups. At median 60 months follow-up, luminal A patients
showed improved survival compared with patients within the other
subtypes (P = 0.02, log rank), with BrCa death in 14% versus 29%,
respectively. DTC in BM were detected in 23.7%. No particular
subtype was associated with DTC, and no particular gene profile was
associated with DTC status, as determined by SAM analysis. However,
when we stratified the patients based on the molecular subtype, and
first considered only the luminal A tumors, we identified 193 genes
(FDR 23%) associated with high expression in tumors from patients
with DTC. Moreover, a considerable number of patients with a luminal
A type of tumor experienced systemic relapse of the disease (28%)
and SAM analysis identified 147 genes associated with different
expression patterns in tumors from relapsed patients versus disease-
free patients
Conclusion This early BrCa study confirms the consistency of the
gene expression profiles and their clinical implications. DTC detection
can further distinguish the clinical outcome in patients with the luminal
A subtype. The gene expression patterns in DTC-positive patients, and
in all patients with systemic relapse, will be further explored.
References
1. Perou CM, Sørlie T, Eisen MB, et al.: Nature 2000, 406:747-752.
2. Sørlie T, Perou CM, Tibshirani R, et al.: Proc Natl Acad Sci USA
2001, 98:10869-10874.
3.Sørlie T, Tibshiranhi R, Parker J, et al.: Proc Natl Acad Sci USA
2003, 100:8418-8423.
4. Wiedswang, Borgen, Kåresen, et al.: J Clin Oncol 2003, 21:
3469-3478.
S.36
Update on HER2-directed therapy
D Slamon
University of California, Los Angeles, California, USA
Breast Cancer Research 2005, 7(Suppl 2):S.36 (DOI 10.1186/bcr1079)
Abstract not submitted.
S.37
Targeting new therapies in combination with
hormonal therapies for ER-positive breast cancer
M Dowsett
Academic Department of Biochemistry, Royal Marsden Hospital,
London, UK
Breast Cancer Research 2005, 7(Suppl 2):S.37 (DOI 10.1186/bcr1080)
Hormonal therapies involving estrogen deprivation or SERMs such as
tamoxifen reduce the risk of relapse and improve the survival of the
>75% of breast cancer patients with ER-positive tumours.
Nonetheless, many of these patients relapse with disease that was
either intrinsically resistant to treatment or that has acquired resistance
to the endocrine treatment. Laboratory studies have revealed that
growth factor receptor pathways form an important route of growth
signalling in both these circumstances, and there is now a series of
agents available that target these pathways at different points. This
provides the opportunity to utilise these agents in combination with
endocrine treatment and the possibility that this may extend the
effectiveness of the hormonal agents. The effective delivery of such
combinations depends on a detailed knowledge of the degree to which
the highly encouraging laboratory findings are translated into the
clinical scenario. We have demonstrated that almost all breast ER-
positive cancer shows some proliferative dependence on oestrogen,
but that this is very variable. We have begun to identify in clinical
samples the key genes whose expression both determines this
variability and are themselves dependent on it. The development of
novel models of drug development that allow the assessment of the
expression of these genes, particularly within the presurgical setting,
offers major opportunities to assess the potential of the various new
targeted agents to be combined with endocrine therapy.
Available online http://breast-cancer-research.com/supplements/7/S2

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S.38
Development of CDK inhibitors as cancer
therapeutics
D Lane
IMCB, Singapore
Breast Cancer Research 2005, 7(Suppl 2):S.38 (DOI 10.1186/bcr1081)
The cyclin-dependent kinases form a large family of enzymes in human
cells that are involved in the control of cell proliferation and
transcription. A large number of small molecule inhibitors of this class
of enzyme have been developed in both the pharmaceutical and
academic communities, and at least two have entered clinical trial,
having shown efficacy in preclinical models. Alterations in the activity of
this class of enzymes is a frequent feature of human cancers, brought
about by altered expression of either the enzymes themselves or their
regulators such as p21, p27 and p16. The exact role of each of the
different kinases has proved hard to determine as knockout mouse
studies have implied a degree of redundancy and the exact substrates
of each enzyme in vivo are still unclear. In addition, most of the current
inhibitors are not specific to a single form of the enzyme and new
regulatory pathways are still being discovered. Intense studies of one
such inhibitor, R-Roscovitine (CYC202), including trials involving more
than 100 patients, have established the potential of the class as non-
genotoxic anti-cancer drugs. In some model systems the activity of this
class of compound is best explained by their activity as inhibitors of
transcriptional elongation, and a link between this mechanism and the
induction of apoptosis has been established. The concept of cyclin-
specific inhibitors as more sophisticated genetic models of target
validation in this field will be discussed.
