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Carcinogenesis Advance Access originally published online on February 10, 2006
Carcinogenesis 2006 27(8):1567-1578; doi:10.1093/carcin/bgi339
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Convergent transcriptional profiles induced by endogenous estrogen and distinct xenoestrogens in breast cancer cells

Tonko Buterin, Caroline Koch and Hanspeter Naegeli*

Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse Winterthurerstrasse 260, 8057 Zürich, Switzerland

*To whom correspondence should be addressed. Tel: +41 1 635 87 63; Fax: +41 1 635 89 10; Email: naegelih{at}vetpharm.unizh.ch


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Estrogen receptors display high levels of promiscuity in accommodating a wide range of ligand structures, but the functional consequence of changing receptor conformations in complex with distinct agonists is highly controversial. To determine variations in the transactivation capacity induced by different estrogenic agonists, we assessed global transcriptional profiles elicited by natural or synthetic xenoestrogens in comparison with the endogenous hormone 17ß-estradiol. Human MCF7 and T47D carcinoma cells, representing the most frequently used model systems for tumorigenic responses in the mammary gland, were synchronized by hormone starvation during 48 h. Subsequently, a 24 h exposure was carried out with equipotent concentrations of the selected xenoestrogens or 17ß-estradiol. Analysis of messenger RNA was performed on high-density oligonucleotide microarrays that display the sequences of 33 000 human transcripts, yielding a total of 181 gene products that are regulated upon estrogenic stimulation. Surprisingly, genistein (a phytoestrogen), bisphenol-A and polychlorinated biphenyl congener 54 (two synthetic xenoestrogens) produced highly congruent genomic fingerprints by regulating the same range of human genes. Also, the monotonous genomic signature observed in response to xenoestrogens is identical to the transcriptional effects induced by physiological concentrations of 17ß-estradiol. This striking functional convergence indicates that the transcription machinery is largely insensitive to the particular structure of estrogen receptor agonists. The occurrence of such converging transcriptional programs reinforces the hypothesis that multiple xenoestrogenic contaminants, of natural or anthropogenic origin, may act in conjunction with the endogenous hormone to induce additive effects in target tissues.

Abbreviations: BrdU, bromodeoxyuridine; DMSO, dimethyl sulfoxide; ER, estrogen receptor; ER{alpha}, estrogen receptor alpha; ERß, estrogen receptor beta; FBS, fetal bovine serum; PCB54, polychlorinated biphenyl congener 54; TCDD, 2,3,7,8-tetrachloro-dibenzo-p-dioxin


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Estrogen signaling is primarily mediated by two members of the nuclear steroid receptor superfamily, i.e. estrogen receptor alpha (ER{alpha}) and beta (ERß). These receptors constitute ligand-stimulated transcription factors that associate with co-regulatory partners to remodel chromatin structure and recruit the general transcription machinery to downstream target genes (1–4). Additional estrogenic responses have been discerned with the discovery of novel ER pools that interact with membrane tyrosine kinases or other components of signal transduction pathways (5–7), which in turn lead to further gene expression changes through activation of transcription factors such as ELK1 or CREB (8–10). Thus, the vast majority of biological responses to estrogenic stimuli culminate in genome-wide transcriptional regulation, even though some of these effects are considered indirect or non-genomic.

Epidemiological studies have linked an increased risk of developing mammary or endometrial malignancies to prolonged estrogen exposure caused by early menarche, late menopause, late first-term pregnancy, oral contraceptives or an estrogen replacement therapy (4,11). There is also widespread concern that chemicals with estrogenic activity, for example bisphenol-A or organochlorine pollutants, may be associated with adverse health effects including cancer or other disorders of the female or male reproductive tract (9,12–16). On the other hand, phytoestrogens have been proposed to confer health benefits because the high dietary intake of plant-derived estrogens, such as genistein, appears to correlate with a lower incidence of breast and prostate cancer (17,18). Why prolonged exposure to synthetic estrogens should increase breast cancer risk, whereas natural phytoestrogens exert an opposite chemopreventive action, is not understood.

