Anti-proliferative effect of estrogen in breast cancer cells that re-express ERα is mediated by aberrant regulation of cell cycle genes

in Journal of Molecular Endocrinology
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  • Syngenta CTL, Alderley Park, Cheshire SK10 4TJ, UK

Estrogen receptor (ER)-negative breast carcinomas do not respond to hormone therapy, making their effective treatment very difficult. The re-expression of ERα 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 17β-estradiol (E2) in ER-positive breast cancer cells, E2 suppresses proliferation in ER-negative breast cancer cells in which ERα 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 ERα 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 ERα has been reintroduced.

Abstract

Estrogen receptor (ER)-negative breast carcinomas do not respond to hormone therapy, making their effective treatment very difficult. The re-expression of ERα 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 17β-estradiol (E2) in ER-positive breast cancer cells, E2 suppresses proliferation in ER-negative breast cancer cells in which ERα 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 ERα 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 ERα has been reintroduced.

Introduction

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)α and ERβ. 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 ERα into ER-negative breast cancer cells results in the suppression of proliferation by 17β-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 ERα. 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

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 ERα in human MDA-MB-231 cells

Full-length human ERα (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 ERα were linearised and transfected into an adenovirus packaging cell line, HEK 293, in order to generate recombinant adenovirus that expresses ERα (Ad-ERα). A control recombinant adenovirus construct containing the E. coli β-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-ERα at a MOI of 2500. Since both Ad-LacZ and Ad-ERα 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 × 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-ERα reproducibly gave between 90 and 100% infectivity of MDA-MB-231 cells under these conditions. Expression of ERα in cells transfected with Ad-ERα 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 ERα 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 ERα

MDA-MB-231 cells infected for 24 h with adenovirus (MOI=2500) encoding either β-galactosidase (control; Ad-LacZ) or ERα (Ad-ERα) 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 ERα

Cells infected for 24 h with adenovirus (MOI=2500) encoding β-galactosidase (control; Ad-LacZ) or ERα (Ad-ERα) 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 β-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-ERα (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 ERα

Cells infected for 24 h with adenovirus (MOI=2500) encoding β-galactosidase (control; Ad-LacZ) or ERα (Ad-ERα) 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-ERα (± 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 1). Each RNA sample was assayed in triplicate and the mean Ct value was calculated. The fold change was determined using the ΔΔCt method. All genes were normalised to the control gene RPLP0/36B4 (Accession number: NM_001002; Laborda 1991).

Results

Reintroduction of functional ERα 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 ERα into ER-negative MDA-MB-231 breast cancer cells confers E2-dependent suppression of proliferation. (He et al. 1998, Fig. 1A). Recombinant adenoviruses were engineered to co-express the full-length human ERα cDNA (amino acids 1–595; Green et al. 1986) and GFP, as described in the Materials and methods (Ad-ERα). Recombinant adenoviruses containing the E. coli LACZ gene in place of the human ERα 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. 1B). The expression of a transcript corresponding to the transfected human ERα cDNA was confirmed by Northern blotting (Fig. 1C). Quantitative real-time PCR analysis of ERα gene expression levels (data not shown) revealed that the reintroduction of ERα into ER-negative MDA-MB-231 breast cancer cells by adenoviral transfection results in higher levels (~5-fold) of ERα expression than those found in MCF-7 breast cancer cells, consistent with previous studies (Lazennec & Katzenellenbogen 1999).

To confirm that ERα 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-ERα, 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. 2A), demonstrating that adenoviral infection resulted in the expression of transcriptionally active ERα. We next examined the ability of adenoviral-encoded ERα 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-ERα 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 ERα (Fig. 2B, lanes 7 to 12), but not in control cells (Fig. 2B, lanes 1 to 6). Therefore, the adenovirus-encoded ERα is capable of activating an endogenous chromosomal gene in the presence of E2.

We next examined the effect of adenoviral transfec-tion of ERα on cell proliferation. The re-expression of human ERα 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 ERα (Fig. 3).

We conclude that adenoviral transfection of ERα 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 ERα.

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

Statistical analysis of genes regulated by E2

We used microarray gene expression profiling to obtain a holistic view of the endogenous transcriptional targets of ERα in our model system. The expression of 22 483 genes in each of four treatment groups (Ad-LacZ, Ad-LacZ+E2, Ad-ERα and Ad-ERα+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. 4). Five of these gene probe sets represented the ERα gene, confirming that this gene was re-expressed in MDA-MB-231 cells infected with the ERα 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-ERα. 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 2. 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 ERα into MDA-MB-231 cells (Lazennec & Katzenellenbogen 1999).

Gene ontology and promoter analysis of ERα-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 2), 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 1).

In order to gain more evidence that these genes were regulated directly by ERα, 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 3). This provides further evidence that the genes we have identified are regulated by ERα 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).

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 2) 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 ERα (Fig. 3). 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. 4), 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 4). 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 4) are summarised in the cell cycle pathway map shown in Fig. 5. The overall effect of changes in the expression of these cell cycle genes is consistent with the observed suppression of proliferation (Fig. 3).

Opposing estrogen-dependent transcriptional regulation of cell cycle genes in MDA-MB-231 cells that re-express ERα and ERα-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 5). 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 ERα in the microarray analysis reported here (Table 5). 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 ERα confirms and extends our observations of opposing transcriptional responses to E2 in these two cell types (Fig. 6). We conclude that the paradoxical anti-proliferative effects of E2 in MDA-MB-231 cells that overexpress ERα may be due to the aberrant regulation of key cell cycle regulators.

Opposing estrogen-dependent transcriptional regulation of growth-related genes in MDA-MB-231 cells that re-express ERα and ERα-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 ERα 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 ERα (Table 5). 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 2), 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 ERα. 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 ERα (Table 2, Fig. 6), consistent with previous observations that AP-1 activity is inhibited by E2 in MDA-MB-231 cells stably transfected with ERα (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 ERα (Lazennec & Katzenellenbogen 1999). The repression of these genes by E2 in cells that re-express ERα is in marked contrast to their induction by E2 in MCF-7 cells (Fig. 6; 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 ERα.

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 ERα, and provide novel mechanistic insights into the anti-proliferative effect of E2 in these cells.

Discussion

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 ERα. 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 ERα 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 ERα (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 ERα has been re-expressed.

An important question arising from our studies is how E2-bound ERα targets the same genes with opposing transcriptional outcomes in ER-negative and ER-positive breast cancer cells. Transfection of functional ERα into MDA-MB-231 cells does not alter gene expression significantly in the absence of exogenous E2 (Fig. 4; Lazennec & Katzenellenbogen 1999), indicating that re-expression of ERα 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 × receptor alpha (RXRα) 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). RXRα 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 ERα (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 ERα in breast cancer cells: silencing of the ERα 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 ERα 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 ERα (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 ERα (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 ERα 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.

