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Journal of Molecular Endocrinology (2005) 34 535-551    DOI: 10.1677/jme.1.01677
© 2005 Society for Endocrinology
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Anti-proliferative effect of estrogen in breast cancer cells that re-express ER{alpha} is mediated by aberrant regulation of cell cycle genes

J G Moggs, T C Murphy, F L Lim, D J Moore, R Stuckey, K Antrobus, I Kimber and G Orphanides

Syngenta CTL, Alderley Park, Cheshire SK10 4TJ, UK

(Requests for offprints should be addressed to J G Moggs; Email: jonathan.moggs{at}syngenta.com)


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Estrogen receptor (ER)-negative breast carcinomas do not respond to hormone therapy, making their effective treatment very difficult. The re-expression of ER{alpha} in ER-negative MDA-MB-231 breast cancer cells has been used as a model system, in which hormone-dependent responses can be restored. Paradoxically, in contrast to the mitogenic activity of 17ß-estradiol (E2) in ER-positive breast cancer cells, E2 suppresses proliferation in ER-negative breast cancer cells in which ER{alpha} has been re-expressed. We have used global gene expression profiling to investigate the mechanism by which E2 suppresses proliferation in MDA-MB-231 cells that express ER{alpha} through adenoviral infection. We show that a number of genes known to promote cell proliferation and survival are repressed by E2 in these cells. These include genes encoding the anti-apoptosis factor SURVIVIN, positive cell cycle regulators (CDC2, CYCLIN B1, CYCLIN B2, CYCLIN G1, CHK1, BUB3, STK6, SKB1, CSE1 L) and chromosome replication proteins (MCM2, MCM3, FEN1, RRM2, TOP2A, RFC1). In parallel, E2-induced the expression of the negative cell cycle regulators KIP2 and QUIESCIN Q6, and the tumour-suppressor genes E-CADHERIN and NBL1. Strikingly, the expression of several of these genes is regulated in the opposite direction by E2 compared with their regulation in ER-positive MCF-7 cells. Together, these data suggest a mechanism for the E2-dependent suppression of proliferation in ER-negative breast cancer cells into which ER{alpha} has been reintroduced.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Estrogens are important regulators of growth and differentiation in the normal mammary gland and participate in the development and progression of breast cancer (Pike et al. 1993). The mitogenic effects of estrogens on breast epithelial cells are mediated, at least in part, via the altered expression of genes involved in cell cycle regulation (Prall et al. 1997). Transcriptional regulation of estrogen-responsive genes is mediated by two members of the nuclear receptor superfamily, estrogen receptor (ER){alpha} and ERß. These ERs function as ligand-activated transcription factors that recruit a variety of coregulator proteins to activate or repress the expression of estrogen-responsive genes (Moggs & Orphanides 2001, Hall et al. 2001, McKenna and O’Malley 2002, Tremblay and Giguere 2002).

ER antagonists are used widely as therapeutic agents in the treatment of ER-positive breast cancers (Vogel 2003). In contrast, ER-negative breast cancers cannot be controlled by hormone therapy, making their effective treatment very difficult. This led to the suggestion that re-introducing ER into these cells would allow them to be controlled using anti-estrogen therapies. However, paradoxically, the reintroduction of ER{alpha} into ER-negative breast cancer cells results in the suppression of proliferation by 17ß-estradiol (E2) (Garcia et al. 1992, Levenson and Jordan 1994). The mechanism underlying this anti-proliferative effect of E2 in these cells is not known.

We have used global gene expression profiling to identify the molecular pathways through which estrogens suppress proliferation in ER-negative MDA-MB-231 breast cancer cells that re-express ER{alpha}. Our data reveal that, in these cells, E2 regulates the expression of a number of genes involved in cell proliferation and survival that have been previously associated with mitogenic stimulation by estrogens. However, strikingly, many of these genes are regulated in the opposite direction compared with their response in ER-positive MCF-7 breast cancer cells exposed to estrogens. Identification of the molecular networks associated with the suppression of proliferation in ER-negative breast cancer cells may allow the development of new strategies to control the growth of ER-negative breast tumours.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture

MDA-MB-231 cells were cultured routinely at 37 °C in humidified chambers at 5% CO2 in Minimal Essential Media (MEM) supplemented with non-essential amino acids, 2 mM glutamine, Penicillin, Streptomycin and 5% charcoal-dextran-treated fetal calf serum. HEK293 cells were cultured as described in He et al.(1998). MCF-7 cells were maintained at 37 °C in humidified chambers at 5% CO2 in RPMI 1640 media containing phenol red, 2 mM glutamine, Penicillin, Streptomycin and 10% heat-inactivated fetal bovine serum. Prior to dosing with vehicle control (ethanol) or E2 (Sigma), MCF-7 cells were incubated for 4 days in RPMI 1640 media without phenol red and containing 2 mM glutamine, Penicillin, Streptomycin and 5% charcoal-dextran-treated fetal bovine serum.