In the normal resting gland, ERs are expressed in only a small proportion of epithelial cells that are largely non-dividing (11). In contrast, enhanced expression of ERs is a critical event during breast cancer development and, accordingly, the growth of malignant tissue is estrogen-regulated in most cases (19,20). To analyze in detail the effects of ER agonists on gene expression, many laboratories have stimulated human breast cancer cells with 17ß-estradiol and determined global transcriptional profiles using oligonucleotide-based microarrays. With few exceptions (21,22) these previous studies were performed at high 17ß-estradiol concentrations of 1 nM (23) or even 10 nM (24–31), thereby exceeding the peak effect level, observed around a concentration of 0.1 nM (22), by one or more orders of magnitude. Previous reports also described transcriptional patterns induced by phytoestrogens, including genistein, at concentrations of 10 µM (29) or 100 µM (27,28), again exceeding by far the saturation level, which for genistein is reached at a concentration of ~1 µM (32). However, molecular insight into the effects of xenoestrogens in a lower subsaturating concentration would be important to assess health hazards or benefits at dose ranges that are more relevant for human exposure. Particular attention has been given to the question of whether the native hormone 17ß-estradiol and exogenous estrogenic agents induce similar or different transactivation functions. This key issue has been analyzed in previous studies with highly conflicting results. For example, a gene expression survey performed on the immature mouse uterus after 3 day exposures to 17ß-estradiol, genistein or diethylstilbestrol (a synthetic estrogen derivative), yielded comparable transcriptional responses (33). Other reports concluded that there are significant differences between the transcriptional effects of 17ß-estradiol and xenoestrogens in the uteri of immature mice (34) or in the reproductive tract of adult rats (35). Several studies came to the conclusion that there is only limited overlap between the expression patterns elicited by 17ß-estradiol and xenoestrogens in human breast cancer cells (27–29,31). These contrasting results led us to undertake a large-scale comparative analysis of early gene expression changes in human MCF7 and T47D breast cancer cells treated with equipotent concentrations of 17ß-estradiol, genistein, bisphenol-A and the polychlorinated biphenyl congener 54 (PCB54). This integrative study revealed that the expression fingerprint induced by many non-physiological estrogens coincides with the known response of these two carcinoma cell lines to the endogenous hormone.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals
Genistein, bisphenol-A, 4-hydroxytamoxifen and 17ß-estradiol were purchased from Fluka (Switzerland). PCB54, PCB126 and PCB153 were obtained from EGT (Switzerland); 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was from the NCI Chemical Carcinogen Reference Standard Repository. The inhibitor ICI 182,780 was purchased from TOCRIS Bioscience (Avonmouth, UK).

Cell culture and treatments
Human T47D.Luc cells (BioDetection Systems, The Netherlands) were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium supplemented with sodium bicarbonate, 1 mM L-glutamine and 7.5% fetal bovine serum (FBS; HyClone Laboratories, USA). The MCF7 cell line subtype BUS (provided by A. M. Soto and C. Sonnenschein, Tufts University, Boston, USA) was grown in DMEM supplemented with 10% FBS. The antibiotics used were 0.1 U/ml penicillin and 0.1 µg/ml streptomycin (Invitrogen). Both cell lines were cultured at 37°C in xenoestrogen-free plastic (Corning, Grand Island, USA) under humidified air containing 5% CO2. Before each experiment, T47D.Luc and MCF7 cells were transferred to phenol red-free medium and cultured for 48 h in the presence of 5% charcoal/dextran-stripped FBS (DCC-FBS). Test compounds were dissolved in dimethyl sulfoxide (DMSO) and added to the culture medium as indicated. The final DMSO concentration was adjusted to 0.1% (v/v).

ER-CALUX assay
The ER-CALUX (estrogen receptor-mediated chemical-activated luciferase expression) assay was carried out following the standard operating procedure provided by BioDetection Systems. Briefly, T47D.Luc cells were seeded in microtiter plates at a density of 5000 cells per well in 0.1 ml phenol red-free medium containing 5% DCC-FBS. After 24 h, the medium was renewed and the cells were incubated for another 24 h followed by the addition of each test compound dissolved in DMSO. Solvent controls and a standard 17ß-estradiol dose–response curve were included on each plate. After the indicated exposure times, cells were harvested, lysed and assayed for luciferase activity (32) on a Dynex microplate luminometer. All values were corrected for background luciferase expression detected in the presence of solvent alone.