Table 1

Taqman Gene expression assays used for quantitative real-time PCR

Affymetrix probe setTaqman gene expression assayGenBank accession numberExon locationCentral nucleotide location
Gene symbol
MCM2202107_s_atHs00170472_m1NM_0045262/3299
MCM3201555_atHs00172459_m1NM_00238812/131938
MCM7201112_s_atHs00428518_m1NM_1827769/101794
CSE1L202870_s_atHs00169158_m1NM_00131614/151614
CDC2210559_s_atHs00176469_m1NM_00178612107
CDC20203213_atHs00415851_g1NM_0012551259
BUB1209642_atHs00177821_m1NM_0043361/273
BIRC5202095_atHs00153353_m1NM_0011683/4393
FEN1204768_s_atHs00748727_s1NM_0041112643
FOSL1204420_atHs00759776_s1NM_00543841103
RPLP0/36B4201033_x_atHs99999902_m1NM_001002267
Table 2

Genes regulated by E2 in MDA-MB-231 cells that re-express ERα

Gene nameBiological pathway/processRatio of gene expression E2:AO (mean±s.d.)
Eighty-three genes demonstrated significantly induced or repressed expression after E2 stimulation of MDA-MB-231 cells that re-express ERa (based on ANOVA using Benjamini and Hochberg multiple testing correction (false positive discovery rate <0.01; Benjamini & Hochberg 1995)). Mean gene expression ratios ±s.d. (E2-treated relative to vehicle control; based on three independent biological replicate experiments) are shown together with Affymetrix GeneChip probe set, gene name, biological pathway/process (as of the NetAffx update on 23 June 2004). Two or more Affymetrix GeneChip probe sets for the same gene indicates that probe sets specific for independent regions of the same gene demonstrated estrogen-regulated expression.
Affymetrix probe set
201531_atzinc finger protein 36ZFP36mRNA catabolism3.8±1.9
212442_s_atLAG1 longevity assurance homolog 6LASS62.2±0.6
201339_s_atsterol carrier protein 2SCP2steroid biosynthesis1.9±0.5
218002_s_atchemokine ligand 14CXCL1430.3±10.7
210517_s_atA kinase anchor protein 12AKAP123.2±0.6
209304_x_atgrowth arrest and DNA damage-inducible, betaGADD45Bcell cycle regulation/apoptosis4.1±1.5
210059_s_atmitogen-activated protein kinase 13MAPK13regulation of translation/signaling3.1±1.3
211168_s_atregulator of nonsense transcripts 1RENT1mRNA catabolism3.2±1.6
219480_atsnail homolog 1SNAI1transcription12.6±6.3
205016_attransforming growth factor, alphaTGFAregulation of cell cycle/signaling4.3±1.0
203058_s_at3′-phosphoadenosine 5′-phosphosulfate synthase 2PAPSS2nucleic acid metabolism8.4±3.8
205206_atKallmann syndrome 1 sequenceKAL1cell adhesion2.6±0.7
40829_atWD and tetratricopeptide repeats 1WDTC11.6±0.3
218322_s_atacyl-CoA synthetase long-chain family member 5ACSL5fatty acid metabolism2.9±0.8
204158_s_atT-cell, immune regulator 1, ATPase, H+ transportingTCIRG1defense response/proliferation5.9±1.8
200884_atcreatine kinaseCKBamino acid metabolism5.4±1.7
218532_s_athypothetical protein FLJ20152FLJ2015210.7±5.5
205105_atmannosidase, alpha 2A1MAN2A1carbohydrate metabolism1.9±0.4
201720_s_atLysosomal-associated multispanning membrane protein-5LAPTM53.9±0.8
205899_atcyclin A1CCNA1regulation of cell cycle5.9±3.1
31637_s_atnuclear receptor subfamily 1, group D, member 1NR1D1transcription/circadian rhythm regulator2.2±0.4
203060_s_at3′-phosphoadenosine 5′-phosphosulfate synthase 2PAPSS2nucleic acid metabolism6.9±1.9
201721_s_atLysosomal-associated multispanning membrane protein-5LAPTM54.3±1.5
218692_athypothetical protein FLJ20366FLJ203663.0±0.6
205009_attrefoil factor 1TFF1cell growth/defense response22.3±4.9
210357_s_atspermine oxidaseSMOXelectron transport4.4±1.2
211429_s_atserine protease inhibitor, clade A, member 1SERPINA1acute-phase response8.5±1.2
220486_x_athypothetical protein FLJ22679FLJ226791.9±0.2
204326_x_atmetallothionein 1XMT1Xresponse to metal ion2.3±0.3
202833_s_atserine protease inhibitor, clade A, member 1SERPINA1acute-phase response10.9±0.3
218749_s_atsolute carrier family 24, member 6SLC24A64.2±1.6
203059_s_at3′-phosphoadenosine 5′-phosphosulfate synthase 2PAPSS2nucleic acid metabolism8.4±1.7
213004_atangiopoietin-like 2ANGPTL2development6.9±2.3
217744_s_atTP53 apoptosis effectorPERPapoptosis2.9±1.2
207935_s_atkeratin 13KRT13cytoskeleton16.8±0.4
212216_atputative amino acid transporterKIAA04361.7±0.3
203071_atsemaphorin 3BSEMA3Bsignaling7.7±1.9
202267_atlaminin, gamma 2LAMC2cell adhesion4.2±1.3
201131_s_atE-cadherinCDH1cell adhesion35.2±35.4
202950_atcrystallin, zetaCRYZ2.4±0.5
216323_x_attubulin, alpha 2TUBA2microtubule4.0±1.8
203661_s_attropomodulin 1TMOD1cytoskeleton7.5±2.9
202053_s_ataldehyde dehydrogenase 3 family, member A2ALDH3A2glycolysis/ascorbate, aldarate and fatty acid metabolism2.0±0.2
204368_atsolute carrier organic anion transporter family, member 2A1SLCO2A1lipid transport8.9±0.8
209035_atmidkineMDKregulation of cell cycle/signaling7.9±4.2
204664_atalkaline phosphataseALPPglycerolipid metabolism/folate biosynthesis129.1±86.8
213308_atSH3 and multiple ankyrin repeat domains 2SHANK2signaling4.1±1.7
214476_attrefoil factor 2TFF2defense response35.1±20.8
213001_atangiopoietin-like 2ANGPTL2development3.9±0.8
217165_x_atmetallothionein 2AMT2Acopper ion homeostasis2.8±0.5
203585_atzinc finger protein 185ZNF1853.0±0.6
210740_s_atinositol 1,3,4-triphosphate 5/6 kinaseITPK1signal transduction2.7±0.8
206461_x_atmetallothionein 1HMT1Hresponse to metal ion2.8±1.2
205068_s_atRho GTPase activating protein 26ARHGAP26growth/cytoskeleton2.6±0.2
212057_atKIAA0182 proteinKIAA01822.6±1.1
202458_atserine protease 23SPUVEproteolysis2.6±0.1
201858_s_atproteoglycan 1PRG12.5±0.5
219369_s_atOUT domain, ubiquitin aldehyde binding 2OTUB24.2±1.5
211474_s_atserine protease inhibitor, clade B, member 6SERPINB6acute-phase response2.2±0.4
213909_atleucine rich repeat containing 15LRRC153.1±1.0
202756_s_atglypican 1GPC1development4.7±2.2
219045_atras homolog gene family, member 5RHOFsignaling4.0±2.1
205148_s_atchloride channel 4CLCN4ion transport3.1±1.3
202976_s_atRho-related BTB domain containing 3RHOBTB32.8±1.3
201349_atsolute carrier family 9, isoform 3 regulator 1SLC9A3R1signaling/cytoskeleton7.5±5.0
221667_s_atheat shock protein 8HSPB87.0±2.5
215189_atkeratin 6KRTHB611.8±8.5
210827_s_atE74-like factor 3ELF3transcription2.1±0.4
212135_s_atATPase, Ca2+ transportingATP2B4ion transport2.2±0.4
210609_s_attumor protein p53 inducible protein 3TP53I3apoptosis3.2±0.8
217190_x_atestrogen receptor alphaESR1transcription/signaling1.5±0.4
215552_s_atestrogen receptor alphaESR1transcription/signaling1.3±0.4
211233_x_atestrogen receptor alphaESR1transcription/signaling1.2±0.5
211234_x_atestrogen receptor alphaESR1transcription/signaling1.2±0.5
211235_s_atestrogen receptor alphaESR1transcription/signaling1.2±0.4
218399_s_atcell division cycle associated 4CDCA4regulation of cell cycle0.6±0.09
202870_s_atcell division cycle 20 homologCDC20regulation of cell cycle0.5±0.1
211519_s_atkinesin family member 2CKIF2Cmitosis/proliferation0.5±0.1
201897_s_atCDC28 protein kinase subunit 1BCKS1Bcell proliferation0.6±0.1
210983_s_atminichromosome maintenance deficient 7MCM7DNA replication0.5±0.1
202503_s_atKIAA0101 gene productKIAA01010.5±0.07
204420_atFOS-like antigen 1FOSL1transcription/proliferation0.4±0.1
208002_s_atacyl-CoA hydrolaseBACHlipid metabolism0.6±0.07
202095_s_atsurvivinBIRC5cell cycle regulation/apoptosis0.5±0.06
204521_atpredicted protein clone 23733HSU797240.4±0.1
220155_s_atbromodomain containing 9BRD90.7±0.08
208994_s_atpeptidyl-prolyl isomerase GPPIGprotein folding/RNA splicing1.0±0.06
210006_atDKFZP564O243 proteinDKFZP564O243aromatic compound metabolism0.9±0.05
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 ERα