S.39
Genes, genomes, and cancer
D Botstein
Princeton University, New Jersey, USA
Breast Cancer Research 2005, 7(Suppl 2):S.39 (DOI 10.1186/bcr1082)
Abstract not submitted.
S.40
The search for low-penetrance breast cancer genes
BAJ Ponder, A Cebrian, AM Dunning, DF Easton, F Lesueur,
C Luccarini, PDP Pharoah
Strangeways Laboratory for Genetic Epidemiology, Department of
Oncology and Department of Public Health, University of Cambridge, UK
Breast Cancer Research 2005, 7(Suppl 2):S.40 (DOI 10.1186/bcr1083)
Background Fifteen per cent to 20% of the familial clustering of breast
cancer is explained by the effects of highly penetrant mutations in
BRCA1 and BRCA2. Modelling based on the patterns of familial
aggregation of breast cancer in the relatives of cases ascertained on a
population basis suggests that much of the remaining familial effect is
due to the combined effects of genetic variants individually of small
effect. The numbers of such variants, their allele frequencies and the
strength of their effects is not known.
Methods We have carried out association studies to search for
common variants (minor allele frequency >5–10%) that contribute to
predisposition,. To date we have studied 400 SNPs in 110 genes
using a two-stage study design, in which a first set of 2300 cases and
controls is analysed and all SNPs with a significance value of P < 0.1
or better are then tested in a second, similar, case/control set.
Results No individual SNP has, to date, given a P value for association
(based on genotype distribution) lower than 10–4. A number of SNPs
give P values between 10–2and 10–4, depending on the genetic model
that is chosen for the analysis. Most of these are probably false
positives, the consequence of multiple testing. However, comparison of
the distribution of P values across the entire study set with that
expected if there were no genetic effect suggests that some of these
are probably true positive associations, representing low-level
predisposing effects.
Conclusions A candidate gene approach is slow and relatively
expensive, and has not so far yielded unequivocal positive results for any
individual gene. The ‘genetic architecture’ of breast cancer — that is, the
number and characteristics of predisposing genetic variants — is still not
known. In an attempt to elucidate this and to hasten the process of gene
discovery, we have initiated (with collaborators in the UK and at
Perlegen Science Inc.) a genome-wide scan. Again we use a two-stage
approach. In the first stage we will evaluate 266,000 SNPs in 400
breast cancer cases and 400 controls. The cases will be ‘enriched’ for
genetic effects by choosing those with a family history, tested negative
for BRCA1/2 mutation. In the second stage, ~5% of SNPs will be
further evaluated in 4600 cases and controls. A final stage of evaluation
for positives from the second stage, and from our earlier studies, will
require analysis of a further, very large (~10,000), case/control set,
which we hope to assemble through international collaboration.
S.41
Functional genomic approaches to breast cancer
R Bernards
Division of Molecular Carcinogenesis, The Netherlands Cancer
Institute, Amsterdam, The Netherlands
Breast Cancer Research 2005, 7(Suppl 2):S.41 (DOI 10.1186/bcr1084)
Background One of the major remaining deficits in our understanding
of the human genome is that information regarding gene function is
available for only one-quarter of the approximately 30,000 genes. Many
of these hitherto anonymous genes are potential targets for the
development of new anti-cancer drugs. It is therefore important to
functionally annotate the tens of thousands of genes for which this
information is currently lacking. My laboratory has developed functional
genetic approaches to obtain information regarding gene function
using high-throughput screens in mammalian cells. We have developed
both gain-of-function genetic screens (using retroviral cDNA
expression libraries) and loss-of-function genetic screens (using vector-
based RNA interference libraries) to carry out large-scale genetic
screens in mammalian cells. We focus on the central growth-regulatory
pathways that are most frequently deregulated in cancer.
Methods We have designed a mammalian expression vector (pSUPER),
which directs the synthesis of short hairpin transcripts (shRNAs) that are
processed intracellularly into siRNA-like molecules. This vector mediates
persistent inhibition of gene expression in a highly specific fashion. We
have used this vector to stably suppress expression of individual
members of several cancer-relevant gene families.