Adenoviral system used to express ER{alpha} in human MDA-MB-231 cells

Full-length human ER{alpha} (1–595; Green et al. 1986) cDNA was cloned into the shuttle vector pAdTrack-cytomegalovirus (CMV). The resulting construct was linearised and cotransformed into E. coli BJ5183 cells, together with an adenoviral backbone plasmid, pAdEasy-1 (He et al. 1998, see also Murphy & Orphanides 2002). Selected recombinants were analysed by restriction endonuclease digestion. Finally, recombinant plasmids encoding ER{alpha} were linearised and transfected into an adenovirus packaging cell line, HEK 293, in order to generate recombinant adenovirus that expresses ER{alpha} (Ad-ER{alpha}). A control recombinant adenovirus construct containing the E. coli ß-galactosidase gene (Ad-LacZ) was constructured in a similar manner. Recombinant adenovirus was harvested from HEK293 cells using Arklone extraction, purified by ultracentrifugation through a caesium chloride gradient and dialysed in a Slide-a-lyser cassette (Perbio Science, Cramlington, Northumbria, UK). The purified adeno-virus was aliquoted and stored at –80 °C. Each virus stock was titered in MDA-MB-231 cells, to determine the multiplicity of infection (MOI). For analysis of E2-dependent transcriptional responses, MDA-MB-231 cells were transfected with either Ad-LacZ or Ad-ER{alpha} at a MOI of 2500. Since both Ad-LacZ and Ad-ER{alpha} were engineered to co-express the green fluorescent protein (GFP), infection levels could be quantified by monitoring the expression of the GFP using fluorescent microscopy (% Infectivity=GFP cells/total cells x 100). Twelve hours after the initial infection, transfected MDA-MB-231 cells were photographed using both light and fluorescent microscopy, to determine the % of GFP-expressing cells. Both Ad-LacZ and Ad-ER{alpha} reproducibly gave between 90 and 100% infectivity of MDA-MB-231 cells under these conditions. Expression of ER{alpha} in cells transfected with Ad-ER{alpha} was confirmed by Northern blot analysis (Sambrook et al. 1989) using 1% denaturing agarose gels containing 10 µg total RNA per lane and a 417 bp 32P-labelled probe generated by PCR of the ER{alpha} cDNA (forward: 5'-ATACGAAA AGACCGAAGAGGAG-‘3; reverse: 5'-CCAGACGA GACCAATCATCA-‘3).

Reporter assay for ER-mediated transcription in MDA-MB-231 cells infected with adenovirus encoding ER{alpha}

MDA-MB-231 cells infected for 24 h with adenovirus (MOI=2500) encoding either ß-galactosidase (control; Ad-LacZ) or ER{alpha} (Ad-ER{alpha}) were co-transfected with a luciferase reporter construct that contained two copies of the vitellogenin estrogen response element (ERE) and also with a CMV-phRenilla plasmid (Promega), to measure transfection efficiency. After 24 h, cells were treated with 0.01% ethanol, as a control, or E2 in fresh medium at the concentrations indicated. Cells were incubated for a further 24 h before harvesting for lysis and luciferase assays using the Dual-luciferase assay system (Promega). Results are expressed in terms of relative luciferase activity after normalisation for renilla luciferase activity ± S.D.

RT-PCR analysis of the endogenous estrogen-responsive gene pS2 in MDA-MB-231 cells infected with adenovirus encoding ER{alpha}

Cells infected for 24 h with adenovirus (MOI=2500) encoding ß-galactosidase (control; Ad-LacZ) or ER{alpha} (Ad-ER{alpha}) were treated for 24, 30 and 50 h with 0.01% ethanol, as a control, or 10–8 M E2. Total RNA was isolated using Trizol reagent (Life Technologies) and purified according to the manufacturer’s instructions. DNA-free RNA was prepared using a DNA-free kit (Ambion, Huntingdon, Cambs, UK) according to the manufacturer’s instructions. DNase-treated RNA (1 µg) was reverse transcribed with oligo-dT using the Superscript II kit (Invitrogen) according to the manufacturer’s instructions. PCR analysis of pS2 gene expression was performed using the oligonucleotide primers 5'-TGACTCGGGGTCGCCT TTGGAG-‘3 and 5'-GTGAGCCGAGGCACAGCTG CAG-‘3. The ß-actin gene (5'-ACCATGGATGATG ATATCGC-‘3 and 5'-ACATGGCTGGGGTGTTG AAG-‘3) was used as a control.