Gene nameNo. of EREsClosest to consensus ERE sequence
Putative EREs within 3000 bp upstream from the transcriptional start site of each gene were identified by comparison to the consensus ERE (GGTCAnnnTGACC) allowing for a maximum of 2 base changes in either half-site. The total number of EREs found within the 3000 bp region is also indicated. Promoter sequences for genes associated with each Affymetrix probe set were extracted from the UCSC mouse genome browser via the NetAffx database (Liu et al. 2003).
Affymetrix probe set
201531_atzinc finger protein 36ZFP363GACCAnnnTGACT
212442_s_atLAG1 longevity assurance homolog 6LASS6no sequence information available
201339_s_atsterol carrier protein 2SCP21GGACAnnnTGACT
218002_s_atchemokine ligand 14CXCL142AGGCAnnnTGACT
210517_s_atA kinase anchor protein 12AKAP123TCTCAnnnTGTCA
209304_x_atgrowth arrest and DNA damage-inducible, betaGADD45B6GGTCAnnnTGGTC (3)
210059_s_atmitogen-activated protein kinase 13MAPK133CGCCAnnnTGACC
211168_s_atregulator of nonsense transcripts 1RENT12GATCAnnnTGAAC
219480_atsnail homolog 1SNAI13TGGCAnnnTGAGC
205016_attransforming growth factor, alphaTGFA4AGCCAnnnTGAGC
203058_s_at3′-phosphoadenosine 5′-phosphosulfate synthase 2PAPSS24AGTCAnnnTGACC
205206_atKallmann syndrome 1 sequenceKAL12TGTCAnnnTGAAG
40829_atWD and tetratricopeptide repeats 1WDTC11GTTCAnnnTGACA
218322_s_atacyl-CoA synthetase long-chain family member 5ACSL54GATCAnnnTGAAC
204158_s_atT-cell, immune regulator 1, ATPase, H+ transportingTCIRG16GGTCAnnnTGACA
200884_atcreatine kinaseCKB3GGGCAnnnTGAGG
218532_s_athypothetical protein FLJ20152FLJ201523TGTCAnnnTGCCC
205105_atmannosidase, alpha 2A1MAN2A12AATCAnnnTGACC
201720_s_atLysosomal-associated multispanning membrane protein-5LAPTM53GGGCAnnnTGACC
205899_atcyclin A1CCNA11TTTCAnnnTGAAC
31637_s_atnuclear receptor subfamily 1, group D, member 1NR1D14AGTCAnnnTGACT
203060_s_at3′-phosphoadenosine 5′-phosphosulfate synthase 2PAPSS24AGTCAnnnTGACC
201721_s_atLysosomal-associated multispanning membrane protein-5LAPTM53GGGCAnnnTGACC
218692_athypothetical protein FLJ20366FLJ203664GTTCAnnnTGAAG
205009_attrefoil factor 1TFF12GGTCAnnnTGGCC
210357_s_atspermine oxidaseSMOX4GACCAnnnTGACC
211429_s_atserine protease inhibitor, clade A, member 1SERPINA12GGGCAnnnTGACT
220486_x_athypothetical protein FLJ22679FLJ226791ACTCAnnnTGAGT
204326_x_atmetallothionein 1XMT1X4CGACAnnnTGACA
202833_s_atserine protease inhibitor, clade A, member 1SERPINA12GGGCAnnnTGACT
218749_s_atsolute carrier family 24, member 6SLC24A66TGTCAnnnTGCCC
203059_s_at3′-phosphoadenosine 5′-phosphosulfate synthase 2PAPSS24AGTCAnnnTGACC
213004_atangiopoietin-like 2ANGPTL21GATGAnnnTGAGG
217744_s_atTP53 apoptosis effectorPERP4GGTCAnnnTGGTC
207935_s_atkeratin 13KRT134TGTCAnnnTGACT
212216_atputative amino acid transporterKIAA0436no sequence information available
203071_atsemaphorin 3BSEMA3B3GTGCAnnnTGACC
202267_atlaminin, gamma 2LAMC22GGTCAnnnTGCCA
201131_s_atE-cadherinCDH11GGCCAnnnTGATG
202950_atcrystallin, zetaCRYZ1AGCCAnnnTGAAG
216323_x_attubulin, alpha 2TUBA2??
203661_s_attropomodulin 1TMOD13CATCAnnnTGATC
202053_s_ataldehyde dehydrogenase 3 family, member A2ALDH3A23TGCCAnnnTGACC
204368_atsolute carrier organic anion transporter family, member 2A1SLCO2A16GATCAnnnTGAGG
209035_atmidkineMDK2TCTCAnnnTGACA
204664_atalkaline phosphataseALPP4GGTCAnnnTGGCA
213308_atSH3 and multiple ankyrin repeat domains 2SHANK23GGTCAnnnTCAGC
214476_attrefoil factor 2TFF23AGTCAnnnTGGCC
213001_atangiopoietin-like 2ANGPTL21GATGAnnnTGAGG
217165_x_atmetallothionein 2AMT2A2TTTCAnnnTGAAA
203585_atzinc finger protein 185ZNF1854GGTCAnnnTGACT
210740_s_atinositol 1,3,4-triphosphate 5/6 kinaseITPK15GGTCAnnnTGGCC
206461_x_atmetallothionein 1HMT1H2GTTCAnnnTGGCA
205068_s_atRho GTPase activating protein 26ARHGAP261GCTCAnnnTGGGC
212057_atKIAA0182 proteinKIAA0182no sequence information available
202458_atserine protease 23SPUVE1AATCAnnnTGTTC
201858_s_atproteoglycan 1PRG16AGTCAnnnTGAGC
219369_s_atOUT domain, ubiquitin aldehyde binding 2OTUB24AGTCAnnnTGCCT
211474_s_atserine protease inhibitor, clade B, member 6SERPINB62AGTGAnnnTGAGC
213909_atleucine rich repeat containing 15LRRC15no sequence information available
202756_s_atglypican 1GPC11GGTCAnnnTGAGG
219045_atras homolog gene family, member 5RHOF1TGACAnnnTGAGC
205148_s_atchloride channel 4CLCN42AGACAnnnTGAGA
202976_s_atRho-related BTB domain containing 3RHOBTB32GGTCAnnnTCACT
201349_atsolute carrier family 9, isoform 3 regulator 1SLC9A3R11GCCCAnnnTGAGG
221667_s_atheat shock protein 8HSPB82GGACAnnnTGAGA
215189_atkeratin 6KRTHB63GGCCAnnnTGACC
210827_s_atE74-like factor 3ELF36GACCAnnnTGAGC
212135_s_atATPase, Ca++ transportingATP2B43ACTCAnnnTGTCT
210609_s_attumor protein p53 inducible protein 3TP53I36TGTCAnnnTGAGA
217190_x_atestrogen receptor alphaESR12AGTCAnnnTGAGA
215552_s_atestrogen receptor alphaESR12AGTCAnnnTGAGA
211233_x_atestrogen receptor alphaESR12AGTCAnnnTGAGA
211234_x_atestrogen receptor alphaESR12AGTCAnnnTGAGA
211235_s_atestrogen receptor alphaESR12AGTCAnnnTGAGA
218399_s_atcell division cycle associated 4CDCA45AATCAnnnTGACC
202870_s_atcell division cycle 20 homologCDC203GTTCAnnnTGATT
211519_s_atkinesin family member 2CKIF2C6GTACAnnnTGACC
201897_s_atCDC28 protein kinase subunit 1BCKS1B1TGTCAnnnTGCCA
210983_s_atminichromosome maintenance deficient 7MCM76AGGCAnnnTGACT
202503_s_atKIAA0101 gene productKIAA01013GGTCAnnnTGGTC
204420_atFOS-like antigen 1FOSL14GATCAnnnTGCCT
208002_s_atacyl-CoA hydrolaseBACH4TTTCAnnnTGAGC
202095_s_atsurvivinBIRC55GGACAnnnTGATT
204521_atpredicted protein clone 23733HSU797243GCCCAnnnTGACC
220155_s_atbromodomain containing 9BRD92GAGCAnnnTGACA
208994_s_atpeptidyl-prolyl isomerase GPPIG4GACCAnnnTGACC
210006_atDKFZP564O243 proteinDKFZP564O2434CGTCAnnnTGCCT
Table 4