DNA synthesis assay
T47D or MCF7 cells were seeded in microtiter plates, at a density of 5000 cells/well, in 0.1 ml phenol red-free medium containing 5% DCC-FBS. After 24 h, the medium was renewed and the cells were incubated for another 24 h followed by the addition of each test compound dissolved in DMSO. Solvent controls were included on each plate. DNA synthesis was measured after 24 h exposures using the Biotrak cell proliferation ELISA system (Amersham Biosciences, Piscataway, NJ). For that purpose, bromodeoxyuridine (BrdU) was added to the culture medium for 2 h and deoxyribonucleotide incorporation was quantified by the addition of peroxidase-labeled anti-BrdU antibodies. The resultant color development, proportional to DNA synthesis, was determined in a LS55 Luminescence Spectrometer (Perkin Elmer, Wellesly, MA) at 450 nm wavelength.

Microarray hybridization, data acquisition and analysis
After a 24 h treatment with the test compounds, cells were collected by trypsinization and total RNA was extracted using the Rneasy kit (Qiagen, Hilden, Germany). Amount, purity and quality of the final RNA fractions were evaluated by UV spectrophotometry (260 and 280 nm wavelength) followed by examination of the probes by capillary electrophoresis on Agilent Bioanalyzers. Double-stranded complementary DNA was synthesized with the SuperScript kit from Invitrogen using a poly(dT)24 primer from Microsynth (Switzerland), which has a T7 RNA polymerase promoter at the 5' end. The synthesis of complementary RNA was performed with the Ambion MEGAScript T7 in vitro transcription kit in the presence of biotinylated CTP and UTP (Logo GmbH, Germany). The resulting biotin-labeled RNA was purified by the Rneasy kit, fragmented by hydrolysis and hybridized to human U-133 GeneChip DNA microarrays (Affymetrix) following the manufacturer's instructions. After hybridization (16 h), the microarrays were processed by an automated washing procedure on the Affymetrix Fluidics Station 400. Staining of the hybridized probes was performed with fluorescent streptavidin–phycoerythrin conjugates (1 mg/ml; Molecular Probes). The subsequent scanning of DNA microarrays was carried out on an Agilent GeneArray laser instrument. Data normalization and filtering were carried out by the dChip software version 1.3 (www.dchip.org). Finally, the results of triplicate experiments were imported into a Microsoft Excel file for SEM calculations, graphical representation and determination of correlation coefficients. The Gene Ontology database (www.geneontology.org) was consulted to verify the predominant molecular function of each transcript.

Real-time RT–PCR
PCR quantifications were carried out to validate the microarray hybridization results. Primers for the selected transcripts were obtained from Applied Biosystems. Briefly, 100 ng of complementary DNA were mixed with 100 nM of forward and reverse primers in a final volume of 25 µl. The reactions were performed in an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) for 45 cycles (95°C for 15 s, 60°C for 1 min) after an initial 10 min incubation at 95°C. The fold change in the expression of each gene was calculated using the 2–{Delta}{Delta}CT method (36), with the abundant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript as an internal standard.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Dose-dependent transactivation from a minimal estrogen-responsive promoter
The dose range of xenoestrogens to be tested in the DNA microarray experiments has been assessed using a standard reporter gene assay. We took advantage of stably transfected T47D carcinoma cells that carry a chromosomally integrated luciferase gene. This synthetic reporter construct is under transcriptional control of a minimal promoter consisting of tandem repeats of palindromic estrogen response elements (5'-GGTCACTGTGACC-3'). To monitor estrogenic actions, cell lysates were examined for luciferase activity after a 24 h treatment with different concentrations of each test compound. In agreement with previous studies (32), the synthetic promoter in T47D cells mediated a detectable reporter gene induction in response to as low as 1 pM 17ß-estradiol added to the cell culture medium. This estrogenic effect reached maximal levels at a hormone concentration of ~60 pM, yielding a nearly 100-fold induction relative to the solvent control (Figure 1A). Both genistein and bisphenol-A induced higher peak values of luciferase expression than the endogenous hormone. Instead, exposure to the estrogen-like PCB congener 54 resulted in lower levels of reporter gene induction compared with 17ß-estradiol (Figure 1A). On the basis of these dose responses, concentrations in the near saturating range were selected for the subsequent transcriptome analyzes, i.e. 30 pM for 17ß-estradiol, 1 µM for genistein as well as PCB54 and 10 µM for bisphenol-A. The induction of luciferase expression by all estrogenic chemicals was suppressed in the presence of the antagonists 4-hydroxytamoxifen or ICI 182,780 at a concentration of 0.1 µM (data not shown). The selectivity of this reporter assay is further demonstrated by the lack of luciferase induction following treatment with the dioxin-like PCB congener 126 (Figure 1A). Similarly, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD; data not shown), as well as the non-dioxin-like PCB congener 153 (data not shown), were completely unable to induce this estrogen-specific reporter system.