Cell proliferation assay

Cells were maintained in MEM containing 5% CDFCS and were seeded at 5000 cells/well in 24-well dishes in the same media. After overnight infection with Ad-LacZ or Ad-ER{alpha} (MOI=2500), the medium was removed and replaced with fresh medium containing either 0.01% ethanol, as a control, or 10–8 M E2 for 24 h. Cells were then incubated with 1 µCi [methyl-3H]thymidine at 37 °C for 4 h. Plates were sequentially washed and fixed with ice cold PBS, 10% TCA, MeOH and the incorporated label was recovered by incubation of the wells in 0.5 M NaOH for 30 min at 37 °C. Lysates were transferred to vials containing Optiphase ‘hi-safe’ 3 scintillation cocktail (PerkinElmer Life Sciences, Bea-consfield, Bucks, UK) and [3H]thymidine incorporation (c.p.m.) was determined in a scintillation counter.

Affymetrix GeneChip transcript profiling and data analysis for MDA-MB-231 cells infected with adenovirus encoding ER{alpha}

Cells infected for 24 h with adenovirus (MOI=2500) encoding ß-galactosidase (control; Ad-LacZ) or ER{alpha} (Ad-ER{alpha}) were treated for 48 h with 0.01% ethanol, as a control, or 10–8 M E2. Total RNA was isolated using Trizol reagent (Life Technologies) and purified according to the manufacturer’s instructions. Biotin-Labeled complementary RNAs were synthesized using the Bioarray HighYield RNA Transcript Labeling Kit (Affymetrix, High Wycombe, Bucks, UK) from 5 µg total RNA and hybridised to Affymetrix human U133A GeneChips as described in the Affymetrix GeneChip Technical Manual. Microarrays were then scanned and the intensities were averaged using Microarray Analysis Suite 5.0 (Affymetrix). The mean signal intensity of each array was globally normalized to 500. Affymetrix pivot files were imported into GeneSpring 6.0 (Silicon Genetics, Redwood City, CA, USA) and normalised to the 50th percentile of each GeneChip and to the median of each gene. Normalised data was filtered to exclude genes that lack a present flag or a raw signal strength >500 in any of the treatment groups. The three independent biological replicates data sets for MDA-MB-231 cells infected Ad-LacZ (± E2) or Ad-ER{alpha} (± E2) were initially filtered using a one-sample Student’s t-test (P<0.05) to identify statistically differentially expressed genes within each treatment group. The resulting 574 genes were subsequently analysed using a one-way ANOVA test with the following conditions: parametric test assuming equal variance, Benjamini and Hochberg false discovery rate <0.01 (Benjamini & Hochberg 1995), Tukey post-hoc testing (see http://www.silicongenetics.com for further details). Using these criteria, less than 1% of the 88 genes selected by ANOVA can be expected to be significant by chance. Genes with similar expression profiles were grouped together using hierarchical clustering (Pearson correlation). Gene names used in this manuscript were derived by homology searching of nucleotide sequence databases (BLASTn) using Affymetrix probe target sequences and the NetAffx (Liu et al. 2003) database. All genes described in the figures and text showed statistically significant alterations in expression in all three replicate studies. MIAME (Minimum Information About a Microarray Experiment)-compliant microarray data for the three independent replicate studies were submitted to the Gene Expression Omnibus (GEO) database (GEO 2004).

Quantitative real-time PCR analysis of gene expression

DNA-free RNA was prepared using ‘DNA-free’ (Ambion) according to the manufacturer’s instructions. DNase-treated RNA (0.7 µg) was reverse transcribed with random hexamers using Superscript III kit (Invitrogen) according to the manufacturer’s instructions. All quantitative real-time PCR reactions were carried out using an ABI Prism 7700 sequence detection system (Applied Biosystems, Warrington, Chester, UK). The thermal cycler conditions were, 2 min at 50 °C and 10 min at 95 °C followed by 15 seconds at 95 °C (denaturation) and 1 min at 60 °C (anneal–extension) for 40 cycles. The total volume for each reaction was 20 µl comprising 9 µl diluted cDNA (0.5 ng/µl), 10 µl Taqman Universal Master mix and 1 µl Taqman gene expression assay (Applied Biosystems). Each Taqman gene expression assay contains forward primer, reverse primer and Taqman MGB probe (primer locations and corresponding gene accession numbers are shown in Table 1Go). Each RNA sample was assayed in triplicate and the mean Ct value was calculated. The fold change was determined using the {Delta}{Delta}Ct method. All genes were normalised to the control gene RPLP0/36B4 (Accession number: NM_001002 [GenBank] ; Laborda 1991).


