help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-0563
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chang, E. C.
Right arrow Articles by Katzenellenbogen, B. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chang, E. C.
Right arrow Articles by Katzenellenbogen, B. S.
Endocrinology Vol. 147, No. 10 4831-4842
Copyright © 2006 by The Endocrine Society

Impact of Estrogen Receptor ß on Gene Networks Regulated by Estrogen Receptor {alpha} in Breast Cancer Cells

Edmund C. Chang, Jonna Frasor, Barry Komm and Benita S. Katzenellenbogen

Departments of Molecular and Integrative Physiology (E.C.C., J.F., B.S.K.), and Cell and Developmental Biology (B.S.K.), University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; and Women’s Health and Musculoskeletal Biology (B.K.), Wyeth Research, Collegeville, Pennsylvania 19426

Address all correspondence and requests for reprints to: Dr. Benita S. Katzenellenbogen, University of Illinois, Department of Molecular and Integrative Physiology, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801-3704. E-mail: katzenel{at}uiuc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two subtypes of the estrogen receptor (ER), ER{alpha} and ERß, mediate the actions of estrogens, and although 70% of human breast cancers express ERß along with ER{alpha}, little is known about the possible comodulatory effects of these two ERs. To investigate this, we have used adenoviral gene delivery to produce human breast cancer (MCF-7) cells expressing different levels of ERß, along with their endogenous ER{alpha}, and have examined the effects of ERß and receptor occupancy, using ER subtype selective ligands, on genome-wide gene expression by microarray and pathway network analysis. ERß had diverse effects on gene expression, enhancing or counteracting ER{alpha} regulation for distinct subsets of estrogen target genes. Strikingly, ERß in the absence of estradiol (E2), elicited the stimulation or suppression of many genes that were normally only regulated by ER{alpha} with E2. In addition, ERß plus E2 elicited the expression of a unique group of genes that were not regulated by ER{alpha} plus E2 alone. The expression of genes in many functional categories were modulated by ERß, with the greatest numbers associated with transcription factors and signal transduction pathways. Regulation of multiple components in the TGFß and semaphorin pathways, and of genes controlling cell cycle progression and apoptosis, may contribute to the suppression of cell proliferation observed with ERß. Our observations suggest that the relative levels of ERß and ER{alpha} in breast cancers are likely to impact cell proliferation and the activities of diverse signaling pathways and their response to ER ligands and endocrine therapies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN SIGNALING IS important in many aspects of reproductive physiology and development, and in the functioning of numerous nonreproductive tissues as well. Estrogen hormones also influence the growth of various cancers (1), including breast and endometrial cancers. Although estrogens were originally thought to signal through only one form of estrogen receptor (ER), the complexity of estrogen physiology was compounded when a second form of the ER (termed ERß, ESR2) was cloned (2, 3). Since then, much effort has gone into investigating the specific roles of the two receptor subtypes in diverse estrogen target tissues (4, 5, 6, 7).

Although ERß is normally coexpressed with ER{alpha} in many tissue types, and the majority of human breast cancers express ERß along with ER{alpha} (8, 9, 10), it is not fully known how the presence of both receptors and their relative levels control cellular responses to estrogen. These two transcription factors have a similar domain structure and very similar DNA binding domains, but have substantial differences in their ligand binding domains and especially in their N-terminal activation function regions (1, 11). Examination of their separate activities in osteosarcoma cells indicated distinct as well as overlapping gene regulatory activities (12, 13). Because these receptors are able to heterodimerize when present in the same cell (14), their joint actions and possible comodulatory effects on gene regulation are issues of importance.

Although it is well documented that ER{alpha}-positive breast cancer cells show enhanced proliferation in response to estrogen (15, 16), the manner in which ERß impacts estrogen mitogenicity and the changes in gene expression that underlie these effects are less clear, although several reports support the role of ERß as a negative regulator of ER{alpha} (17, 18, 19).

To better understand the role of ERß in influencing estrogen action, we have used adenoviral gene delivery of ERß and gene expression microarray analyses to investigate gene regulatory effects of ERß in breast cancer cells expressing ER{alpha}, as well as to distill the information into gene networks and pathways responsible for controlling estrogen activities. Our results indicate that ERß can modulate ER{alpha} gene expression in both an enhancing and a suppressing fashion, and, strikingly, that it can have a modulatory effect even in the absence of ligand and it can regulate additional gene targets in the presence of ER{alpha} that are not regulated by ER{alpha} alone. We present evidence from analysis of the functional categories of the genes impacted by ERß, and from cell proliferation studies, to suggest that ERß acts to counter some of the important actions of ER{alpha} in breast cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture, adenovirus infection, and RNA extraction
MCF-7 cells were maintained in MEM (Sigma, St Louis, MO) supplemented with 5% calf serum (HyClone, Logan, UT), 100 µg/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA), and 25 µg/ml gentamicin (Invitrogen). For estrogen-free experiments, MCF-7 cells were grown in phenol red-free MEM plus 5% charcoal-dextran-treated calf serum for at least 5 d, then seeded at a density of 3 x 105 cells per 10-cm tissue culture dish (Corning, Corning, NY) for 2 d before adenovirus infection. Cells were infected with either control empty adenovirus or control adenovirus expressing ß-galactosidase (AdGal), or with ERß-containing adenovirus (AdERß) at different multiplicities of infection (moi) for 8 h before excess virus was removed with media change. Cell incubation with virus continued for 48 h before estrogen treatment was initiated for the last 24 h of the 72-h adenoviral infection period. Greater than 90% of the cells were observed to be infected at moi of 5 or higher, using AdERß, or Ad-GFP or AdGal for cell transfection analysis. Total RNA was prepared using TRIzol (Invitrogen) according to the manufacturer’s recommendations and was further purified using RNeasy columns (Qiagen, Valencia, CA) and ribonuclease-free deoxyribonuclease I (Qiagen).

GeneChip microarrays and statistical analysis
Total RNA was harvested for cRNA labeling and hybridization to Affymetrix HGU133A GeneChips as described previously (20). After washing, the arrays were scanned and analyzed using the GeneChip Operating Software (Affymetrix, Santa Clara, CA). CEL files were processed and quantile normalized using the "affy" and "gcrma" package protocols in R/Bioconductor (20, 21). To find genes differentially regulated by estradiol (E2) with high confidence (≥1.8-fold change and <5% false discovery rate), we used the Significance Analysis of Microarray application (22) to compare expression levels between vehicle-treated and E2-treated samples of Ad-infected cells and separately for AdERß-infected cells. We then used two-way ANOVA to identify genes significantly modulated by ERß in the presence and absence of E2 (≥1.5-fold change and <5% false discovery rate).

Functional categorization of target genes
Gene functions were curated based on available published data in PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed) and theNetAffx databases (http://www.affymetrix.com), and were loaded into the GeneSpring software version 7 (Silicon Genetics, Redwood City, CA). Global analysis for overrepresented GenMAPPs (23), KEGG pathways (24), and Gene Ontology Biological Function categories was performed using the Expression Analysis Systematic Explorer annotation tool developed by the National Institutes of Health (25) on our set of 1072 E2-regulated genes identified in this study compared with the entire genome of the Affymetrix GeneChip U133A. A similar enrichment analysis for the 222 E2-regulated genes significantly modulated by ERß over the 1072 E2-regulated genes was also performed.