Figure 1
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  		Fig. 1 Luciferase reporter gene expression. Stably transfected T47D cells were incubated with the indicated concentrations of each compound. ER activation was determined by measuring the luciferase induction from a minimal promoter containing repeats of estrogen response elements. (A) Dose dependence (mean values of 5–6 independent experiments done at different times). (B) Time course (mean values of three independent experiments ± SD). Results are shown as the fold induction relative to the solvent control.

 
Time course of synthetic promoter activation
The progression of reporter gene induction during the exposure period was determined in time course experiments. For that purpose, stably transfected T47D cells carrying the estrogen-dependent reporter construct were treated with 17ß-estradiol (30 pM), genistein (1 µM), bisphenol-A (10 µM) or PCB54 (1 µM) and luciferase activity was measured in cell lysates after various time intervals (Figure 1B). In view of the steep increase of luciferase induction observed after 24 h estrogen exposures, this time point was used to compare in detail early transcriptional changes in response to ER stimuli. The 24 h period also corresponds to the time of maximal induction of most estrogen-regulated genes according to previous experiments with 17ß-estradiol (21), although transcripts that may be subjected to upregulation exclusively during a very early phase of the estrogenic response would be under-represented (27).

Induction of DNA synthesis
Breast cancer cells that are deprived of estrogens or other growth factors accumulate early in the G1 stage of the division cycle. Treatment of estrogen-dependent cells with 17ß-estradiol triggers cell cycle progression such that, 24 h after addition of the hormone, the majority of cells undergo DNA replication (21,37). To test whether all xenoestrogens, at the selected concentrations, elicit the same replicative response as the endogenous hormone, growth-arrested MCF7 or T47D cells were exposed to 17ß-estradiol (30 pM), genistein (1 µM), bisphenol-A (10 µM) or PCB54 (1 µM) for 24 h. Entry into S phase was recorded by measuring DNA synthesis through the addition of BrdU to the culture medium. Specific antibodies directed against BrdU were employed to monitor incorporation of the deoxyribonucleoside analog following another 2 h of incubation. This quantitative assessment confirmed that all treatments induce comparable levels of DNA synthesis in both cell lines (Figure 2). Even PCB54 resulted in a similar rate of DNA polymerization as the other ER agonists despite the fact that this compound was much less efficient than 17ß-estradiol in the induction of luciferase expression from the synthetic promoter (Figure 1). In contrast to PCB54, the dioxin-like PCB congener 126 (1 µM; Figure 2), TCDD (data not shown) and the non-dioxin-like PCB congener 153 (data not shown) were unable to stimulate DNA synthesis in human breast cancer cells.


Figure 2
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  		Fig. 2 Stimulation of DNA synthesis. MCF7 or T47D cells were incubated with near saturating concentrations of each test compound for 24 h. DNA synthesis rates were measured by monitoring the incorporation of BrdU (mean values of three independent determinations ± SD).

 
Global expression profiles
MCF-7 or T47D cells were treated in triplicate experiments with equipotent concentrations of the estrogenic test compounds as outlined in the previous section. After 24 h of exposure, a fraction of RNA from each sample was analyzed using Affymetrix microarrays that display the sequences of 33 000 human transcripts. A total of ~35% of the surveyed gene products were called to be present at detectable levels by the Microarray Suite version 5.0 software. To identify transcripts that are susceptible to ER regulation, these results were normalized and subjected to statistical evaluation using the DNA-Chip Analyzer (dChip) open-source software (37). In a first step, all hybridization data were filtered using, as cut-offs, a fold change of >2.5 and a statistical significance of P < 0.05 (ANOVA) in at least one of the treatment groups. The number of genes whose transcription was regulated by estrogenic chemicals was considerably higher in MCF7 than in T47D cells. To facilitate direct comparisons between the two cell lines, a more stringent cut-off with a fold change of 3.0 was then applied to the positively regulated transcripts in MCF7 cells. Overall, this dChip analysis yielded a total of 134 transcripts in MCF7 cells, but only 76 transcripts in T47D cells, that are susceptible to estrogenic stimuli. The expression pattern in MCF7 and T47D cell lines is partially overlapping, resulting in a total of 181 ER-regulated human transcripts.