Results We used a retroviral derivative of the pSUPER siRNA vector
to generate a large collection of siRNA vectors that each target a
single gene for suppression. In total, we constructed a set of 23,742
siRNA vectors that together target 7914 human genes for suppression
by RNA interference. Furthermore, we developed a very efficient way to
identify biologically active shRNA vectors in a large population of
vectors, a technology that we named ‘siRNA bar code screening’. We
will present two applications of this technology to study major
questions in breast cancer. First, we have used the RNAi library to
identify genes whose suppression causes resistance to anti-hormonal
therapy (tamoxifen resistance). In addition, we have used RNAi
technology to ask how clinical resistance to the Her2/neu/ErbB2-
targeted therapeutic Herceptin can arise.
Conclusion RNA interference is a powerful technology to identify
genes that are causally involved in disease processes. Application of
this technology to breast cancer may greatly expedite the development
of novel diagnostics and therapeutics for the treatment of this disease.
References
1. Brummelkamp TR, Nijman SMB, Dirac AMG, Bernards R: Loss of
the cylindromatosis tumour suppressor inhibits apoptosis by
activating NF-kB. Nature 2003, 424:797-801.
2. Brummelkamp TR, Bernards R: New tools for functional mam-
malian cancer genetics. Nat Rev Cancer 2003, 3:781-789.
3. Berns K, Hijmans EM, Mullenders J, Brummelkamp TR, Velds A,
Kerkhoven RMH, Madiredjo M, Nijkamp W, Weigelt B, Agami R,
et al.: A large-scale RNAi screen in human cells identifies new
components of the p53 pathway. Nature 2004, 428:431-437.
Breast Cancer Research Vol 7 Suppl 2 Third International Symposium on the Molecular Biology of Breast Cancer

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S.42
Proteomic approaches to early detection of breast
cancer
JE Celis1,2, P Gromov1,2, JMA Moreira1,2, T Cabezón1,2, E Friis1,3,
F Rank1,4, I Gromova1,2
1The Danish Centre for Translational Breast Cancer Research
(DCTB), Copenhagen, Denmark; 2Department of Proteomics in
Cancer, Institute of Cancer Biology, Danish Cancer Society, Denmark;
3Department of Breast and Endocrine Surgery, Rigshospitalet,
Denmark; 4Department of Pathology, The Centre of Diagnostic
Investigations, Rigshospitalet, Denmark
Breast Cancer Research 2005, 7(Suppl 2):S.42 (DOI 10.1186/bcr1085)
The completion of the human genome as well as the explosion of novel
technologies within genomics, proteomics and functional genomics
promise to have a major impact on clinical practice, as these
technologies are expected to accelerate the translation of basic
discoveries to the clinical practice. In particular, proteomic
technologies are expected to play a key role in the study and treatment
of cancer as they provide invaluable resources to define and
characterize regulatory and functional networks, to investigate the
precise molecular defect in diseased tissues and biological fluids, and
to develop specific reagents to precisely pinpoint a particular disease
or stage of a disease. For drug discovery, proteomics assist with
powerful tools for identifying new clinically relevant drug targets, and
provide functional insight for drug development.
Today, the application of novel technologies from proteomics and
functional genomics to the study of cancer is rapidly shifting to the
analysis of clinically relevant samples such as fresh biopsy specimens
and fluids, as their use will accelerate the translation of basic
discoveries. Being a patient-oriented organisation, The Danish Cancer
Society catalysed in 2002 the creation of a multidisciplinary research
environment, the DCTB, to fight breast cancer. The DCTB hosts
scientists working in various areas of preclinical cancer research (cell
cycle control, invasion and microenvironmental alterations, apoptosis,
cell signalling, and immunology) with clinicians (surgeons, oncologists)
and pathologists in an integrated, mission-oriented, discovery-driven
translational research environment. The unifying concept behind our
experimental strategy is the use of multiple experimental paradigms for
the prospective analysis of clinically relevant samples obtained from the
same patient, along with the systematic integration of the biological
and clinical data.
Here I will describe our efforts to apply proteomics approaches to
search for markers for early detection of breast cancer using the newly
characterized interstitial fluids recovered from fresh tissue biopsies of
both normal (NIF) and tumour (TIF) origin. The protein composition of
the fluids is strikingly different to that of serum and cyst fluids, although
they share some of their major components. The TIF is highly enriched
in proteins that are either secreted via the classic endoplasmic
reticulum/Golgi pathway, shed by membrane vesicles (membrane
blebbing), or externalized by plasma membrane transporter. Hundreds
of primary translation products, as well as post-translational
modifications, have so far been identified using a combination of
procedures that include mass spectrometry, two-dimensional gel
immunoblotting, and cytokine and signalling pathway-specific antibody
arrays. The workflow to biomarker discovery as well as recent
developments will be discussed.