Recombinant adenovirus preparation
Recombinant adenoviruses were constructed and prepared as described (26). Briefly, replication-deficient adenovirus serotype 5 were propagated in HEK293 cells (Microbix, Toronto, Canada) and then harvested. Approximately 60 µl of this stock was used to reinfect HEK293 cells at 70% confluence in 100-mm dishes, and within 2–3 d, more than 90% of the 293 cells detached from the dish surface. The cells were harvested as described earlier and used to reinfect up to 20 150-mm dishes of cells using 500 µl of 107 expression forming units per milliliter per 150-mm dish. Again, in 2–3 d, >90% of the 293 cells will be detached from the plate surface. Virus was concentrated, up to 1012 expression forming units per milliliter, using a CsCl gradient protocol (http://www.coloncancer.org/adeasy).

RT-PCR and quantitative PCR
Total RNA was isolated from MCF-7 cells using TRIzol (Invitrogen) following the manufacturer’s recommendations. RNA samples were reverse transcribed by SuperScript II reverse transcriptase (Invitrogen) in a 20-µl volume and subsequently diluted to 400 µl with sterile water. Real-time PCR was performed on an ABI Prism 7900HT instrument using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations. Briefly, each PCR contained: 1x master mix, 4 µl of the diluted cDNA reaction, and 62.5 nM forward and reverse primers designed to yield 80- to 125-bp amplicons. Relative expression levels were calculated using the {Delta}-{Delta} CT method as described previously (27).

Protein extraction and Western blot analysis
Whole cell extracts were prepared using lysis buffer containing 20 mM Tris, 150 mM NaCl, 1% NP-40, 1% SDS, 5% glycerol, and the Complete-Mini protease inhibitor cocktail tablet (Roche, Nutley, NJ). Samples were boiled in 2x Laemlli buffer and run on the mini-blot SDS-PAGE gels (Bio-Rad, Hercules, CA). Proteins were transferred onto nitrocellulose membrane overnight (20 V constant) and blocked with 5% nonfat milk in Tris-buffered saline solution (10 mM Tris, pH 7.4; 150 mM NaCl; and 0.5% Tween 20) before incubation with primary antibody. Anti-ER{alpha} antibodies (polyclonal rabbit HC-20) were bought from Santa Cruz Biotechnologies (Santa Cruz, CA). Anti-ERß antibodies (monoclonal mouse, CWK-F12) were produced by our lab (28). Secondary antibodies to the respective primary antibodies were purchased from Zymed Antibodies (South San Francisco, CA).

Cell proliferation
MCF-7 cells were infected with empty adenovirus (or with AdGal, which gave similar results) or AdERß in 100-mm dishes (Corning) for 8 h before seeding in MEM phenol red-free medium supplemented with 5% charcoal-dextran-treated calf serum at 1000 cells per well, in 96-well plates (BD Falcon, Franklin Lakes, NJ). After 18 h, cells received fresh media and vehicle or E2 (10 nM), and this was renewed every 72 h over the cell proliferation time course. Cell growth was followed over time using the MTS tetrazolium colorimetric CellTiter 96 AQueous One Solution Cell Proliferation Assay according to the manufacturer’s recommendation (Promega, Madison, WI).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To study the effects of ERß in ER{alpha}-positive breast cancer cells, we used adenovirus-mediated gene delivery of ERß, an efficient gene delivery method we had used previously to express dominant-negative ERs in MCF-7 cells (26). To obtain different levels of ERß in cells, ER{alpha}-expressing MCF-7 breast cancer cells were infected with recombinant ERß adenovirus (AdERß), or with control adenovirus, at different moi (number of viruses used per cell) ranging from 5–50, and ER mRNA and protein expression were examined (Fig. 1Go). To quantify ER{alpha} and ERß protein levels in MCF-7 cells, we generated standard curves from Western blots using serial dilutions of known amounts of recombinant ER proteins. After 72 h of AdERß infection at moi 5, concentrations of ERß and ER{alpha} protein were approximately 0.6 and 2.0 fmol/µg cell protein, respectively (Fig. 1Go, A and B). This ratio of ER{alpha} to ERß, where ERß is approximately 30% that of ER{alpha}, approximates that seen in many breast tumors where ER{alpha} is usually the more abundant receptor (9, 10, 29), and hence we used this moi of 5 for most of the work reported here. For some comparative studies we used an moi of 25 or 50, where ERß levels exceeded ER{alpha} levels severalfold (Fig. 1BGo). Notably, endogenous ER{alpha} levels were unaffected by low levels of ERß (moi = 5), but ER{alpha} diminished progressively at higher ERß mois (Fig. 1Go, A and B). The observed levels of ERß and ER{alpha} proteins correlated well with their respective mRNA levels (Fig. 1CGo). In all cases, addition of E2 resulted in some reduction in ER{alpha} and ERß protein and mRNA (Fig. 1Go, A-C), as is well documented for ER{alpha} in MCF-7 cells (30).


Figure 1
View larger version (13K):
[in this window]
[in a new window]
  		FIG. 1. Adenovirus-delivered ERß effectively transduces ERß mRNA and protein in MCF-7 cells. A, ERß and ER{alpha} proteins were monitored by Western blot of whole cell protein extracts (30 µg) from AdERß-infected cells (moi 5, 25, and 50) or control empty adenovirus-infected cells (moi 50) that were treated with vehicle (–, left lanes) or 10 nM E2 (+, right lanes) for 24 h during the last 24 h of the 72-h adenovirus infection period. Samples were blotted for ER{alpha} (HC-20; Santa Cruz Biotechnologies), ERß (CWK-F12; described in Ref. 28 ), and ß-actin (AC-20; Sigma). B, Cellular ER{alpha} and ERß levels were quantitated from standard curves we prepared (data not shown) using purified ER{alpha} and ERß recombinant proteins (PanVera) and Western blots with ER{alpha} and ERß antibodies. Quantitation of the ER{alpha} and ERß protein levels and normalization (to actin levels) were done on ImageQuant TL 2005 software (GE Biosciences). ER{alpha} and ERß levels are expressed in femtomoles per 30 µg of whole cell protein. C, ER{alpha} and ERß mRNA levels in cells after infection with empty adenovirus (Ad) or ERß adenovirus (ERß) at the indicated moi. Treatment was with vehicle (veh) or 10 nM E2 for the last 24 h of the 72-h time period.

 
Estrogen regulates distinct sets of genes in MCF-7 cells expressing ER{alpha} and in cells coexpressing ER{alpha} and ERß
Two distinct effects impact ER{alpha} activity in our study: estrogen (E2) ligand treatment (Fig. 2AGo, comparisons 1 and 2) and ERß presence (Fig. 2AGo, comparisons 3 and 4). To identify genes significantly regulated by E2 (comparisons 1 and 2; fold change ≥1.8x, false discovery rate < 0.05), we used the Significance Analysis of Microarrays application (22) to compare E2-treated and untreated samples in the two ER cell backgrounds (Fig. 2Go, Venn circles C+E2 and +ERß+E2; where C denotes control virus and +ERß denotes adenovirus expressing ERß). Interestingly, we found that only half of the total genes regulated by E2 in either ER cell background (524 of 1072) were shared (Fig. 2Go). This indicates that introduction of ERß can significantly alter the gene expression profile of estrogen-treated ER{alpha}-containing cells.