Concordance with real-time PCR values
Real-time RT–PCR was carried out on 14 sequences to validate the microarray hybridization results. The following transcripts were subjected to PCR analysis after exposure to bisphenol-A (10 µM): AREG (coding for amphiregulin), CCNG2 (cyclin G2), CDC2 (cell division cycle 2), CTSD (cathepsin D), CYFIP2 (cytoplasmic binding partner of fragile X protein), E2F1 (E2F transcription factor 1), IER3 (immediate early response 3), MYC (myelocytomatosis oncogene), OLFM1 (olfactomedin 1), RRM2 (ribonucleotide reductase M2 polypeptide), SDF1 (stromal cell-derived factor 1, also known as chemokine ligand 12), SNK (serum-inducible kinase), TFF1 (trefoil factor 1, also known as pS2) and KIAA0101 (predicted protein with unknown function). After normalization using the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcript, expression values were transformed as the ratio of messenger levels between estrogen-treated and solvent-treated cells. A direct comparison of the microarray hybridization data with the respective RT–PCR values showed a high degree of correlation for transcripts that were significantly up- or downregulated by estrogenic stimuli (Figure 3). In some cases, the hybridization data tend to underestimate the fold changes induced by ER activation. For example, the most abundant transcript induced by xenoestrogens codes for TFF1, which is a primary marker of ER-positive breast tumors (38,39). According to the microarray hybridizations, TFF1 was 6.3-fold induced in MCF7 cells, but the subsequent analysis by RT–PCR yielded a 13.5-fold increase for the same transcript. In T47D cells, the transcription level of SDF1, another prominent marker of ER signaling (40), was 10.5-fold induced according to the microarray hybridizations but the subsequent PCR quantification revealed a 17.1-fold increment. No induction of SDF1 occurred in MCF7 cells in agreement with the report of Coser et al. (22), who showed that this transcript is increased in MCF7 cells only at highly saturating concentrations of the estrogenic stimulus. The upregulation of KIAA0101, which is pronounced in MCF7 cells although it can be detected in both cell lines (Figure 3), has not been reported before and hence represents a novel estrogenic response.


Figure 3
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  		Fig. 3 Comparison between hybridization results and the corresponding RT–PCR values. Fold changes of messenger levels according to normalized and filtered hybridization data (dChip) were plotted against the corresponding real-time RT–PCR quantifications. This analysis was performed with RNA extracted from MCF7 (A) and T47D cells (B) exposed to 10 µM bisphenol-A.

 
Estrogen-dependent transcripts in MCF7 and T47D cells
Most human transcripts that were significantly regulated by estrogenic treatment encode protein products with either a known or an inferred biological function. Accordingly, these transcripts were grouped in functional categories involving cell cycle, DNA metabolism and apoptosis (Tables I and II),growth stimulation, transcription and cell adhesion (Tables III and IV), as well as metabolism and transport systems (Tables V and VI). There were only few transcripts (included in Tables V and VI) that encode for predicted proteins with unknown function such as, for example, KIAA0101 or GREB1 (gene regulated by estrogen in breast cancer 1), which has been identified by virtue of its overexpression in estrogen-responsive breast carcinomas (41).


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  		Table I Estrogen-regulated transcripts in MCF7 cells

 

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  		Table II Estrogen-regulated transcripts in T47D cells

 

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  		Table III Estrogen-regulated transcripts in MCF7 cells

 

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  		Table IV Estrogen-regulated transcripts in T47D cells

 

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  		Table V Estrogen-regulated transcripts in MCF7 cells

 

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  		Table VI Estrogen-regulated transcripts in T47D cells