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  		Table 1 Taqman Gene expression assays used for quantitative real-time PCR
 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Reintroduction of functional ER{alpha} into ER-negative MDA-MB-231 breast cancer cells by adenoviral transfection

We used a recombinant adenoviral delivery system to examine the molecular mechanisms through which the reintroduction of ER{alpha} into ER-negative MDA-MB-231 breast cancer cells confers E2-dependent suppression of proliferation. (He et al. 1998, Fig. 1AGo). Recombinant adenoviruses were engineered to co-express the full-length human ER{alpha} cDNA (amino acids 1–595; Green et al. 1986) and GFP, as described in the Materials and methods (Ad-ER{alpha}). Recombinant adenoviruses containing the E. coli LACZ gene in place of the human ER{alpha} gene were used as a control (Ad-LacZ). Quantification of infection levels in MDA-MB-231 cells by fluorescent microscopy revealed that GFP was expressed in >90% of cells after infection (Fig. 1BGo). The expression of a transcript corresponding to the transfected human ER{alpha} cDNA was confirmed by Northern blotting (Fig. 1CGo). Quantitative real-time PCR analysis of ER{alpha} gene expression levels (data not shown) revealed that the reintroduction of ER{alpha} into ER-negative MDA-MB-231 breast cancer cells by adenoviral transfection results in higher levels (~5-fold) of ER{alpha} expression than those found in MCF-7 breast cancer cells, consistent with previous studies (Lazennec & Katzenellenbogen 1999).



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  		Figure 1 Reintroduction of ER{alpha} into ER-negative MDA-MB-231 breast cancer cells. (A) Recombinant adenovirus engineered to co-express both GFP and human ER{alpha} (Ad-ER{alpha}) was used to infect MDA-MB-231 cells. Recombinant adenovirus containing the E. coli LACZ gene in place of ER{alpha} (Ad-LacZ) was used as a control. (B) MDA-MB-231 cell infection efficiencies of greater than 90% were measured routinely using the adenovirus constructs at a MOI of 2500. Left panel: light microscopy of MDA-MB-231 cells 24 hr after infection with Ad-ER{alpha}. The efficiency of viral infection was determined by measuring the proportion of cells that exhibit GFP fluorescence (right panel). (C) Northern blot analysis of ER{alpha} expression 24 h after Ad-LacZ (lane 1) or Ad-ER{alpha} (lane 2) infection of MDA-MB-231 cells.

 
To confirm that ER{alpha} re-expression in MDA-MB-231 cells was functional, we used a reporter-based transfec-tion assay that measures the ability of ligand-activated ERs to regulate transcription via a consensus multimer-ised ERE present on a transiently transfected plasmid. In cells infected with Ad-ER{alpha}, but not in cells infected with Ad-LacZ, the addition of E2 (10–8 M and 10–7 M) increased reporter gene expression (4.8- and 3.3-fold respectively; Fig. 2AGo), demonstrating that adenoviral infection resulted in the expression of transcriptionally active ER{alpha}. We next examined the ability of adenoviral-encoded ER{alpha} to regulate endogenous (i.e. chromosomal) genes. For this purpose, we selected the classical estrogen responsive gene, pS2 (also known as TFF1; Davidson et al. 1986), which contains a consensus ERE in its promoter region and is regulated directly by ERs (Berry et al. 1989). Cells infected with Ad-LacZ or Ad-ER{alpha} were treated with 10–8 M E2 or vehicle (ethanol) for 24, 30 or 50 h prior to RT-PCR analysis of pS2 gene expression. As expected, pS2 gene expression was induced by E2 in cells expressing ER{alpha} (Fig. 2BGo, lanes 7 to 12), but not in control cells (Fig. 2BGo, lanes 1 to 6). Therefore, the adenovirus-encoded ER{alpha} is capable of activating an endogenous chromosomal gene in the presence of E2.



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  		Figure 2 MDA-MB-231 cells infected with adenovirus encoding ER{alpha} contain transcriptionally active ERs. (A) Cells were infected with adenovirus encoding ß-galactosidase (Ad-LacZ control) or ER{alpha} (Ad-ER{alpha}) and were co-transfected with a luciferase reporter construct, that contained two copies of the vitellogenin ERE, and with the CMV-phRenilla plasmid (to measure transfection efficiency). After 24 h, cells were treated with 0.01% ethanol, as a control, or estradiol at the concentrations indicated. Results are expressed as relative luciferase activities after normalisation for Renilla luciferase activity +S.D. (n=6). (B) MDA-MB-231 cells infected with Ad-LacZ or Ad-ER{alpha} were treated with vehicle (ethanol) or E2 (10–8 M) for the times indicated and the expression of the endogenous pS2 gene was analysed by RT-PCR. The ß-actin gene was used as a control.

 
We next examined the effect of adenoviral transfec-tion of ER{alpha} on cell proliferation. The re-expression of human ER{alpha} in MDA-MB-231 breast cancer cells restores hormone responsiveness, but leads to the inhibition of proliferation by E2 (Garcia et al., 1992, Levenson and Jordan 1994, Lazennec and Katzenellenbogen 1999). Consistent with these observations, E2 caused a 3-fold decrease in proliferation in MDA-MB-231 cells that re-express ER{alpha} (Fig. 3Go).