Figure 2
View larger version (22K):
[in this window]
[in a new window]
  		FIG. 2. Estrogen-regulated genes are differentially modulated by ERß in ER{alpha}-positive MCF-7 cells. A, To find significant E2-regulated genes, we made pairwise comparisons using Significance Analysis of Microarrays (Stanford University, Stanford, CA) between vehicle-treated and E2-treated samples of adenvirus-infected (no. 1) and AdERß-infected cells (no. 2). The settings used were equal to or more than 1.8 times the cutoff thresholds and less than or equal to 5% false discovery rate. B, A total of 1072 genes (491 stimulated; 581 genes repressed) were found to be E2 regulated in either adenvirus-infected (no. 1) or AdERß-infected cells (no. 2). Two-way ANOVA was used to find genes significantly modulated by ERß (nos. 3 and 4) in either enhancing or attenuating fashions. Pie charts for E2-stimulated genes and E2-repressed genes depicting categories of genes modulated by ERß in MCF-7 cells. C, The percentage of E2-stimulated genes modulated by ERß increased from 32–61% when a higher dose of ERß adenovirus (moi 50) was used. The percentage of E2-inhibited genes modulated also increased from 11–46%, when the moi was 50 compared with moi of 5.

 
To find genes that were significantly up- or down-modulated by ERß within the set of estrogen-regulated genes, we used two-way ANOVA to compare the vehicle- or E2-treated samples of the two ER backgrounds (Fig. 2AGo). Only genes with expression levels changed at least 1.5-fold between C+E2 and +ERß+E2 (false discovery rate < 0.05) were deemed "ERß-modulated." We observed significantly different expression levels for 222 genes between ER{alpha}/ERß coexpressing and ER{alpha}-only cells (Fig. 2BGo) at the ERß moi of 5, where ERß is approximately 30% the level of ER{alpha}. Expressed as a percent of total genes regulated by estrogen, 32% of all E2-stimulated genes were modulated by ERß (158 of 491), whereas only 11% of all E2-repressed genes were modulated by ERß (64 of 581) (Fig. 2BGo). The several subcategories of E2-regulated genes modulated by ERß are shown in the pie charts (Fig. 2BGo).

We also asked whether higher cellular levels of ERß would lead to even greater ERß modulation of ER{alpha}-regulated genes. Therefore, we compared genome-wide gene expression in MCF-7 cells infected with 10 times more AdERß virus (moi 50) and performed similar statistical analysis (as described in Materials and Methods) for differentially expressed genes between treatment groups. Our results indicate that this approximately 10-fold higher ERß expression led to an even greater percentage of E2-regulated genes being modulated (61% of all E2-up-regulated genes and 46% of all E2-down-regulated genes; Fig. 2CGo). It is worth noting again that ER{alpha} levels are substantially reduced in cells containing these high levels of ERß (Fig. 1Go and see Discussion).

ERß modulates ER{alpha}-mediated transcriptional activity in both positive and negative fashions
Because ERß is a weaker transcriptional activator relative to ER{alpha} in reporter gene transfection studies (31, 32), we expected that ERß might mostly attenuate ER{alpha} activity, dampening E2-stimulation and reversing E2-repression. However, we found that ERß enhanced as well as dampened gene expression stimulated through ER{alpha} (Fig. 3AGo, clusters I and II, respectively). Similarly, for ER{alpha}-mediated repression, ERß exerted an enhanced repressive effect on some genes (cluster III) and reversal of repressive effects on other genes (cluster IV). These findings suggest that ERß does not act unilaterally as a moderator of the effects of ER{alpha}, but rather modulates (positively or negatively) ER{alpha} activity in a gene-specific manner. Of note, ERß presence was necessary for the E2-induced stimulation of 37 genes (cluster V "ERß-driven up-regulation"; Fig. 3CGo) that were not significantly regulated by E2 in MCF-7 cells expressing only ER{alpha}.


Figure 3
View larger version (28K):
[in this window]
[in a new window]
  		FIG. 3. ERß modulates transcriptional activity of ER{alpha} in several distinct ways. A, Heatmap showing expression profile for E2-regulated genes (10 nM E2 for 24 h) modulated by ERß (moi 5). Genes were grouped based on expression into distinct clusters. Cluster I, ERß enhances the ER{alpha} stimulation (71 genes). Cluster II, ERß reduces the ER{alpha} stimulation (33 genes). Cluster III, ERß reduces the ER{alpha} repression (15 genes). Cluster IV, ERß enhances the ER{alpha} repression (41 genes). B, Specific types of ERß-modulation were verified over a range of E2 concentrations using real-time RT-PCR. All data are mean (±SD) of three independent samples and are expressed as fold change relative to vehicle control adenovirus-infected cells. Examples are shown for each cluster. Cluster I, IL17RB and BIRC3. Cluster II, SDF1 and NPYY1. Cluster III, CSRP2 and BMP7. Cluster IV, DKK1 and CLDN1. C and D, cluster V, genes exhibiting no significant stimulation by ER{alpha} plus E2 that become up-regulated in the presence of ERß and E2 (44 genes). Two examples, S100P and OTUB2, are shown.

 
We examined ERß modulation of ER{alpha} activity for representative genes in these five categories as a function of E2 concentration (Fig. 3Go, B and D). Our findings indicate that ERß modulation of ER{alpha} activity was E2 dose-dependent for most genes and was greatest at E2 concentrations of 1 nM and higher.

Ligand occupancy of ERß determines the magnitude of ERß modulatory effects on gene expression
We have reported previously on the activities of the ER{alpha}-selective ligand [propyl-pyrazole-triol (PPT)] on a number of defined estrogen target gene sites (33, 34, 35). Using this ER{alpha}-selective ligand, we reasoned that cell treatment with PPT might not evoke the same ERß modulatory effect on gene expression as observed with E2, because PPT does not bind to ERß. Indeed, we found that to be true for some genes where E2 and PPT had similar effects in MCF-7 ER{alpha}-containing cells, but PPT did not elicit the same response as seen with E2 in cells coexpressing ER{alpha} and ERß (Fig. 4AGo, left panels): BIRC3 (enhanced by ERß), NPYY1 (dampened by ERß), CSRP2 (E2-repression reversed by ERß), and S100P (ERß-driven up-regulation).


Figure 4
View larger version (19K):
[in this window]
[in a new window]
  		FIG. 4. Ligand occupancy of ERß determines the degree of ERß modulatory effects on specific gene expression. Adenovirus-infected (control) and AdERß-infected (+ERß moi 5) MCF-7 cells were treated either with 0.1% ethanol vehicle, 10 nM E2, or 10 nM PPT (an ER{alpha}-selective ligand) for 24 h beginning after 48 h of adenovirus infection. Genes were assayed by real-time RT-PCR. A, Modulatory effects of ERß on these genes were observed with E2 but not with PPT treatment (left panels, BIRC3, NPYY1, CSRP2, and S100P). B, Modulatory effects of ERß on these genes were observed for both E2 and PPT treatment (right panels, IL17RB, SDF1, BMP7, and RGS10). All values are mean (+SD) of three independent samples and are expressed as fold change relative to vehicle control adenovirus-infected cells.