Transcriptional responses associated with cell proliferation and survival in E2 stimulated MDA-MB-231 cells that re-express ERα

Gene nameBiological pathway/processRatio of gene expression E2:AO (mean±s.d.)
Gene ontology classifications for DNA replication, cell cycle, cell proliferation and cell survival were used to interrogate 574 genes exhibiting a significant (students t-test P<0.05) alteration in their expression due to estrogen stimulation. Mean gene expression ratios ± s.d. (E2-treated relative to vehicle control; based on 3 independent biological replicate experiments) are shown together with Affymetrix GeneChip probe set, gene name, biological pathway/process (as of the NetAffx update on 23 June 2004). Two or more Affymetrix GeneChip probe sets for the same gene indicates that probe sets specific for independent regions of the same gene demonstrated estrogen-regulated expression.
Affymetrix probe set
205899_atcyclin A1CCNA1cell cycle6.0±3.0
201621_atneuroblastoma suppression of tumorigenicity 1NBL1cell cycle5.1±2.8
219534_x_atcyclin-dependent kinase inhibitor 1C (p57, Kip2)CDKN1Ccell cycle3.9±3.1
217744_s_atTP53 apoptosis effectorPERPapoptosis2.9±1.2
213348_atcyclin-dependent kinase inhibitor 1C (p57, Kip2)CDKN1Ccell cycle2.3±1.0
210538_s_atbaculoviral IAP repeat-containing 3BIRC3apoptosis3.1±1.9
209304_x_atgrowth arrest and DNA damage-inducible, betaGADD45Bcell cycle/apoptosis4.1±1.5
201482_atquiescin Q6QSCN6cell cycle/cell proliferation2.2±0.9
222036_s_atminichromosome maintenance deficient 4MCM4DNA replication0.7±0.4
210559_s_atcell division cycle 2 (CDK1)CDC2cell cycle0.5±0.08
204092_s_atserine/threonine kinase 6STK6cell cycle0.7±0.2
213677_s_atpostmeiotic segregation increased 1PMS1cell cycle0.8±0.02
209642_atbudding inhibited by benzimidazoles 1 homologBUB1cell cycle0.6±0.07
205394_atCHK1 checkpoint homologCHEK1cell cycle/cell proliferation0.7±0.07
204768_s_atflap structure-specific endonuclease 1FEN1DNA replication0.8±0.1
202107_s_atminichromosome maintenance deficient 2MCM2DNA replication/cell cycle0.7±0.05
218399_s_atcell division cycle associated 4CDCA4cell cycle0.6±0.09
218355_atkinesin family member 4AKIF4Acell cycle0.5±0.09
202705_atcyclin B2CCNB2cell cycle0.6±0.05
211519_s_atkinesin family member 2CKIF2Ccell cycle0.5±0.1
203213_atcell division cycle 2 (CDK1)CDC2cell cycle0.5±0.2
202870_s_atcell division cycle 20 homologCDC20cell cycle0.5±0.1
218009_s_atprotein regulator of cytokinesisPRC1cell cycle0.5±0.1
202095_s_atbaculoviral IAP repeat-containing 5 (survivin)BIRC5cell cycle/apoptosis0.5±0.06
201112_s_atchromosome segregation 1-likeCSE1Lcell proliferation/apoptosis0.7±0.1
214710_s_atcyclin B1CCNB1cell cycle0.5±0.07
201897_s_atCDC28 protein kinase regulatory subunit 1BCKS1Bcell proliferation0.6±0.1
209773_s_atribonucleotide reductase M2 polypeptideRRM2DNA replication0.5±0.07
217786_atSKB1 homologSKB1cell cycle/cell proliferation0.7±0.1
201291_s_atDNA topoisomerase II alphaTOP2ADNA replication0.5±0.2
212563_atblock of proliferation 1BOP1cell proliferation0.6±0.03
210983_s_atminichromosome maintenance deficient 7MCM7DNA replication/cell cycle0.5±0.1
208795_s_atminichromosome maintenance deficient 7MCM7DNA replication/cell cycle0.5±0.1
208796_s_atcyclin G1CCNG1cell cycle0.7±0.2
200920_s_atB-cell translocaiton gene 1BTG1cell cycle/cell proliferation/apoptosis0.7±0.09
204240_s_atstructural maintenance of chromosomes 2-like 1SMC2L1cell cycle0.6±0.05
210766_s_atchromosome segregation 1-likeCSE1Lcell proliferation/apoptosis0.9±0.3
201555_atminichromosome maintenance deficient 3MCM3DNA replication/cell cycle0.8±0.2
219350_s_atdiable homologDIABLOapoptosis0.7±0.06
211036_x_atanaphase promoting complex subnit 5ANAPC5cell cycle0.8±0.08
201457_x_atbudding inhibited by benzimidazoles 3 homologBUB3cell cycle0.8±0.07
208021_s_atreplication factor C 1RFC1DNA replication0.6±0.04
Table 5