 
Tables 1 and 2 display the estrogen-responsive transcripts whose protein products are involved in cell cycle, DNA metabolism and apoptosis. The majority of these genes have been identified before as being susceptible to transcriptional regulation by ERs (21–31), including HCAP-G (chromosome condensation protein G), the cell division cycle factors CDC2, CDC6, CDC20 and CCNA2 (cyclin A2), RRM2 (ribonucleotide reductase M2 polypeptide), PRIM1 (primase 1), TK1 (thymidine kinase 1), PCNA (proliferating cell nuclear antigen), DTYMK (deoxythymidylate kinase), FEN1 (flap structure-specific endonuclease 1), H2AFX (histone 2A family X) and UNG (uracil-DNA glycosylase). We also found that several members of the minichromosome maintenance deficient family are upregulated by estrogen treatment, including MCM2, MCM6, MCM7 and MCM10, all of which have roles in promoting DNA replication. As noted in a previous study (26), the induction of transcripts that drive cell proliferation was accompanied in MCF7 cells by the upregulation of survivin (BIRC5), an inhibitor of apoptosis. This antiapoptotic response was accompanied in MCF7 cells by the induction of DAD1 (defender against apoptotic death 1). In addition to these known responses, an appreciable number of novel targets were identified, including RAD51, RAD51C (RAD51 homolog C) and RAD51AP1 (RAD51 interacting protein 1), which are subunits of the homologous recombination machinery. Another cluster of estrogen-induced factors comprises the centromeric proteins CENPA, CENPF, CENPJ and TACC3 (transforming acidic coiled–coil 3) (42). On the other hand, GADD45A (growth arrest and DNA damage inducible 45A), BTG1 (B cell translocating gene 1) and cyclin-dependent kinase inhibitors CDKN1, CDKN2C and CDKN3, known to inhibit cell cycle progression, were downregulated. In addition, we identified transcripts involved in chromosome segregation that have not been associated with estrogenic signaling in previous studies, as for example PRC1 (protein regulator of cytokinesis 1), RAB6KIFL (kinesin family member 20A), KNSL5 (kinesin family member 23), KIF4A (kinesin family member 4A), KIF14 (kinesin family member 14), KIF11 (kinesin family member 11) and KNSL7 (kinesin like 7).

Estrogen-responsive transcripts involved in growth stimulation, transcription and cell adhesion are listed in Tables III and IV. In agreement with former DNA microarray studies (21–28,31), the production of TFF1, TFF3 (trefoil factor 3), IGFBP4 (insulin-like growth factor binding protein 4), SDF1, STC2 (stanniocalcin 2), AREG, OLFM1 and OLFML3 (olfactomedin-like 3) was induced upon estrogen treatment. THBS1 (thrombospondin 1) is also upregulated in an estrogen-dependent manner in T47D cells. Concomitantly, the expression of inhibitors of cell growth such as TGFB2 (transforming growth factor beta 2) or EFNB2 (ephrin B2) is suppressed (Table III). GFRA1 (glial cell line-derived neurotrophic factor family receptor alpha 1) represents a growth factor receptor that is susceptible to positive estrogenic regulation in both cell lines, whereas PTGER3 (prostaglandin E receptor 3) was upregulated only in T47D cells (Table IV). The induction of signal transduction pathways is illustrated by an increased level of transcripts coding for ITPK1 (inositol 1,3,4-triphosphate 5/6 kinase). As noted previously (30), the estrogen-dependent reprogramming of breast cancer cells was further characterized by the downregulation of tight junction and adhesion molecules including CLDN4 (claudin 4), L1CAM (L1 cell adhesion molecule) and JUP (junction plakoglobin), implying that ER activation may predispose to anchorage loss. Cell adhesion could be further reduced by the suppression of TIMP3 (tissue inhibitor of metalloproteinase 3), leading to increased metalloproteinase activity. We observed that many transcription factors and proto-oncogenes were suppressed following estrogenic stimulation (Tables III and IV). In fact, ATF3 (activating transcription factor), ETS2 (erythoblastosis E26 oncogene homolog 2), KRAS (Kirsten rat sarcoma oncogene), MAXD4 (MAX dimerization protein 4), NCOA3 (nuclear receptor coactivator 3) and other related transcripts were downregulated in MCF7 cells. The SOX4 (SRY-box 4) transcript was suppressed in both MCF7 and T47D cells. The tumor suppressor DOC-1R (deleted in oral cancer-related 1) was suppressed only in T47D cells. The downregulation of several proto-oncogenes is compensated by the induction of MYBL2 (also known as B-Myb) in both cell types. Other prominent transcripts that were induced in the course of this estrogen-dependent program include the MYC (myelocytomatosis), MYB (myeloblastosis) and ECT2 (epithelial cell transforming 2) oncogenes, as well as TMPO (thymopoietin), HEC (highly expressed in cancer), LAP18 (stathmin 1) and MLF1 (myeloid leukemia factor), which are known markers of malignant cell proliferation (43).