 
However, to our surprise, we also found that for some other genes, ERß modulatory effects were still observed in the presence of PPT (Fig. 4BGo). Genes such as: IL17RB (ERß enhances stimulation), SDF1 (ERß dampens stimulation), BMP7 (ERß reverses E2-repression), and RGS10 (ERß-driven up-regulation) (Fig. 4BGo). Because PPT has very low affinity for ERß (36), it is unlikely to transactivate ERß at the 10 nM concentration used in these studies. This suggests that in the context of ligand-occupied ER{alpha}, the modulatory effects of unoccupied ERß on some genes (Fig. 4BGo) can be partial (e.g. IL17RB) or full (e.g. RGS10).

Therefore, we conclude that, for genes regulated by agonist-bound ER{alpha}, modulation by ERß falls into two distinct categories. In the first, ERß modulation of ligand-occupied ER{alpha} requires ligand occupancy of ERß (e.g. BIRC3, NPYY1, CSRP2, and S100P; Fig. 4AGo); in the second, ERß modulation of ligand-occupied ER{alpha} does not require ligand occupancy of ERß (e.g. IL17RB, SDF1, BMP7, and RGS10; Fig. 4BGo).

Coexpression of ERß with ER{alpha} in the absence of added ligand elicited an E2 plus ER{alpha}-like effect on some genes
Remarkably, introduction of ERß in itself (i.e. without E2 treatment) elicited some changes in gene expression similar to estrogenic effects mediated by ER{alpha} (Fig. 5Go). Expression of ERß in MCF-7 cells in the absence of added E2 induced the up-regulation of 27 E2-stimulated genes and the down-regulation of 19 E2-inhibited genes. In some cases, the ERß-induced changes in basal gene expression were so great that they essentially eliminated response to added E2 (EPB41L3, KRT13, and CXCR4), whereas other genes retained some response to added E2 (WISP2, RAB31, and ERBB2). It is also interesting that for some genes the ERß-mediated basal level change did not affect the final E2-stimulated/ repressed levels of genes such as WISP2, RAB31, and CXCR4 (Fig. 5Go), but compounded the E2-mediated effect for other genes such as EPB41L3 and ERBB2 (Fig. 5Go), and DKK1 and CLDN1 (Fig. 3Go).


Figure 5
View larger version (38K):
[in this window]
[in a new window]
  		FIG. 5. ERß can regulate a subset of E2-responsive genes in the absence of ligand. A, Heatmap showing microarray expression profiles for 42 genes whose basal expression levels were modulated by ERß. MCF-7 cells were infected with either empty control adenovirus (C) or two different mois of ERß adenovirus (+ERß5 or +ERß50). B and C, Real-time RT-PCR data as means (+SD) of three independent samples and are expressed as relative fold change to vehicle control adenovirus-infected cells. B, Examples of genes stimulated by E2 plus ER{alpha} that were up-regulated by ERß addition in the absence of E2 (veh, open bars; WISP2, EPB41L3, RAB31, KRT13). Basal levels increased with higher ERß expression levels. C, Examples of E2 plus ER{alpha} inhibited genes that were down-regulated by ERß addition in the absence of E2 (ERBB2 and CXCR4). Basal gene expression levels (veh, open bars) decreased with higher ERß.

 
ERß expression attenuates the growth-promoting activity of ER{alpha} in MCF-7 cells
Several labs have reported on growth suppression by ERß in breast cancer cells (18, 19). We find that ERß expression (+ERß+veh) reduced the basal MCF-7 growth rate (Fig. 6AGo), suggesting that ERß expression by itself can modulate genes involved in cell proliferation independent of estrogen ligand. Treatment with 10 nM E2 was able to increase proliferation of the ER{alpha}/ERß-coexpressing cells, but the rate of proliferation was much less than that of MCF-7 cells expressing only ER{alpha} (i.e. C+E2).


Figure 6
View larger version (11K):
[in this window]
[in a new window]
  		FIG. 6. Introduction of ERß into MCF-7 cells slows cell proliferation. A, Control adenovirus-infected cells or AdERß-expressing cells (moi 5) were assayed for growth over time in the presence of vehicle or 10 nM E2 using the cell proliferation assay as described in Materials and Methods. Data represent the mean (±SD) of four samples. B, Genes involved in cell cycle control are modulated by ERß. ERß down-regulates genes essential for cell cycle progression (FOXM1, CDC25A, E2F1) and down-regulates the antiapoptosis gene survivin/BIRC5, while up-regulating an inhibitor of the cell cycle (p21WAF1). Gene expression measured by real-time RT-PCR and expressed as fold change relative to vehicle control Ad-infected cells. C, Western immunoblot for FoxM1 in vehicle-treated (–) and 10 nM E2-treated (+E2) control (adenovirus moi 5) or ERß (AdERß moi 5)-expressing MCF-7 cells. Treatment with E2 or vehicle was for the times indicated.

 
To explore the basis for this reduction in cell numbers with ERß, we examined several genes involved in regulation of cell cycle progression and apoptosis by real-time PCR, as the Affymetrix GeneChip microarray analyses indicated their modulation by ERß. As seen in Fig. 6BGo, ERß down-modulated FOXM1, CDC25A, E2F1, and survivin/BIRC5 mRNA expression in the absence and presence of E2. FoxM1 protein levels, examined by Western blot, were also markedly reduced by ERß (Fig. 6CGo). Because FoxM1 is a known regulator of CDC25A, the down-modulation of FoxM1, in turn, would be expected to result in attenuated CDC25A expression, as was observed. In contrast, the levels of p21WAF1, a cell cycle inhibitor, were higher in ER{alpha}/ERß-coexpressing cells relative to ER{alpha}-only cells and were less down-regulated by E2 treatment (Fig. 6BGo). ERß also markedly suppressed the control and E2-stimulated level of the antiapoptosis gene survivin/BIRC5 (Fig. 6BGo). Taken together, these results provide evidence that ERß may attenuate MCF-7 growth by repressing positive proliferative and antiapoptotic factors (FOXM1 and CDC25A, E2F1, and survivin/BIRC5) and up-regulating negative proliferation-regulating factors (p21WAF1).

Global analysis of functional categories and cellular processes regulated by ER{alpha} and ERß
To better understand the physiological impact of E2 effects as mediated through ER{alpha} and ERß, we assessed the enrichment of GenMAPP pathways, KEGG pathways, and Gene Ontology Biological Process categories for the set of 1072 E2-regulated genes (supplemental Table 1, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). We note that GO Biological Process categories relating to cell cycle regulation, energy/metabolism, and ion homeostasis were well represented. In addition, two GenMAPP pathways were found to be significantly regulated by E2 treatment (TGFß signaling and cell cycle), based on their Expression Analysis Systematic Explorer score (<0.05, supplemental Table 1). These findings are consonant with the mitogenic effect of E2 on ER-positive breast cancer cells.