Opposing transcriptional responses to E2 in ER-positive MCF-7 cells versus MDA-MB-231 cells that re-express ERα

Response to E2 in MDA-MB-231 cells that re-express ERαResponse to E2 in ER-positive MCF-7 cells
1Quantitative gene expression data are shown in Table 4.
Gene name
cell division cycle 20 homologCDC20down-regulated1up-regulated (Lobenhofer et al. 2002Frasor et al. 2003)
cell division cycle 2 (CDK1)CDC2down-regulated1up-regulated (Frasor et al. 2003)
CDC28 protein kinase regulatory subunit 1BCKS1Bdown-regulated1up-regulation of CDC28 protein kinase 2 (Lobenhofer et al. 2002)
budding inhibited by benzimidazoles 1 homologBUB1down-regulated1up-regulated (Frasor et al. 2003)
minichromosome maintenance deficient 2MCM2down-regulated1up-regulated (Frasor et al. 2003)
minichromosome maintenance deficient 3MCM3down-regulated1up-regulated (Lobenhofer et al. 2002; Frasor et al. 2003)
minichromosome maintenance deficient 7MCM7down-regulated1up-regulated (Lobenhofer et al. 2002)
flap structure-specific endonuclease 1FEN1down-regulated1up-regulated (Lobenhofer et al. 2002)
replication factor C 1RFC1down-regulated1up-regulation of RFC3 (Lobenhofer et al. 2002) and RFC4 (Frasor et al. 2003)
chromosome segregation 1-likeCSE1Ldown-regulated1up-regulated (Lobenhofer et al. 2002)
serine/threonine kinase 6STK6down-regulated1up-regulated (Frasor et al. 2003)
baculoviral IAP repeat-containing 5 (survivin)BIRC5down-regulated1up-regulated (Frasor et al. 2003)
E-cadherinCDH1up-regulated1down-regulated (Osterreich et al. 2003)
Figure 1
Figure 1

Reintroduction of ERα into ER-negative MDA-MB-231 breast cancer cells. (A) Recombinant adenovirus engineered to co-express both GFP and human ERα (Ad-ERα) was used to infect MDA-MB-231 cells. Recombinant adenovirus containing the E. coli LACZ gene in place of ERα (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-ERα. The efficiency of viral infection was determined by measuring the proportion of cells that exhibit GFP fluorescence (right panel). (C) Northern blot analysis of ERα expression 24 h after Ad-LacZ (lane 1) or Ad-ERα (lane 2) infection of MDA-MB-231 cells.

Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01677

Figure 2
Figure 2

MDA-MB-231 cells infected with adenovirus encoding ERα contain transcriptionally active ERs. (A) Cells were infected with adenovirus encoding β-galactosidase (Ad-LacZ control) or ERα (Ad-ERα) 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-ERα 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 β-actin gene was used as a control.

Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01677

Figure 3
Figure 3

E2 inhibits proliferation in MDA-MB-231 breast cancer cells that re-express ERα after transfection with Ad-ERα. MDA-MB-231 cells were infected with either Ad-LacZ or Ad-ERα. 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.

Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01677

Figure 4
Figure 4

Microarray analysis of estrogen-responsive genes in MDA-MD-231 breast cancer cells transfected with Ad-LacZ or Ad-ERα. 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-ERα 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 2.

Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01677

Figure 5
Figure 5

E2-responsive cell cycle genes in MDA-MB-231 cells that re-express ERα. 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.

Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01677

Figure 6
Figure 6

Quantitative real-time PCR analysis of opposing transcriptional responses in MCF-7 versus MDA-MD-231 breast cancer cells that re-express ERα. MDA-MB-231 cells were transfected with either Ad-LacZ or Ad-ERα 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. ΔΔCt 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.

Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01677

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 ERα 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.

References

  • Bard JB & Rhee SY 2004 Ontologies in biology: design, applications and future challenges. Nature Reviews Genetics 5 213–222.

  • Benjamini Y & Hochberg Y 1995 Controlling the False Discovery Rate: a Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society: Series B 57 289–300.

    • Search Google Scholar
    • Export Citation
  • Berry M, Nunez AM & Chambon P 1989 Estrogen-responsive element of the human pS2 gene is an imperfectly palindromic sequence. PNAS 86 1218–1222.

    • Search Google Scholar
    • Export Citation
  • Blow JJ & Hodgson B 2002 Replication licensing – defining the proliferative state? Trends in Cell Biology 12 72–78.

  • Bourdeau V, Deschenes J, Metivier R, Nagai Y, Nguyen D, Bretschneider N, Gannon F, White JH & Mader S 2004 Genome-wide identification of high-affinity estrogen response elements in human and mouse. Molecular Endocrinology 18 1411–1427.

    • Search Google Scholar
    • Export Citation
  • Coppock DL, Cina-Poppe D & Gilleran S 1998 The quiescin Q6 gene (QSCN6) is a fusion of two ancient gene families: thioredoxin and ERV1. Genomics 54 460–468.

    • Search Google Scholar
    • Export Citation
  • van der Burg B, van Selm-Miltenburg AJ, de Laat SW & van Zoelen EJ 1989 Direct effects of estrogen on c-fos and c-myc protooncogene expression and cellular proliferation in human breast cancer cells. Molecular and Cellular Endocrinology 64 223–228.