Tables V and VI display the estrogen-responsive transcripts involved in metabolism and transport sytems. Interestingly, CYP1A1 (cytochrome P4501A1) is downregulated in MCF7 cells (Figure 9A) but strongly induced in T47D cells. CYP1B1 is another P450 enzyme induced in T47D cells. Also, several genes associated with carrier function were susceptible to estrogen-dependent regulation. Induction was observed for the solute carrier family members SLC6A14 and SLC39A8 while the transcript coding for SLC7A11 was repressed. Finally, different factors involved in protein ubiquitylation, including FBX05 (F-box only protein 5), UBCH10 and HSPC150 (coding for ubiquitin conjugating enzymes) were positively regulated after estrogen stimulation. An estrogen-dependent induction of the immunophilin FKBP4 (FK506 binding protein 4), a factor involved in protein folding, has already been described before (30). Another estrogen-inducible factor with a function in protein folding is the chaperone ATAD2 (ATPase family, AAA domain containing 2).

Convergence of estrogenic transcriptional profiles
The only criterion for the inclusion of transcripts in Tables I–VI was their significant up or downregulation in at least one treatment group, without any bias for overlaps with the response to the other tested estrogenic agents. Surprisingly, this extensive comparison performed with two distinct cell lines revealed a high degree of similarity between the expression profiles elicited by natural and synthetic estrogenic compounds. Transcripts that were regulated by a 17ß-estradiol stimulus turned out to be modulated in the same direction, and to a similar extent, by genistein, bisphenol-A and PCB54. Conversely, transcriptional induction or repression mediated by these xenoestrogens was accompanied by a corresponding change in the level of the same transcripts following exposure to the native hormone. This striking similarity between the responses to diverse estrogenic agents is illustrated for example by the RRM2 (ribonucleotide reductase M2 polypeptide) messenger, which encodes the rate-limiting enzyme for deoxyribonucleotide production during DNA synthesis (22). The RRM2 transcript was induced in MCF7 cells 13.5-fold after treatment with 17ß-estradiol and between 12.8- and 14.9-fold after treatment with the different xenoestrogens (Table I). The RRM2 transcript was also increased (between 3.1- and 4.6-fold) in T47D cells following all kinds of estrogenic stimuli (Table II). Another prominent example is the cyclin A2 transcript, whose induction level ranges from 2.5- to 7.4-fold in both MCF7 and T47D cells following treatment with 17ß-estradiol as well as the different xenoestrogens (Tables I and II). To delineate the degree of similarity in quantitative terms, the messenger RNA profiles induced by the tested xenoestrogens in MCF7 cells were plotted against the corresponding values obtained for 17ß-estradiol. In these comparisons, all 134 data points representing estrogen-dependent transcripts cumulated along the diagonal axis of the graphs (Figure 4) and the resulting correlation coefficients were in the range of R = 0.98–0.99. This quantitative analysis thus confirms that the transcriptional responses induced by the distinct estrogenic agents are nearly identical. When the levels of these estrogen-dependent transcripts were plotted against the amount of the same transcripts following treatment with TCDD (0.1 µM), the resulting correlation coefficient was reduced to R = 0.11, reflecting the distinct transactivation pattern elicited by this aromatic hydrocarbon receptor agonist.


Figure 4
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  		Fig. 4 Relationship between gene expression profiles resulting from exposure to different estrogenic agents. Transcriptional fingerprints after treatment with xenoestrogens were compared with the expression pattern induced by 17ß-estradiol in scatter blot graphs. The axes indicate log10 expression levels of transcripts in units of light intensity. The R-values were calculated for each relationship on the basis of the linear regression between each pair of data. (A) Comparison between genistein and 17ß-estradiol. (B) Comparison between bisphenol-A and 17ß-estradiol. (C) Comparison between PCB54 and 17ß-estradiol. (D) Comparison between 17ß-estradiol and TCDD (0.1 µM). Higher concentrations of TCDD (≥1 µM) exerted cytotoxic effects.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Estrogenic regulation plays an important role not only in the development of normal mammary glands but also in the initiation and progression of breast cancer, which has become the most common malignancy among American and European women (11). Epidemiological studies suggest a positive correlation between blood levels of chemicals with estrogenic activity, such as organochlorine pollutants, and breast cancer incidence among women, implying that the growing risk of contracting mammary cancer may be linked to the wide distribution of synthetic xenoestrogens (12,13,44–47). On the other hand, beneficial health effects have been attributed to the dietary intake of natural phytoestrogens in food of plant origin (17,18).