Next, we evaluated whether any functional categories were enriched within the 222 genes significantly modulated by ERß. GO Biological Process categories relating to chemokine/growth factor signaling (expression of genes encoding CXCL1, CXCL2, CXCL10, CXCL20) and ion homeostasis were significantly enriched in the ERß-modulated gene set (supplemental Table 2). This suggests that regulation of autocrine/paracrine signaling may be a key difference in cells expressing both ER{alpha} and ERß vs. ER{alpha} only.

Estrogen down-regulates the TGFß signaling pathway in MCF-7 cells
TGFß signaling is important for the regulation of cell proliferation and differentiation, embryonic development, and pathogenesis of certain tumors (37, 38). Although TGFß signaling is known to up-regulate p21WAF1 and down-regulate c-MYC, E2 has the opposite effect on both genes. We find that E2 treatment significantly up- or down-regulated four and 12 genes involved in TGFß signaling, respectively (Fig. 7Go). It is worth noting that most of the E2-down-regulated genes functionally activate the TGFß pathway (TGFß1, TGFß2, TGFß3, INHBB, BMP4, BMP7, THBS1, ITGB6, BAMBI, SMAD3, SMAD6, SMAD7), whereas the up-regulated genes negatively impact TGFß signaling [SKIL, TIEG, FST, TGIF2: SKIL/SNO (negative regulator of Smad4) (39); TIEG/KLF10 (repressor of Smad7 transcription) (40); follistatin (inhibitor of TGFß ligand), and TGIF2 (homeobox transcriptional corepressor that recruits HDAC1) (41)].


Figure 7
View larger version (38K):
[in this window]
[in a new window]
  		FIG. 7. Multiple components of the TGFß pathway are down-regulated by E2 treatment and modulated by ERß presence in MCF-7 cells. A, Heatmap of the expression profile of TGFß pathway genes after 10 nM E2 treatment for 24 h. ERß significantly modulated the expression of many genes including TGFß2, THBS1, BMP7, and ITGB6. B, Interaction schematic between members of the TGFß signaling pathway (adapted from GenMAPP). TGFß ligands are down-regulated by E2 treatment, whereas negative regulators of the TGFß signaling pathways are up-regulated (e.g. FST, TGIF2, and SKIL). Genes up-regulated by E2 treatment are highlighted in red; down-regulated genes are highlighted in blue.

 
Taken together, these results suggest that E2 may promote cell proliferation by inhibiting TGFß production and coordinately regulating several points in the TGFß pathway. Furthermore, ERß modulated ER{alpha} regulation of TGFß signaling by reversing the repression of BMP7 by estrogen as well as by repressing thrombospondin expression (Fig. 7Go), which may represent novel mechanisms by which ERß acts as a tumor-suppressive protein.

Estrogen down-regulates the semaphorin and SDF1 signaling pathways in MCF-7 cells
Signaling pathways involving semaphorins provide directional cues during axon guidance, mainly through interactions with transmembrane proteins, plexins, and neuropilins (42, 43). However, the activities of semaphorins in breast cancer are virtually unknown. We observed that estrogen regulated class 3 and 4 semaphorin ligands (Sema3B, Sema3C, Sema3F, Sema4C, Sema4D, Sema4F), their cognate receptors (PLXNA3, PLXNC1), and downstream adaptors (MICAL2) (Fig. 8Go).


Figure 8
View larger version (28K):
[in this window]
[in a new window]
  		FIG. 8. Semaphorin pathway genes (classes 3 and 4) are significantly regulated by E2 treatment in MCF-7 cells. A, Heatmap depicting the expression profile of semaphorin pathway genes after 10 nM E2 treatment for 24 h. ERß significantly modulated the expression of Sema3B (ERß-driven stimulation), MICAL2 (ERß enhances stimulation), SDF1 (ERß dampens stimulation), and CXCR4 (ERß further enhances the repression). B, Interaction schematic between members of the semaphorin signaling pathway. Genes up-regulated by E2 treatment are highlighted in red; down-regulated genes are highlighted in blue.

 
In particular, ERß modulated genes associated with class 3 semaphorin signaling by selectively up-regulating SEMA3B and MICAL2. SEMA3B is a known tumor suppressor that is regulated by p53 (44, 45), whereas MICAL is a flavomonooxygenase necessary for mediating class 3 semaphorin signaling (46). ERß also down-modulated the E2-ER{alpha}-mediated enhanced expression of the chemokine, SDF1/CXCL12, an autocrine growth factor for breast cancer cells (47) that is also reported to interfere with semaphorin signaling (48). Taken together, our findings suggest that modulation of class 3 semaphorin signaling represents another means by which ERß impacts MCF-7 cell activity and might suppress breast cancer cell proliferation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings document that ERß has a significant impact on the pattern of gene expression in breast cancer cells containing ER{alpha}. The effect of ERß is diverse, either enhancing or repressing the expression of genes regulated by ER{alpha}, as well as inducing the expression of new genes that are not regulated by ER{alpha} alone. This orchestrated gene regulation by ER{alpha} and ERß may be, at least in part, responsible for the overall reduction in cell proliferation observed when ERß is expressed in these cells.

Our observations suggest that the level of ERß relative to ER{alpha} in breast cancers is likely to impact the activities of multiple cell signaling pathways. Although the expression of components in many signal transduction and transcription factor regulatory pathways were affected by the presence of ERß, it is noteworthy in particular that multiple components in the TGFß signaling and semaphorin pathways were affected. The first of these is consistent with observations that TGFß is normally associated with the suppression of breast cancer cell proliferation (49, 50).

In parental MCF-7 cells, E2 treatment inhibited TGFß signaling by down-regulating TGFß ligands (TGFß 1, 2 and 3) and up-regulating negative factors such as follistatin and SKIL. Because TGFß is known to be anti-proliferative in breast cancer cells, at least in part by repressing c-myc and up-regulating p21WAF1, down-regulation of TGFß signaling might contribute to the acceleration of cell growth by estrogen. In addition, ER{alpha} has been demonstrated to inhibit both TGFß and BMP signaling by physically interacting with Smad proteins (51, 52). Therefore, estrogen appears capable of inhibiting TGFß signaling both by regulating expression of TGFß-signaling-related genes and also physically interfering with transducers of TGFß signaling.

ERß modulated the estrogen-mediated repression of thrombospondin 1 (THBS1; repression enhanced by ERß), integrin ß6 (ITGB6; repression reduced by ERß) and bone morphogenetic protein7 (BMP7; repression reduced by ERß). THBS1 and ITGB6 are both important in the release of extracellular, latent TGFß (53, 54). Although little is known for BMP7 actions on breast cancer cells, it has been shown that BMP7 counters TGFß1-mediated epithelial-to-mesenchymal transitions in renal tubular cells by increasing the expression of E-cadherins (55). Our quantitative PCR data confirmed that BMP7 gene expression regulation by E2 was reversed by the presence of ERß.