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  		Figure 3 E2 inhibits proliferation in MDA-MB-231 breast cancer cells that re-express ER{alpha} after transfection with Ad-ER{alpha}. MDA-MB-231 cells were infected with either Ad-LacZ or Ad-ER{alpha}. The cells were then treated with control vehicle (0.01% ethanol) or E2 at the concentrations indicated for 24 h. Proliferation was measured by [methyl-3H]thymidine incorporation. Values are the mean+ S.D. of three determinations. Similar results were obtained in two independent experiments.

 
We conclude that adenoviral transfection of ER{alpha} into the ER-negative MDA-MB-231 breast cancer cell line confers both transcriptional and anti-proliferative responses to E2. Therefore, this model system is suitable for investigating the mechanism by which E2 suppresses proliferation in ER-negative cells that re-express ER{alpha}.

Changes in gene expression associated with estrogen-induced suppression of proliferation in ER-negative MDA-MB-231 breast cancer cells that re-express ER{alpha}

Statistical analysis of genes regulated by E2
We used microarray gene expression profiling to obtain a holistic view of the endogenous transcriptional targets of ER{alpha} in our model system. The expression of 22 483 genes in each of four treatment groups (Ad-LacZ, Ad-LacZ+E2, Ad-ER{alpha} and Ad-ER{alpha}+E2) was measured using the Affymetrix human GeneChip U133A, and the resulting data were subjected to rigorous statistical analyses (Materials and methods). 574 gene probe sets were selected as being significantly (P<0.05) under- or overexpressed in one or more of the 4 treatment groups using a Student’s t-test, based on data from three independent biological replicates. A stringent ANOVA test (Benjamini and Hochberg multi-testing correction, false positive rate <0.01; Benjamini & Hochberg 1995) was then applied, resulting in the identification of 88 gene probe sets showing differential expression between one or more of the 4 treatment groups (Fig. 4Go). Five of these gene probe sets represented the ER{alpha} gene, confirming that this gene was re-expressed in MDA-MB-231 cells infected with the ER{alpha} adenoviral construct. None of the 88 gene probe sets were E2-responsive in MDA-MB-231 cells transfected with the control adenovirus construct (Ad-LacZ), consistent with the ER-negative status of this cell line. In contrast, 83 probe sets showed a transcriptional response to E2 in MDA-MB-231 cells transfected with Ad-ER{alpha}. The magnitude of E2-dependent alterations in gene expression for the 83 gene probe sets, together with their gene ontology descriptions and functional classifications, are shown in Table 2Go. These genes include the classical E2-responsive gene pS2/TFF1 (Davidson et al. 1986, Berry et al. 1989) and TGFA, both of which have previously been observed to be up-regulated by E2 after adenoviral transfection of ER{alpha} into MDA-MB-231 cells (Lazennec & Katzenellenbogen 1999).



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  		Figure 4 Microarray analysis of estrogen-responsive genes in MDA-MD-231 breast cancer cells transfected with Ad-LacZ or Ad-ER{alpha}. Statistical analysis of Affymetrix HG-U133A GeneChip data was performed on three independent biological replicate studies of MDA-MB-231 cells infected with either Ad-LacZ or Ad-ER{alpha} prior to 48 h incubation with either vehicle control (0.01% ethanol) or 10–8M E2. Differentially expressed genes within each treatment group were identified using a one sample Student’s t-test (P<0.05). The resulting 547 genes were subsequently filtered using a stringent one-way ANOVA test combined with Benjamini and Hochberg multiple testing correction (false discovery rate<0.01; Benjamini & Hochberg 1995). Using these criteria, less than 1% of the 88 genes shown can be expected to be significant by chance. Genes with similar expression profiles were grouped together using hierarchical clustering (Pearson correlation) and the resulting gene tree is shown. The magnitude of fold-induction or -repression for each gene (relative to the median of its expression across all experimental samples) is indicated by the colour bar. Data shown are based on three replicate studies. Quantitative data for the magnitude of each gene expression change, together with gene descriptions and Affymetrix probe set IDs are shown in Table 2Go.

 

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  		Table 2 Genes regulated by E2 in MDA-MB-231 cells that re-express ER{alpha}
 
Gene ontology and promoter analysis of ER{alpha}-dependent estrogen-responsive genes.
The molecular functions of the 83 E2-responsive genes we identified fall into a broad range of gene ontology classifications (Liu et al. 2003, Bard & Rhee 2004), including cell cycle control, signalling, growth factors, transporters, defense responses and cell adhesion (Table 2Go), highlighting the diverse gene networks and metabolic and cell regulatory pathways through which E2 exerts its effects on breast cancer cells. Many of these genes have not previously been identified as being E2-responsive in breast cancer cells, and include genes whose function is unknown (e.g. FLJ20152, FLJ20366, FLJ22679; Table 1Go).