S.43
Dissection of molecular pathways of cancer by high-
throughput biochip technologies and RNA interference
O Kallioniemi
Medical Biotechnology, VTT Technical Research Centre of Finland;
University of Turku, Finland
Breast Cancer Research 2005, 7(Suppl 2):S.43 (DOI 10.1186/bcr1086)
Objective Our aim is to identify new molecular targets and
mechanisms for therapeutic intervention in cancer. To achieve this aim,
we develop and apply multiple high-throughput technologies including
‘in silico’ screening as well as technologies for molecular, cellular and
clinical discovery research. Finally, data integration from these
technology platforms is applied to facilitate interpretation and
prioritization of the findings.
In silico screening In order to make use of the exponential increase of
published data on gene expression arrays, we have launched a project
to acquire and make use of these data as a discovery resource. We
currently have data on 5700 samples analyzed on the Affymetrix gene
expression platform stored in our relational database. These samples
include, for example, 64 normal tissues/cell types, 43 tumor types,
many other diseases as well as functional experiments; altogether
84 million data points. We have developed methods to mine these data
to identify tissue-specific and disease-specific expression patterns of
transcripts, to identify gene coexpression profiles, to explore networks
of gene regulation as well as methods to interpret new microarray
experiments. In silico transcriptomic screening makes it possible to
generate dozens of testable hypotheses for laboratory analysis based
on datasets that are much larger and more extensive than any single
academic laboratory can afford to generate. Analysis of gene
expression profiles across hundreds of tissue and tumor types,
diseases and experimental manipulations generates novel, often
unexpected, insights of gene function as well as of the underlying
biology and medicine.
Molecular screening Large cohorts of clinical samples are now being
investigated not only at the RNA level by gene expression profiling, but
also at the DNA-level using comparative genomic hybridization (CGH)
arrays for analysis of somatic genetic alterations or SNP arrays for
studies of allelic gains and losses. There is also an emerging interest
for large-scale proteomic and metabolic profiling. It will be increasingly
important to integrate multiple levels of molecular profiling data to gain
new insights and comprehensive views on mechanisms of cancer
development. We are applying single-gene resolution oligo-CGH
arrays and integrating these data with gene expression information on
the same samples. The increased CGH resolution has highlighted
several microdeletions as well as small amplifications, whose impact on
gene expression can be substantial and highly specific. This has led to
an opportunity for rapid identification of genes that may be targets of
genetic alterations in cancer. As demonstrated by several recently
approved drugs for cancer, such mutated genes represent attractive
targets for the development of effective cancer-specific therapeutics.
Functional screening using RNA interference The molecular profiling
of DNA expression patterns, RNA expression patterns or protein
expression patterns in patient samples is not sufficient for implicating
these molecules or molecular mechanisms as therapeutic targets. It is
also necessary to generate functional information on such genes and
pathways. Towards this aim, we have developed a high-throughput
screening system that is composed of a robotic, automated platform for
the analysis of up to 20,000 functional experiments with living cells at a
time using the 384-well microplate format. Cells are dispensed into
culture wells, exposed to siRNAs or small molecule compounds,
incubated for 1–3 days, washed, and stained with phenotype-specific
markers for cell growth, cell cycle distribution or induction of apoptosis.
The results are read by plate readers or cell cytometers. Functional
studies with RNAi libraries (e.g. 1000–10,000 siRNAs) have
implicated genes whose targeting by RNAi is lethal to specific cancer
types, such as breast cancer. Integration of such functional RNAi data
with gene expression and aCGH data has enabled us to identify genes
that are targets of genetic alterations and whose expression is required
for the maintenance of the malignant phenotype. Such genes represent
attractive candidate drug targets.
Clinical screening Data on molecular targets arising from functional in
vitro studies need to be corroborated in studies of large-scale clinical
sample cohorts in order to verify that such molecular targets are
relevant in clinical patient samples. A number of technologies are being
developed towards this aim. First, the in silico
transcriptomics database with 5700 samples has made it possible to
develop an approach for ‘in silico clinical validation’. It is possible to
determine the expression levels of any gene across a very large number
of tumor types and normal sample types. Second, more established
screening
Available online http://breast-cancer-research.com/supplements/7/S2