It is increasingly clear that some metastatic cancers have coopted the signaling paradigms used by migrating neuronal cells (56). Hence it is of interest that a second pathway impacted by ERß involved several estrogen regulated genes involved in class 3 and 4 semaphorin signaling. Class 3 semaphorins are reported to suppress growth of breast cancer cells (44) and regulate vascular morphogenesis of spreading endothelial cells (57). In our studies, we found that ERß selectively up-regulated expression of SEMA3B, a known tumor suppressor. We also note that ERß down-modulated the E2-mediated enhanced expression of stromal cell-derived factor 1 (SDF-1), an autocrine growth factor for breast cancer cells (47). Because SDF-1 has been demonstrated to interfere with semaphorin signaling (48), it is possible that the ERß-mediated up-regulation of SEMA3B and MICAL2, coupled with ERß down-modulation of SDF-1, might contribute to the suppressed cell growth observed in cells coexpressing ERß and ER{alpha}.

Although ER{alpha} levels appear to be generally higher than those of ERß in most breast cancers, ERß is estimated to be present in over 70% of breast cancers (10, 58). Hence, our studies provide some data to assist in elucidating ERß activities in gene regulation. Perhaps of most interest were our observations that unoccupied ERß alone, in the absence of added E2, could elicit the regulation of some genes normally regulated by ER{alpha} only in the presence of E2. This is likely a reflection of the significant ligand-independent activity of the N-terminal activation function-1 region of ERß (32, 59). The modulatory effects of ERß might stem from differential usage of regulatory surfaces of ER{alpha} and ERß (60), and their heterodimerization resulting in differential coregulator recruitment, an aspect requiring further study.

In most of our studies, we examined the impact of ERß on the gene regulatory actions of estrogen using a level of ERß (moi 5) that might typically be found in breast cancers, namely an ER{alpha} to ERß ratio of 3 (Fig. 1Go). Although studied less extensively, we also used expression of higher levels of ERß (moi 50). These resulted in many of the same changes in gene expression, but also some differences (Fig. 2CGo). It is of note that high ERß expression lowered ER{alpha} at both the mRNA and protein levels. This reduction in ER{alpha} levels might further enhance effects in which ERß is opposing the actions of ER{alpha}, which appears to be illustrated by the data shown in Fig. 2CGo where a larger proportion of genes were repressed by the high, moi 50 vs. the moi 5 level of ERß.

ERß at the lower level (moi 5) reduced the rate of MCF-7 cell proliferation in both the absence and presence of E2. The ability of ERß to down-regulate the expression of some genes associated with enhancement of cell proliferation such as FOXM1, may contribute to the growth suppression of ERß. This forkhead transcription factor facilitates G1/S and G2/M transitions by decreasing p21WAF1 levels and regulating CDC25A and CDC25B transcription, as well as other G2-specific genes (61, 62). Because CDC25A and p21WAF1 were previously shown to be repressed and up-regulated, respectively, by ERß (18, 19), it is possible that ERß down-regulation of FOXM1 represents the upstream event leading to decreased CDC25A expression and increased p21WAF1 expression. Our findings are concordant with the observations of reduced proliferation of T47D breast cancer cells expressing ERß, as well as reduced tumorigenesis of MCF-7 cell xenografts expressing ERß (17, 18, 19).

Most, but not all, clinical outcome studies indicate that the copresence of ERß in ER{alpha}-containing primary breast cancers is associated with a better outcome on endocrine therapy (10). In this regard, it is of interest that ERß markedly reduced expression of the antiapoptosis gene, survivin (Fig. 6BGo), because several studies have shown that high expression of survivin is associated with poor-prognosis breast tumors (i.e. tumors from women showing only a short period of relapse-free survival) (63). Also, survivin is one of the few proliferation/antiapoptosis signature genes found to be of value and, hence, currently used in the recurrence score prediction for ER-positive, lymph node-negative tamoxifen-treated breast cancer patients (64), implying that reduced survivin expression with ERß might in part account for the more favorable clinical outcome in ERß-containing breast cancers.

Because ER-positive human breast cancers usually contain both ER{alpha} and ERß, our gene transcriptional profiling at different levels of ERß provide insights in helping to elucidate the activities and impact of ERß in breast cancer. Our work suggests that the levels and ratios of ER{alpha} to ERß will be significant factors in determining estrogen actions in breast cancer and the likely response of ER-positive breast cancers to endocrine therapies involving ER suppression with selective ER modulators such as tamoxifen or estrogen deprivation with aromatase inhibitors.


   Acknowledgments
 
We thank Ken Chang for his assistance in these studies and appreciate advice from Lei Liu and Mark Band of the University of Illinois Carver Biotechnology Center.


   Footnotes
 
This work was supported by grants from the National Institutes of Health (R01CA18119 and P01AG024387) and The Breast Cancer Research Foundation. E.C. received support from National Institutes of Health Training Grant T32 ES07326 and the Mary Landfield Fellowship in Cancer Biology.

Disclosure statement: E.C.C., J.F., and B.S.K. have nothing to declare. B.K. is employed by Wyeth Research.

First Published Online June 29, 2006

Abbreviations: AdERß, ERß-containing adenovirus; AdGal, adenovirus expressing ß-galactosidase; E2, 17ß-estradiol; ER, estrogen receptor; moi, multiplicity of infection; PPT, propyl-pyrazole-triol; SDF-1, stromal cell-derived factor 1.

Received April 28, 2006.