In order to gain more evidence that these genes were regulated directly by ER{alpha}, we searched their regulatory regions for EREs, based on similarity to the consensus ERE sequence (Klinge 2001). In silico analysis of the sequences found 3000 bp immediately upstream of the transcriptional start site of each of the 83 genes and revealed the presence of one or more candidate EREs (Table 3Go). This provides further evidence that the genes we have identified are regulated by ER{alpha} directly. A number of these ERE motifs have also been identified independently in a recent genome-wide screen for ERE motifs in the human and mouse genomes (Bourdeau et al. 2004).


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  		Table 3 Identification of putative estrogen-response elements (EREs) in E2-responsive genes identified by microarray analysis of MDA-MD-231 cells that re-express ER{alpha}
 
Estrogen represses the expression of genes that promote cell proliferation and survival
Several of the E2-responsive genes we have identified (e.g. MCM7, CDC20, CKSB1, SURVIVIN; Table 2Go) have been shown previously to be involved in the regulation of cell proliferation and survival. Among these genes is: (1) the MCM7 (minichromosome maintenance deficient 7) gene, encoding a DNA replication licensing factor that functions to limit a cell to a single round of replication per cell cycle (Blow & Hodgson 2002); (2) the WD-repeat protein CDC20, essential for progression through mitosis (Vodermaier 2001); (3) the CKS1B gene, encoding a substrate-targeting subunit of the SCF ubiquitin ligase complex that regulates the entry into S phase (Reed 2003) and (4) SURVIVIN, is a member of the inhibitor of apoptosis (IAP) family that is involved in the regulation of cell division (Kobayashi et al. 1999). Importantly, the repression of these genes by E2 is consistent with the suppression of proliferation observed in E2-treated MDA-MB-231 cells that re-express ER{alpha} (Fig. 3Go). We speculate, therefore, that these transcriptional responses are associated directly with the anti-proliferative effects of E2 observed in these cells.

In order to identify additional E2-responsive regulators of cell proliferation and survival that may have been missed by our initial stringent ANOVA analysis, in which multi-testing correction was employed to mini-mise the false discovery rate (Fig. 4Go), we re-interrogated our transcript profiling data. We found 34 additional E2-responsive probe sets (Student’s t-test P<0.05) whose gene ontology classifications were consistent with a role in the regulation of cell cycle progression, proliferation or survival (Table 4Go). This analysis revealed that E2 down-regulated the expression of many additional genes involved in cell cycle progression (CDC2, CYCLIN B1, CYCLIN B2, CYCLIN G1, CHK1, BUB3, STK6, SKB1, CSE1) and chromosome replication (MCM2, MCM3, FEN1, RRM2, TOPII, RFC1). A number of negative regulators of the cell cycle were also induced by E2, including KIP2, NBL1 (neuroblastoma suppressor of tumorigenicity 1, also known as DAN; Ozaki et al. 1995) and QUIESCIN Q6 (Coppock et al. 1998). The functional relationships between the numerous estrogen-responsive cell cycle regulators identified in this study (Table 4Go) are summarised in the cell cycle pathway map shown in Fig. 5Go. The overall effect of changes in the expression of these cell cycle genes is consistent with the observed suppression of proliferation (Fig. 3Go).


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  		Table 4 Transcriptional responses associated with cell proliferation and survival in E2 stimulated MDA-MB-231 cells that re-express ER{alpha}
 


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  		Figure 5 E2-responsive cell cycle genes in MDA-MB-231 cells that re-express ER{alpha}. The cell cycle pathway map was originally adapted from KEGG and was obtained from www.GenMAPP.org (Dahlquist et al. 2002). Red and green boxes indicate up- and down-regulation of gene expression by estrogen, respectively.

 
Opposing estrogen-dependent transcriptional regulation of cell cycle genes in MDA-MB-231 cells that re-express ER{alpha} and ER{alpha}-positive MCF-7 breast cancer cells
Further evidence for the involvement of the genes described above in the suppression of proliferation in our model system is provided by previous reports showing that the expression of many of the same genes is regulated in the opposite direction in ER-positive MCF-7 breast cancer cells treated with E2 (Table 5Go). Transcript profiling previously revealed that FEN1, MCM2, MCM3, MCM7, CDC2, CDC20, BUB1, STK6, CSE1 L and SURVIVIN are up-regulated during E2-induced proliferation of MCF-7 cells (Lobenhofer et al. 2002, Frasor et al. 2003). This is in contrast to the repression of these genes by E2 in MDA-MB-231 cells that re-express ER{alpha} in the microarray analysis reported here (Table 5Go). Quantitative real-time PCR analysis of the E2-responsiveness of these genes in both MCF-7 cells and MDA-MB-231 cells that re-express ER{alpha} confirms and extends our observations of opposing transcriptional responses to E2 in these two cell types (Fig. 6Go). We conclude that the paradoxical anti-proliferative effects of E2 in MDA-MB-231 cells that overexpress ER{alpha} may be due to the aberrant regulation of key cell cycle regulators.