    • Search Google Scholar
    • Export Citation
  • Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC & Conklin BR 2002 GenMAPP, a new tool for viewing and analyzing microarray data on biological pathways. Nature Genetics 31 19–20.

    • Search Google Scholar
    • Export Citation
  • Davidson NE, Bronzert DA, Chambon P, Gelmann EP & Lippman ME 1986 Use of two MCF-7 cell variants to evaluate the growth regulatory potential of estrogen-induced products. Cancer Research 46 1904–1908.

    • Search Google Scholar
    • Export Citation
  • Ferguson AT, Lapidus RG, Baylin SB & Davidson NE 1995 Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression. Cancer Research 55 2279–2283

    • Search Google Scholar
    • Export Citation
  • Frasor J, Danes JM, Komm B, Chang KC, Lyttle CR & Katzenellenbogen BS 2003 Profiling of estrogen up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology 144 4562–4574.

    • Search Google Scholar
    • Export Citation
  • Fujita N, Jaye DL, Kajita M, Geigerman C, Moreno CS & Wade PA 2003 MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell 113 207–219.

    • Search Google Scholar
    • Export Citation
  • Garcia M, Derocq D, Freiss G & Rochefort H 1992 Activation of estrogen receptor transfected into a receptor-negative breast cancer cell line decreases the metastatic and invasive potential of the cells. PNAS 89 11538–11542.

    • Search Google Scholar
    • Export Citation
  • GEO 2004 Gene Expression Omnibus Homepage, Bethesda, MD: National Center for Biotechnology Information, National Library of Medicine, Available: http://www.ncbi.nlm.nih.gov/geo/.

  • Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P & Chambon P 1986 Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320 134–139.

    • Search Google Scholar
    • Export Citation
  • Hall JM, Couse JF & Korach KS 2001 The multifaceted mechanisms of estradiol and estrogen receptor signaling. Journal of Biological Chemistry 276 36869–36872.

    • Search Google Scholar
    • Export Citation
  • He TC, Zhou S, da Costa LT, Yu J, Kinzler KW & Vogelstein B 1998 A simplified system for generating recombinant adenoviruses. PNAS 95 2509–2514.

    • Search Google Scholar
    • Export Citation
  • Klinge CM 2001 Estrogen receptor interaction with estrogen response elements. Nucleic Acids Research 9 2905–2919.

  • Kobayashi K, Hatano M, Otaki M, Ogasawara T & Tokuhisa T 1999 Expression of a murine homologue of the inhibitor of apoptosis protein is related to cell proliferation. PNAS 96 1457–1462.

    • Search Google Scholar
    • Export Citation
  • Laborda J 1991 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein P0. Nucleic Acids Research 19 3998.

    • Search Google Scholar
    • Export Citation
  • Lazennec G & Katzenellenbogen BS 1999 Expression of human estrogen receptor using an efficient adenoviral gene delivery system is able to restore hormone-dependent features to estrogen receptor-negative breast carcinoma cells. Molecular and Cellular Endocrinology 149 93–105.

    • Search Google Scholar
    • Export Citation
  • Levenson AS & Jordan C 1994 Transfection of Human Estrogen Receptor (ER) cDNA into ER-negative Mammalian Cell Lines. Journal of Steroid Biochemistry and Molecular Biology 51 229–239.

    • Search Google Scholar
    • Export Citation
  • Levenson AS, Svoboda KM, Pease KM, Kaiser SA, Chen B, Simons LA, Jovanovic BD, Dyck PA & Jordan VC 2002 Gene expression profiles with activation of the estrogen receptor alpha-selective estrogen receptor modulator complex in breast cancer cells expressing wild-type estrogen receptor. Cancer Research 62 4419–4426.

    • Search Google Scholar
    • Export Citation
  • Liu G, Loraine AE, Shigeta R, Cline M, Cheng J, Valmeekam V, Sun S, Kulp D & Siani-Rose MA 2003 NetAffx: Affymetrix probesets and annotations. Nucleic Acids Research 31 82–86

    • Search Google Scholar
    • Export Citation
  • Lobenhofer EK, Bennett L, Cable PL, Li L, Bushel PR & Afshari CA 2002 Regulation of DNA replication fork genes by 17 beta-estradiol. Molecular Endocrinology 16 1215–1229.

    • Search Google Scholar
    • Export Citation
  • McKenna NJ & O’Malley BW 2002 Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108 465–474

  • Menard S, Pupa SM, Campiglio M & Tagliabue E 2003 Biologic and therapeutic role of HER2 in cancer. Oncogene 22 6570–6578.

  • Moggs JG & Orphanides G 2001 Estrogen receptors: orchestrators of pleiotropic cellular responses. EMBO Reports 2 775–781.

  • Murphy TC & Orphanides G 2002 Characterisation of the molecular responses to xenoestrogens using gene expression profiling. Phytochemistry Reviews 1 199–208.

    • Search Google Scholar
    • Export Citation
  • Oesterreich S, Deng W, Jiang S, Cui X, Ivanova M, Schiff R, Kang K, Hadsell DL, Behrens J & Lee AV 2003 Estrogen-mediated Down-regulation of E-cadherin in Breast Cancer Cells. Cancer Research 63 5203–5208.

    • Search Google Scholar
    • Export Citation
  • Ottaviano YL, Issa JP, Parl FF, Smith HS, Baylin SB & Davidson NE 1994 Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells. Cancer Research 54 2552–2555.

    • Search Google Scholar
    • Export Citation
  • Ozaki T, Nakamura Y, Enomoto H, Hirose M & Sakiyama S 1995 Overexpression of DAN gene product in normal rat fibroblasts causes a retardation of the entry into the S phase. Cancer Research 55 895–900.

    • Search Google Scholar
    • Export Citation
  • Philips A, Teyssier C, Galtier F, Rivier-Covas C, Rey JM, Rochefort H & Chalbos D 1998 FRA-1 expression level modulates regulation of activator protein-1 activity by estradiol in breast cancer cells. Molecular Endocrinology 12 973–985.

    • Search Google Scholar
    • Export Citation
  • Pike MC, Spicer DV, Dahmoush L & Press MF 1993 Estrogens, progestogens, normal breast cell proliferation, and breast cancer risk. Epidemiologic Reviews 15 17–35.

    • Search Google Scholar
    • Export Citation
  • Prall OW, Sarcevic B, Musgrove EA, Watts CK & Sutherland RL 1997 Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2. Journal of Biological Chemistry 272 10882–10894.

    • Search Google Scholar
    • Export Citation
  • Reed SI 2003 Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover. Nature Reviews Molecular Cell Biology 4 855–864.

  • Rochefort H, Glondu M, Sahla ME, Platet N & Garcia M 2003 How to target estrogen receptor-negative breast cancer? Endocrine Related Cancer 10 261–266.

    • Search Google Scholar
    • Export Citation
  • Sambrook J, Fritsch EF & Maniatis T 1989 Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

  • Shang Y & Brown M 2002 Molecular determinants for the tissue specificity of SERMs. Science 295 2380–2381.