Accepted for publication June 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G, Enmark E, Pettersson K, Warner M, Gustafsson JA 2001 Mechanisms of estrogen action. Physiol Rev 81:1535–1565[Abstract/Free Full Text]
  2. Kuiper GJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930[Abstract/Free Full Text]
  3. Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  4. Coleman KM, Dutertre M, El-Gharbawy A, Rowan BG, Weigel NL, Smith CL 2003 Mechanistic differences in the activation of estrogen receptor-{alpha} (ER{alpha})- and ERß-dependent gene expression by cAMP signaling pathway(s). J Biol Chem 278:12834–12845[Abstract/Free Full Text]
  5. Hall JM, McDonnell DP 1999 The estrogen receptor ß-isoform (ERß) of the human estrogen receptor modulates ER{alpha} transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566–5578[Abstract/Free Full Text]
  6. Liu MM, Albanese C, Anderson CM, Hilty K, Webb P, Uht RM, Price Jr RH, Pestell RG, Kushner PJ 2002 Opposing action of estrogen receptors {alpha} and ß on cyclin D1 gene expression. J Biol Chem 277:24353–24360[Abstract/Free Full Text]
  7. Paech K, Webb P, Kuiper GGJM, Nilsson S, Gustafsson J-A, Kushner PJ, Scanlan TS 1997 Differential ligand activation of estrogen receptors ER{alpha} and ERß at AP1 sites. Science 277:1508–1510[Abstract/Free Full Text]
  8. Katzenellenbogen BS, Frasor J 2004 Therapeutic targeting in the estrogen receptor hormonal pathway. Semin Oncol 31:28–38[Medline]
  9. Kurebayashi J, Otsuki T, Kunisue H, Tanaka K, Yamamoto S, Sonoo H 2000 Expression levels of estrogen receptor-{alpha}, estrogen receptor-ß, coactivators, and corepressors in breast cancer. Clin Cancer Res 6:512–518[Abstract/Free Full Text]
  10. Saji S, Hirose M, Toi M 2005 Clinical significance of estrogen receptor ß in breast cancer. Cancer Chemother Pharmacol 56(Suppl 1):21–26
  11. Katzenellenbogen BS, Montano MM, Ediger TR, Sun J, Ekena K, Lazennec G, Martini PG, McInerney EM, Delage-Mourroux R, Weis K, Katzenellenbogen JA 2000 Estrogen receptors: selective ligands, partners, and distinctive pharmacology. Recent Prog Horm Res 55:163–193; discussion 194–195[Medline]
  12. Monroe DG, Getz BJ, Johnsen SA, Riggs BL, Khosla S, Spelsberg TC 2003 Estrogen receptor isoform-specific regulation of endogenous gene expression in human osteoblastic cell lines expressing either ER{alpha} or ERß. J Cell Biochem 90:315–326[CrossRef][Medline]
  13. Stossi F, Barnett DH, Frasor J, Komm B, Lyttle CR, Katzenellenbogen BS 2004 Transcriptional profiling of estrogen-regulated gene expression via estrogen receptor (ER) {alpha} or ER ß in human osteosarcoma cells: distinct and common target genes for these receptors. Endocrinology 145:3473–3486[Abstract/Free Full Text]
  14. Ali S, Coombes RC 2002 Endocrine-responsive breast cancer and strategies for combating resistance. Nat Rev Cancer 2:101–112[CrossRef][Medline]
  15. Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS 1986 Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc Natl Acad Sci USA 83:2496–2500[Abstract/Free Full Text]
  16. Dickson RB, Lippman ME 1995 Growth factors in breast cancer. Endocr Rev 16:559–589[CrossRef][Medline]
  17. Lindberg MK, Moverare S, Skrtic S, Gao H, Dahlman-Wright K, Gustafsson JA, Ohlsson C 2003 Estrogen receptor (ER)-ß reduces ER{alpha}-regulated gene transcription, supporting a "ying yang" relationship between ER{alpha} and ERß in mice. Mol Endocrinol 17:203–208[Abstract/Free Full Text]
  18. Paruthiyil S, Parmar H, Kerekatte V, Cunha GR, Firestone GL, Leitman DC 2004 Estrogen receptor ß inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest. Cancer Res 64:423–428[Abstract/Free Full Text]
  19. Strom A, Hartman J, Foster JS, Kietz S, Wimalasena J, Gustafsson J-A 2004 Estrogen receptor ß inhibits 17ß-estradiol-stimulated proliferation of the breast cancer cell line T47D. Proc Natl Acad Sci USA 101:1566–1571[Abstract/Free Full Text]
  20. 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[Abstract/Free Full Text]
  21. Wu Z, Irizarry RA, Gentleman R, Martinez-Murillo F, Spencer F 2004 A model-based background adjustment for oligonucleotide expression arrays. J Amer Stat Assoc 99:909–917[CrossRef]
  22. Tusher VG, Tibshirani R, Chu G 2001 Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98:5116–5121[Abstract/Free Full Text]
  23. Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC, Conklin BR 2002 GenMAPP, a new tool for viewing and analyzing microarray data on biological pathways. Nat Genet 31:19[CrossRef][Medline]
  24. Kanehisa M, Goto S 2000 KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28:27–30[Abstract/Free Full Text]
  25. Hosack DA, Dennis Jr G, Sherman BT, Lane HC, Lempicki RA 2003 Identifying biological themes within lists of genes with EASE. Genome Biol 4:R70
  26. Lazennec G, Alcorn JL, Katzenellenbogen BS 1999 Adenovirus-mediated delivery of a dominant negative estrogen receptor gene abrogates estrogen-stimulated gene expression and breast cancer cell proliferation. Mol Endocrinol 13:969–980[Abstract/Free Full Text]
  27. Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(–{Delta}{Delta}C(T)) method. Methods 25:402–408[CrossRef][Medline]
  28. Choi I, Ko C, Park-Sarge OK, Nie R, Hess RA, Graves C, Katzenellenbogen BS 2001 Human estrogen receptor ß-specific monoclonal antibodies: characterization and use in studies of estrogen receptor ß protein expression in reproductive tissues. Mol Cell Endocrinol 181:139–150[CrossRef][Medline]
  29. Roger P, Sahla ME, Makela S, Gustafsson JA, Baldet P, Rochefort H 2001 Decreased expression of estrogen receptor ß protein in proliferative preinvasive mammary tumors. Cancer Res 61:2537–2541[Abstract/Free Full Text]
  30. Cho H, Ng PA, Katzenellenbogen BS 1991 Differential regulation of gene expression by estrogen in estrogen growth-independent and -dependent MCF-7 human breast cancer cell sublines. Mol Endocrinol 5:1323–1330[CrossRef][Medline]
  31. Hall JM, McDonnell DP, Korach KS 2002 Allosteric regulation of estrogen receptor structure, function, and coactivator recruitment by different estrogen response elements. Mol Endocrinol 16:469–486[Abstract/Free Full Text]
  32. McInerney EM, Weis KE, Sun J, Mosselman S, Katzenellenbogen BS 1998 Transcription activation by the human estrogen receptor subtype ß (ERß) studied with ERß and ER{alpha} receptor chimeras. Endocrinology 139:4513–4522[Abstract/Free Full Text]
  33. Harrington WR, Sheng S, Barnett DH, Petz LN, Katzenellenbogen JA, Katzenellenbogen BS 2003 Activities of estrogen receptor {alpha}- and ß-selective ligands at diverse estrogen responsive gene sites mediating transactivation or transrepression. Mol Cell Endocrinol 206:13–22[CrossRef][Medline]
  34. Kraichely DM, Sun J, Katzenellenbogen JA, Katzenellenbogen BS 2000 Conformational changes and coactivator recruitment by novel ligands for estrogen receptor-{alpha} and estrogen receptor-ß: correlations with biological character and distinct differences among SRC coactivator family members. Endocrinology 141:3534–3545[Abstract/Free Full Text]