The two ER subtypes (ER{alpha} and ERß) are unique among the steroid receptor family in their ability to interact with a wide variety of ligands that exhibit remarkably diverse structural features (48). Several lines of evidence apparently support the view that the biological action of different ER agonists may diverge significantly. First, the endogenous hormone and various xenoestrogens display differences in the binding affinity for ER{alpha} and ERß (48). Second, ER{alpha} and ERß expression levels vary among different cells, indicating that the biological activity of estrogens is modulated by tissue-specific ER patterns (49). Third, ER{alpha} and ERß have been shown to exert, at least in part, antagonistic biological effects (50,51). Finally, a conformational change in the ER protein is required for activation or repression of responsive genes but it has been observed that 17ß-estradiol and genistein induce distinct changes in the receptor fold (52), prompting the hypothesis that different ER agonists may exert distinct transactivation functions. In apparent agreement with this expectation, numerous studies reported that the transcriptional patterns induced by genistein or bisphenol-A in human breast cancer cells are only in part similar to the characteristic expression fingerprint of 17ß-estradiol (27–29,31).

Previous comparative analyses have been performed with highly saturating levels of estrogenic agents, reaching concentrations of up to 10 nM for 17ß-estradiol and 100 1 µM for genistein. Therefore, the goal of our study was to employ subsaturating and equipotent levels of each ER agonist to determine transactivation patterns in MCF7 and T47D cells. The resulting expression signatures have been compared with the emerging transcriptional profile elicited by the endogenous hormone 17ß-estradiol in the same cancer cell lines. Indeed, the gene expression changes that we observed in response to 17ß-estradiol include a large number of transcripts that were previously known to be susceptible to estrogenic regulation, thus substantiating the validity of our transcriptomic analysis. In contrast to previous reports (27–29,31), we unexpectedly found that the transcriptional machineries of MCF7 and T47D breast cancer cells respond in a very monotonous manner to estrogenic stimuli. Presumably, the differential transcription profiles documented in previous studies arise from dose-dependent variations in the magnitude of gene expression, rather than from distinct mechanisms of gene regulation. For example, it has been demonstrated that some estrogen-responsive transcripts are induced only when the hormone level is raised to concentrations that exceed the saturation range (22). On the other hand, the induction of similar expression patterns in response to distinct ER ligands, including 17ß-estradiol and genistein, has already been reported for the mouse uterus (33). Thus, there is growing evidence that, at least in some susceptible target tissues, phytoestrogens and synthetic estrogenic chemicals elicit the same monotonous transcriptional program as the endogenous hormone.

The existence of congruent expression profiles help to explain the discrepancy between the high concentrations of estrogenic chemicals that are needed in most cases to elicit an effect and the low level of these compounds in the diet or environment. In fact, it has been demonstrated in a simple experimental set-up, consisting of a reporter gene assay in yeast transfected with human ER{alpha}, that the multiple components of xenoestrogen mixtures can act together to yield measurable responses when combined at concentrations which individually produce undetectable effects (53). Thus, the induction of identical transcriptional signatures, both with respect to the precise endpoints (gene targets) and the quality of response (gene induction or repression), supports the view that distinct estrogens can act in a cumulative manner even in complex systems covering a multitude of genomic targets at higher levels of biological organization. On the basis of our results, it appears that non-saturating concentrations of 17ß-estradiol, genistein, bisphenol-A, PCB54 and other xenoestrogens may cooperate to transactivate or repress the same spectrum of genes, thereby inducing an additive transcriptional response that is characteristic for ER agonists. Therefore, we propose that the multitude of estrogenic chemicals to which the population is exposed involuntarily, in conjunction to changes in endogenous hormone levels, may constitute the cumulative cause for an increased risk of breast cancer or other malignancies of estrogen-dependent tissues.


   Acknowledgments
 
We thank A.M. Soto and C. Sonnenschein for the gift of MCF7 cells. This research was supported by the Swiss National Science Foundation grant 4050-066572.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 6, 2005; revised December 12, 2005; accepted January 3, 2006.


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