  • Tanaka T, Dancheck BL, Trifiletti LC, Birnkrant RE, Taylor BJ, Garfield SH, Thorgeirsson U & De Luca LM 2004 Altered localization of retinoid X receptor alpha coincides with loss of retinoid responsiveness in human breast cancer MDA-MB-231 cells. Molecular and Cellular Biology 24 3972–3982.

    • Search Google Scholar
    • Export Citation
  • Thiery JP 2002 Epithelial-mesenchymal transitions in tumour progression. Nature Reviews Cancer 2 442–454.

  • Tremblay GB & Giguere V 2002 Coregulators of estrogen receptor action. Critical Reviews in Eukaryotic Gene Expression 12 1–22

  • Vodermaier HC 2001 Cell cycle: Waiters serving the Destruction machinery. Current Biology 11 R834–R837.

  • Vogel VG & Lo S 2003 Preventing hormone-dependent breast cancer in high-risk women. Journal of the National Cancer Institute 95 91–93.

  • Weisz A, Cicatiello L, Persico E, Scalona M & Bresciani F 1990 Estrogen stimulates transcription of c-jun protooncogene. Molecular Endocrinology 4 1041–1050.

    • Search Google Scholar
    • Export Citation
  • Yang X, Ferguson AT, Nass SJ, Phillips DL, Butash KA, Wang SM, Herman JG & Davidson NE 2000 Transcriptional activation of estrogen receptor alpha in human breast cancer cells by histone deacetylase inhibition. Cancer Research 60 6890–6894.

    • Search Google Scholar
    • Export Citation
  • Yang X, Phillips DL, Ferguson AT, Nelson WG, Herman JG & Davidson NE 2001 Synergistic activation of functional estrogen receptor (ER)-alpha by DNA methyltransferase and histone deacetylase inhibition in human ER-alpha-negative breast cancer cells. Cancer Research 61 7025–7029.

    • Search Google Scholar
    • Export Citation

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  • View in gallery

    Reintroduction of ERα into ER-negative MDA-MB-231 breast cancer cells. (A) Recombinant adenovirus engineered to co-express both GFP and human ERα (Ad-ERα) was used to infect MDA-MB-231 cells. Recombinant adenovirus containing the E. coli LACZ gene in place of ERα (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-ERα. The efficiency of viral infection was determined by measuring the proportion of cells that exhibit GFP fluorescence (right panel). (C) Northern blot analysis of ERα expression 24 h after Ad-LacZ (lane 1) or Ad-ERα (lane 2) infection of MDA-MB-231 cells.

  • View in gallery

    MDA-MB-231 cells infected with adenovirus encoding ERα contain transcriptionally active ERs. (A) Cells were infected with adenovirus encoding β-galactosidase (Ad-LacZ control) or ERα (Ad-ERα) 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-ERα 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 β-actin gene was used as a control.

  • View in gallery

    E2 inhibits proliferation in MDA-MB-231 breast cancer cells that re-express ERα after transfection with Ad-ERα. MDA-MB-231 cells were infected with either Ad-LacZ or Ad-ERα. 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.

  • View in gallery

    Microarray analysis of estrogen-responsive genes in MDA-MD-231 breast cancer cells transfected with Ad-LacZ or Ad-ERα. 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-ERα 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 2.

  • View in gallery

    E2-responsive cell cycle genes in MDA-MB-231 cells that re-express ERα. 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.

  • View in gallery

    Quantitative real-time PCR analysis of opposing transcriptional responses in MCF-7 versus MDA-MD-231 breast cancer cells that re-express ERα. MDA-MB-231 cells were transfected with either Ad-LacZ or Ad-ERα 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. ΔΔCt 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.

  • Bard JB & Rhee SY 2004 Ontologies in biology: design, applications and future challenges. Nature Reviews Genetics 5 213–222.

  • Benjamini Y & Hochberg Y 1995 Controlling the False Discovery Rate: a Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society: Series B 57 289–300.

    • Search Google Scholar
    • Export Citation
  • Berry M, Nunez AM & Chambon P 1989 Estrogen-responsive element of the human pS2 gene is an imperfectly palindromic sequence. PNAS 86 1218–1222.

    • Search Google Scholar
    • Export Citation
  • Blow JJ & Hodgson B 2002 Replication licensing – defining the proliferative state? Trends in Cell Biology 12 72–78.

  • Bourdeau V, Deschenes J, Metivier R, Nagai Y, Nguyen D, Bretschneider N, Gannon F, White JH & Mader S 2004 Genome-wide identification of high-affinity estrogen response elements in human and mouse. Molecular Endocrinology 18 1411–1427.

    • Search Google Scholar
    • Export Citation
  • Coppock DL, Cina-Poppe D & Gilleran S 1998 The quiescin Q6 gene (QSCN6) is a fusion of two ancient gene families: thioredoxin and ERV1. Genomics 54 460–468.

    • Search Google Scholar
    • Export Citation
  • van der Burg B, van Selm-Miltenburg AJ, de Laat SW & van Zoelen EJ 1989 Direct effects of estrogen on c-fos and c-myc protooncogene expression and cellular proliferation in human breast cancer cells. Molecular and Cellular Endocrinology 64 223–228.

    • Search Google Scholar
    • Export Citation
  • Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC & Conklin BR 2002 GenMAPP, a new tool for viewing and analyzing microarray data on biological pathways. Nature Genetics 31 19–20.

    • Search Google Scholar
    • Export Citation
  • Davidson NE, Bronzert DA, Chambon P, Gelmann EP & Lippman ME 1986 Use of two MCF-7 cell variants to evaluate the growth regulatory potential of estrogen-induced products. Cancer Research 46 1904–1908.

    • Search Google Scholar
    • Export Citation
  • Ferguson AT, Lapidus RG, Baylin SB & Davidson NE 1995 Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression. Cancer Research 55 2279–2283

    • Search Google Scholar
    • Export Citation
  • Frasor J, Danes JM, Komm B, Chang KC, Lyttle CR & Katzenellenbogen BS 2003 Profiling of estrogen up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology 144 4562–4574.

    • Search Google Scholar
    • Export Citation
  • Fujita N, Jaye DL, Kajita M, Geigerman C, Moreno CS & Wade PA 2003 MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell 113 207–219.

    • Search Google Scholar
    • Export Citation
  • Garcia M, Derocq D, Freiss G & Rochefort H 1992 Activation of estrogen receptor transfected into a receptor-negative breast cancer cell line decreases the metastatic and invasive potential of the cells. PNAS 89 11538–11542.

    • Search Google Scholar
    • Export Citation
  • GEO 2004 Gene Expression Omnibus Homepage, Bethesda, MD: National Center for Biotechnology Information, National Library of Medicine, Available: http://www.ncbi.nlm.nih.gov/geo/.

  • Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P & Chambon P 1986 Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320 134–139.

    • Search Google Scholar
    • Export Citation
  • Hall JM, Couse JF & Korach KS 2001 The multifaceted mechanisms of estradiol and estrogen receptor signaling. Journal of Biological Chemistry 276 36869–36872.