  35. Sun J, Meyers MJ, Fink BE, Rajendran R, Katzenellenbogen JA, Katzenellenbogen BS 1999 Novel ligands that function as selective estrogens or antiestrogens for estrogen receptor-{alpha} or estrogen receptor-ß. Endocrinology 140:800–804[Abstract/Free Full Text]
  36. Stauffer SR, Coletta CJ, Tedesco R, Nishiguchi G, Carlson K, Sun J, Katzenellenbogen BS, Katzenellenbogen JA 2000 Pyrazole ligands: structure-affinity/activity relationships and estrogen receptor-{alpha}-selective agonists. J Med Chem 43:4934–4947[CrossRef][Medline]
  37. Kang Y, Siegel PM, Shu W, Drobnjak M, Kakonen SM, Cordon-Cardo C, Guise TA, Massague J 2003 A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 3:537–549[CrossRef][Medline]
  38. Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, Viale A, Olshen AB, Gerald WL, Massague J 2005 Genes that mediate breast cancer metastasis to lung. Nature 436:518–524[CrossRef][Medline]
  39. Stroschein SL, Wang W, Zhou S, Zhou Q, Luo K 1999 Negative feedback regulation of TGF-ß signaling by the SnoN oncoprotein. Science 286:771–774[Abstract/Free Full Text]
  40. Johnsen SA, Subramaniam M, Katagiri T, Janknecht R, Spelsberg TC 2002 Transcriptional regulation of Smad2 is required for enhancement of TGFß/Smad signaling by TGFß inducible early gene. J Cell Biochem 87:233–241[CrossRef][Medline]
  41. Melhuish TA, Gallo CM, Wotton D 2001 TGIF2 interacts with histone deacetylase 1 and represses transcription. J Biol Chem 276:32109–32114[Abstract/Free Full Text]
  42. Kolodkin AL, Levengood DV, Rowe EG, Tai YT, Giger RJ, Ginty DD 1997 Neuropilin is a semaphorin III receptor. Cell 90:753–762[CrossRef][Medline]
  43. Tamagnone L, Artigiani S, Chen H, He Z, Ming GI, Song H, Chedotal A, Winberg ML, Goodman CS, Poo M, Tessier-Lavigne M, Comoglio PM 1999 Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. Cell 99:71–80[CrossRef][Medline]
  44. Castro-Rivera E, Ran S, Thorpe P, Minna JD 2004 Semaphorin 3B (SEMA3B) induces apoptosis in lung and breast cancer, whereas VEGF165 antagonizes this effect. Proc Natl Acad Sci USA 101:11432–11437[Abstract/Free Full Text]
  45. Ochi K, Mori T, Toyama Y, Nakamura Y, Arakawa H 2002 Identification of semaphorin3B as a direct target of p53. Neoplasia 4:82–87[CrossRef][Medline]
  46. Terman JR, Mao T, Pasterkamp RJ, Yu HH, Kolodkin AL 2002 MICALs, a family of conserved flavoprotein oxidoreductases, function in plexin-mediated axonal repulsion. Cell 109:887–900[CrossRef][Medline]
  47. Hall JM, Korach KS 2003 Stromal cell-derived factor 1, a novel target of estrogen receptor action, mediates the mitogenic effects of estradiol in ovarian and breast cancer cells. Mol Endocrinol 17:792–803[Abstract/Free Full Text]
  48. Chalasani SH, Sabelko KA, Sunshine MJ, Littman DR, Raper JA 2003 A chemokine, SDF-1, reduces the effectiveness of multiple axonal repellents and is required for normal axon pathfinding. J Neurosci 23:1360–1371[Abstract/Free Full Text]
  49. Brünner N, Frandsen TL, Holst-Hansen C, Bei M, Thompson EW, Wakeling AE, Lippman ME, Clarke R 1993 MCF7/LCC2: a 4-hydroxytamoxifen resistant human breast cancer variant that retains sensitivity to the steroidal antiestrogen ICI 182,780. Cancer Res 53:3229–3232[Abstract/Free Full Text]
  50. Herman ME, Katzenellenbogen BS 1996 Response-specific antiestrogen resistance in a newly characterized MCF-7 human breast cancer cell line resulting from long-term exposure to trans-hydroxytamoxifen. J Steroid Biochem Mol Biol 59:121–134[CrossRef][Medline]
  51. Paez-Pereda M, Giacomini D, Refojo D, Nagashima AC, Hopfner U, Grubler Y, Chervin A, Goldberg V, Goya R, Hentges ST, Low MJ, Holsboer F, Stalla GK, Arzt E 2003 Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk. Proc Natl Acad Sci USA 100:1034–1039[Abstract/Free Full Text]
  52. Yamamoto T, Saatcioglu F, Matsuda T 2002 Cross-talk between bone morphogenic proteins and estrogen receptor signaling. Endocrinology 143:2635–2642[Abstract/Free Full Text]
  53. Munger JS, Huang X, Kawakatsu H, Griffiths MJ, Dalton SL, Wu J, Pittet JF, Kaminski N, Garat C, Matthay MA, Rifkin DB, Sheppard D 1999 The integrin {alpha}vß6 binds and activates latent TGFß1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96:319–328[CrossRef][Medline]
  54. Yee KO, Streit M, Hawighorst T, Detmar M, Lawler J 2004 Expression of the type-1 repeats of thrombospondin-1 inhibits tumor growth through activation of transforming growth factor-ß. Am J Pathol 165:541–552[Abstract/Free Full Text]
  55. Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F, Kalluri R 2003 BMP-7 counteracts TGF-ß1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med 9:964–968[CrossRef][Medline]
  56. Chedotal A, Kerjan G, Moreau-Fauvarque C 2005 The brain within the tumor: new roles for axon guidance molecules in cancers. Cell Death Differ 12:1044–1056[CrossRef][Medline]
  57. Serini G, Valdembri D, Zanivan S, Morterra G, Burkhardt C, Caccavari F, Zammataro L, Primo L, Tamagnone L, Logan M, Tessier-Lavigne M, Taniguchi M, Puschel AW, Bussolino F 2003 Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function. Nature 424:391–397[CrossRef][Medline]
  58. Speirs V, Carder PJ, Lane S, Dodwell D, Lansdown MR, Hanby AM 2004 Oestrogen receptor ß: what it means for patients with breast cancer. Lancet Oncol 5:174–181[CrossRef][Medline]
  59. Tremblay A, Tremblay GB, Labrie F, Giguere V 1999 Ligand-independent recruitment of SRC-1 to estrogen receptor ß through phosphorylation of activation function AF-1. Mol Cell 3:513–519[CrossRef][Medline]
  60. Heldring N, Nilsson M, Buehrer B, Treuter E, Gustafsson JA 2004 Identification of tamoxifen-induced coregulator interaction surfaces within the ligand-binding domain of estrogen receptors. Mol Cell Biol 24:3445–3459[Abstract/Free Full Text]
  61. Laoukili J, Kooistra MR, Bras A, Kauw J, Kerkhoven RM, Morrison A, Clevers H, Medema RH 2005 FoxM1 is required for execution of the mitotic programme and chromosome stability. Nat Cell Biol 7:126–136[CrossRef][Medline]
  62. Wang X, Kiyokawa H, Dennewitz MB, Costa RH 2002 The forkhead box m1b transcription factor is essential for hepatocyte DNA replication and mitosis during mouse liver regeneration. Proc Natl Acad Sci USA 99:16881–16886[Abstract/Free Full Text]
  63. Oh DS, Troester MA, Usary J, Hu Z, He X, Fan C, Wu J, Carey LA, Perou CM 2006 Estrogen-regulated genes predict survival in hormone receptor–positive breast cancers. J Clin Oncol 24:1656–1664[Abstract/Free Full Text]
  64. Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, Baehner FL, Walker MG, Watson D, Park T, Hiller W, Fisher ER, Wickerham DL, Bryant J, Wolmark N 2004 A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 351:2817–2826[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
S. A. Ochsner, D. L. Steffen, S. G. Hilsenbeck, E. S. Chen, C. Watkins, and N. J. McKenna
GEMS (Gene Expression Metasignatures), a Web Resource for Querying Meta-analysis of Expression Microarray Datasets: 17{beta}-Estradiol in MCF-7 Cells
Cancer Res., January 1, 2009; 69(1): 23 - 26.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Chen, I. Hsu, A. Wolfe, S. Radovick, K. Huang, S. Yu, C. Chang, E. M. Messing, and S. Yeh
Defe