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  		Table 5 Opposing transcriptional responses to E2 in ER-positive MCF-7 cells versus MDA-MB-231 cells that re-express ER{alpha}
 


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  		Figure 6 Quantitative real-time PCR analysis of opposing transcriptional responses in MCF-7 versus MDA-MD-231 breast cancer cells that re-express ER{alpha}. MDA-MB-231 cells were transfected with either Ad-LacZ or Ad-ER{alpha} before treatment with either vehicle control (0.01% ethanol) or 10–8 M E2 for 48 h. MCF-7 cells were treated with either vehicle control (0.1% ethanol) or 10–9 M E2 for 4, 8, 24 and 48 h. E2-dependent changes in gene expression are shown relative to time-matched vehicle controls. {Delta}{Delta}Ct was calculated by normalising to the control gene RPLP0/36B4 (Accession number: NM_001002; Laborda 1991) and comparative Ct values are shown as log2 fold changes.

 
Opposing estrogen-dependent transcriptional regulation of growth-related genes in MDA-MB-231 cells that re-express ER{alpha} and ER{alpha}-positive MCF-7 breast cancer cells
In addition to the aberrant regulation of cell cycle genes, we also found that a number of growth-related genes were regulated by E2 in the opposite direction in ER-negative MDA-MB-231 cells that re-express ER{alpha} compared with ER-positive MCF7 cells. These include E-CADHERIN (CDH1), an important mediator of cell–cell interactions that acts as a tumour suppressor gene and whose loss of expression is associated with invasive growth (Thiery 2002). CDH1 is down-regulated by E2 in ER-containing breast cancer cells (Oesterreich et al. 2003), but is up-regulated in MDA-MD-231 cells transfected with ER{alpha} (Table 5Go). This suggests that the negative growth response to E2 in these cells may involve alteration of epithelial cell architecture. Surprisingly, the SNAIL gene, a known negative transcriptional regulator of CDH1, was also up-regulated by E2 in these cells (Table 2Go), an event that is normally associated with the loss of expression of CDH1 (Fujita et al. 2003). Nevertheless, our data reveal the altered expression in these cells by E2 of two genes associated with the invasive growth pathway in breast cancer.

Components of the c-myc and AP-1 transcription factors also show opposing transcriptional responses in breast cancer cells containing endogenous versus transfected ER{alpha}. The gene encoding the AP-1 transcription factor, Fos-like antigen 1 (FOSL1; also known as FRA-1), is repressed by E2 in MDA-MB-231 cells transfected with ER{alpha} (Table 2Go, Fig. 6Go), consistent with previous observations that AP-1 activity is inhibited by E2 in MDA-MB-231 cells stably transfected with ER{alpha} (Philips et al. 1998). Furthermore, c-myc has previously been reported to be repressed by E2 in MDA-MB-231 cells transfected with adenovirally encoded ER{alpha} (Lazennec & Katzenellenbogen 1999). The repression of these genes by E2 in cells that re-express ER{alpha} is in marked contrast to their induction by E2 in MCF-7 cells (Fig. 6Go; van der Burg et al. 1989, Weisz et al. 1990) and suggests that the negative regulation of transcription factors that control growth and differentiation may be a key event leading to the E2-dependent suppression of proliferation in MDA-MB-231 cells that re-express ER{alpha}.

Overall, these data reveal the diverse gene networks and metabolic and cell regulatory pathways through which E2 exerts its effects on MDA-MB-231 breast cancer cells that re-express ER{alpha}, and provide novel mechanistic insights into the anti-proliferative effect of E2 in these cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We have used gene expression profiling to obtain a holistic view of the transcriptional responses associated with the effects of estrogen in ER-negative MDA-MB-231 breast cancer cells that re-express ER{alpha}. The genes we have identified are likely to be regulated directly by E2. Evidence for this comes from: (1) the dependence of their regulation on E2; (2) the requirement for ER{alpha} and (3) the presence of consensus EREs within 3000 bp upstream of their transcriptional start sites. Moreover, the molecular functions of many of the E2-responsive genes that we have identified, including chromosome replication, cell cycle regulation, cell survival and growth factor signalling, provide novel insights into the mechanisms underlying the E2-induced suppression of proliferation in ER-negative breast cancer cells that re-express ER{alpha} (Garcia et al. 1992, Levenson & Jordan 1994). Importantly, our data reveal that several key regulators of cell proliferation and survival are regulated in opposite directions when compared with their behaviour in ER-positive MCF-7 breast cancer cells. Therefore, these data go some way towards explaining the paradoxical effects of estrogens in ER-negative breast cancer cells in which ER{alpha} has been re-expressed.