    • Search Google Scholar
    • Export Citation
  • He TC, Zhou S, da Costa LT, Yu J, Kinzler KW & Vogelstein B 1998 A simplified system for generating recombinant adenoviruses. PNAS 95 2509–2514.

    • Search Google Scholar
    • Export Citation
  • Klinge CM 2001 Estrogen receptor interaction with estrogen response elements. Nucleic Acids Research 9 2905–2919.

  • Kobayashi K, Hatano M, Otaki M, Ogasawara T & Tokuhisa T 1999 Expression of a murine homologue of the inhibitor of apoptosis protein is related to cell proliferation. PNAS 96 1457–1462.

    • Search Google Scholar
    • Export Citation
  • Laborda J 1991 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein P0. Nucleic Acids Research 19 3998.

    • Search Google Scholar
    • Export Citation
  • Lazennec G & Katzenellenbogen BS 1999 Expression of human estrogen receptor using an efficient adenoviral gene delivery system is able to restore hormone-dependent features to estrogen receptor-negative breast carcinoma cells. Molecular and Cellular Endocrinology 149 93–105.

    • Search Google Scholar
    • Export Citation
  • Levenson AS & Jordan C 1994 Transfection of Human Estrogen Receptor (ER) cDNA into ER-negative Mammalian Cell Lines. Journal of Steroid Biochemistry and Molecular Biology 51 229–239.

    • Search Google Scholar
    • Export Citation
  • Levenson AS, Svoboda KM, Pease KM, Kaiser SA, Chen B, Simons LA, Jovanovic BD, Dyck PA & Jordan VC 2002 Gene expression profiles with activation of the estrogen receptor alpha-selective estrogen receptor modulator complex in breast cancer cells expressing wild-type estrogen receptor. Cancer Research 62 4419–4426.

    • Search Google Scholar
    • Export Citation
  • Liu G, Loraine AE, Shigeta R, Cline M, Cheng J, Valmeekam V, Sun S, Kulp D & Siani-Rose MA 2003 NetAffx: Affymetrix probesets and annotations. Nucleic Acids Research 31 82–86

    • Search Google Scholar
    • Export Citation
  • Lobenhofer EK, Bennett L, Cable PL, Li L, Bushel PR & Afshari CA 2002 Regulation of DNA replication fork genes by 17 beta-estradiol. Molecular Endocrinology 16 1215–1229.

    • Search Google Scholar
    • Export Citation
  • McKenna NJ & O’Malley BW 2002 Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108 465–474

  • Menard S, Pupa SM, Campiglio M & Tagliabue E 2003 Biologic and therapeutic role of HER2 in cancer. Oncogene 22 6570–6578.

  • Moggs JG & Orphanides G 2001 Estrogen receptors: orchestrators of pleiotropic cellular responses. EMBO Reports 2 775–781.

  • Murphy TC & Orphanides G 2002 Characterisation of the molecular responses to xenoestrogens using gene expression profiling. Phytochemistry Reviews 1 199–208.

    • Search Google Scholar
    • Export Citation
  • Oesterreich S, Deng W, Jiang S, Cui X, Ivanova M, Schiff R, Kang K, Hadsell DL, Behrens J & Lee AV 2003 Estrogen-mediated Down-regulation of E-cadherin in Breast Cancer Cells. Cancer Research 63 5203–5208.

    • Search Google Scholar
    • Export Citation
  • Ottaviano YL, Issa JP, Parl FF, Smith HS, Baylin SB & Davidson NE 1994 Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells. Cancer Research 54 2552–2555.

    • Search Google Scholar
    • Export Citation
  • Ozaki T, Nakamura Y, Enomoto H, Hirose M & Sakiyama S 1995 Overexpression of DAN gene product in normal rat fibroblasts causes a retardation of the entry into the S phase. Cancer Research 55 895–900.

    • Search Google Scholar
    • Export Citation
  • Philips A, Teyssier C, Galtier F, Rivier-Covas C, Rey JM, Rochefort H & Chalbos D 1998 FRA-1 expression level modulates regulation of activator protein-1 activity by estradiol in breast cancer cells. Molecular Endocrinology 12 973–985.

    • Search Google Scholar
    • Export Citation
  • Pike MC, Spicer DV, Dahmoush L & Press MF 1993 Estrogens, progestogens, normal breast cell proliferation, and breast cancer risk. Epidemiologic Reviews 15 17–35.

    • Search Google Scholar
    • Export Citation
  • Prall OW, Sarcevic B, Musgrove EA, Watts CK & Sutherland RL 1997 Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2. Journal of Biological Chemistry 272 10882–10894.

    • Search Google Scholar
    • Export Citation
  • Reed SI 2003 Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover. Nature Reviews Molecular Cell Biology 4 855–864.

  • Rochefort H, Glondu M, Sahla ME, Platet N & Garcia M 2003 How to target estrogen receptor-negative breast cancer? Endocrine Related Cancer 10 261–266.

    • Search Google Scholar
    • Export Citation
  • Sambrook J, Fritsch EF & Maniatis T 1989 Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

  • Shang Y & Brown M 2002 Molecular determinants for the tissue specificity of SERMs. Science 295 2380–2381.

  • Tanaka T, Dancheck BL, Trifiletti LC, Birnkrant RE, Taylor BJ, Garfield SH, Thorgeirsson U & De Luca LM 2004 Altered localization of retinoid X receptor alpha coincides with loss of retinoid responsiveness in human breast cancer MDA-MB-231 cells. Molecular and Cellular Biology 24 3972–3982.

    • Search Google Scholar
    • Export Citation
  • Thiery JP 2002 Epithelial-mesenchymal transitions in tumour progression. Nature Reviews Cancer 2 442–454.

  • Tremblay GB & Giguere V 2002 Coregulators of estrogen receptor action. Critical Reviews in Eukaryotic Gene Expression 12 1–22

  • Vodermaier HC 2001 Cell cycle: Waiters serving the Destruction machinery. Current Biology 11 R834–R837.

  • Vogel VG & Lo S 2003 Preventing hormone-dependent breast cancer in high-risk women. Journal of the National Cancer Institute 95 91–93.

  • Weisz A, Cicatiello L, Persico E, Scalona M & Bresciani F 1990 Estrogen stimulates transcription of c-jun protooncogene. Molecular Endocrinology 4 1041–1050.

    • Search Google Scholar
    • Export Citation
  • Yang X, Ferguson AT, Nass SJ, Phillips DL, Butash KA, Wang SM, Herman JG & Davidson NE 2000 Transcriptional activation of estrogen receptor alpha in human breast cancer cells by histone deacetylase inhibition. Cancer Research 60 6890–6894.

    • Search Google Scholar
    • Export Citation
  • Yang X, Phillips DL, Ferguson AT, Nelson WG, Herman JG & Davidson NE 2001 Synergistic activation of functional estrogen receptor (ER)-alpha by DNA methyltransferase and histone deacetylase inhibition in human ER-alpha-negative breast cancer cells. Cancer Research 61 7025–7029.

    • Search Google Scholar
    • Export Citation