An important question arising from our studies is how E2-bound ER{alpha} targets the same genes with opposing transcriptional outcomes in ER-negative and ER-positive breast cancer cells. Transfection of functional ER{alpha} into MDA-MB-231 cells does not alter gene expression significantly in the absence of exogenous E2 (Fig. 4Go; Lazennec & Katzenellenbogen 1999), indicating that re-expression of ER{alpha} per se does not alter the transcriptional status of these genes. Since ER-mediated transcriptional regulation involves a plethora of coregu-lator proteins (Moggs & Orphanides 2001, Hall et al. 2001, McKenna & O’Malley 2002, Tremblay & Giguere 2002), it is possible that cell type-specific differences in transcriptional responses to estrogens are due to differences in the expression levels, accessibility, or localisation of critical cofactors. Precedent exists for this mechanism: higher levels of steroid receptor coactivator 1 (SRC-1) expression in Ishikawa endome-trial cells, compared with MCF-7 breast cancer cells, result in opposing cellular responses to the selective estrogen receptor modulator tamoxifen (Shang & Brown 2002). Furthermore, the altered localisation of Retinoid x receptor alpha (RXR{alpha}) in MDA-MB-231 cells versus MCF-7 cells has been associated with the differential responsiveness of these cell lines to retinoids (Tanaka et al. 2004). RXR{alpha} is localized throughout the nucleoplasm in the retinoid-responsive MCF-7 breast cancer cell line, whereas it is found in the splicing factor compartment of the retinoid-resistant MDA-MB-231 breast cancer cell line. Interestingly, previous studies have shown that hydroxytamoxifen can reverse the suppression of proliferation by E2 in MDA-MB-231 cells that re-express ER{alpha} (Garcia et al. 1992, Lazennec & Katzenellenbogen 1999). Since hydroxytamoxifen normally suppresses proliferation in ER-containing breast cancer cells, these observations are consistent with MDA-MB-231 cells lacking the full complement of cofactors that are required for appropriate regulation of proliferation by E2 and anti-estrogens.

Another factor that may contribute to the contrasting ER-mediated transcriptional effects seen in MDA-MB-231 and MCF-7 cells is the DNA methylation status and chromatin structure of the gene regulatory regions. Indeed, DNA methylation status determines the expression levels of ER{alpha} in breast cancer cells: silencing of the ER{alpha} gene in MDA-MB-231 cells occurs through epigenetic alterations that include the hypermethylation of CpG island DNA sequences in the gene promoter region (Ottaviano et al. 1994). Consistent with the existence of an epigenetic silencing mechanism in MDA-MB-231 cells, the ER{alpha} gene can be reactivated by the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (Ferguson et al. 1995), and the histone deacetylase inhibitor, trichostatin A (Yang et al. 2000), and a combination of these inhibitors results in the synergistic reactivation of ER{alpha} (Yang et al. 2001). It is, therefore, likely that differences in the direction of E2-induced gene regulation between MDA-MB-231 and MCF-7 cells may be due to differences in the epigenetic status of target genes.

The poor prognosis of ER-negative breast cancers, together with their unresponsiveness to anti-estrogen therapy, creates an urgent need for novel targeted therapies that do not rely on inhibition of ER{alpha} (Rochefort et al. 2003). Alternative approaches have met with some success. For example, over-expression of the human epidermal growth fator-2 (HER2) oncogene in human breast cancers has been associated with a more aggressive progression of disease, and a monoclonal antibody (trastuzumab) directed against the extracellular domain of HER2 is therapeutically active in a proportion of HER2-positive breast tumours (Menard et al. 2003). We have identified the transcriptional networks through which ER{alpha} is able to inhibit the proliferation of an ER-negative cell line. Targeting of these, or similar, pathways may lead to the development of novel approaches for the control of ER-negative breast tumours.


   Acknowledgements
 
We would like to thank K Bundell (AstraZeneca Pharmaceuticals, Macclesfield, UK) for the generous gift of adenovirus DNA constructs for the expression of LacZ and human ER{alpha} and also J Edmunds for generating the MCF-7 cell RNA samples. We would also like to thank T Barlow and B Jeffery from the Food Standards Agency and our colleagues at Syngenta CTL for their guidance and advice throughout the course of this project. This work was partially supported by a grant from the UK Food Standards Agency. The authors declare that they have no conflict of interest.


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Received 22 November 2004
Accepted 13 December 2004



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