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[Cancer Research 63, 3546-3555, July 1, 2003]
© 2003 American Association for Cancer Research

Endocrinology

Induction of Aromatase (CYP19) Expression in Breast Cancer Cells through a Nongenomic Action of Estrogen Receptor {alpha}1

Yoshiyuki Kinoshita and Shiuan Chen2

Department of Surgical Research, Beckman Research Institute of the City of Hope, Duarte, California 91010


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aromatase plays a critical role in breast cancer development by converting androgen to estrogen. In this report, results are presented to demonstrate that estrogen, the product of aromatase, can up-regulate its expression. Estrogen receptor (ER) transient transfection experiments were performed using the SK-BR-3 breast cancer cell line, which is ER negative and expresses aromatase. When SK-BR-3 cells were transfected with the expression plasmid pCI-ER{alpha}, but not pCI-ERß, aromatase activity was elevated by 17ß-estradiol (E2) in a dose-dependent manner. The E2 induction could be enhanced by cotransfection with the coactivator GRIP1 and suppressed by antiestrogens such as tamoxifen and ICI 182,780. The aromatase activity in the ER{alpha}-transfected SK-BR-3 cells could also be induced by environmental chemicals that were known to have an estrogen-like activity. Using aromatase gene exon Is-specific reverse transcription-PCR, the level of promoter I.1-driven transcripts was found to be elevated in E2-treated ER{alpha}-transfected cells. This suggested that E2 induced aromatase expression through the up-regulation of promoter I.1. Using DNA deletion analysis of the 5'-flanking region of promoter I.1, the section between -300 and -280 bp upstream from exon I.1 was identified to be important for mediating E2 induction. However, a direct binding of ER{alpha} to this 20-bp region could not be demonstrated. It was found that E2 induction could be suppressed by the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor, PD98059, and the epidermal growth factor receptor tyrosine kinase inhibitor, PD153035 hydrochloride. A significant induction of aromatase expression was also detected in ER-positive MCF-7 breast cancer cells after transfection with pCI-ER{alpha} and E2 treatment. Furthermore, after ER{alpha} transfection and E2 treatment, the aromatase activity in Her-2-overexpressing MCF-7 cells was drastically higher than that of the wild-type MCF-7 cells. In addition, aromatase induction in MCF-7 cells could also be suppressed by PD153035 hydrochloride. These results suggest that E2 up-regulates aromatase expression by a nongenomic action of ER{alpha} via cross-talk with growth factor-mediated pathways.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogens play an important role in breast cancer development. Approximately 60% of premenopausal and 75% of postmenopausal patients have estrogen-dependent carcinomas. Aromatase, a cytochrome P450, is the enzyme synthesizing estrogens by converting C19 androgens to aromatic C18 estrogenic steroids. The expression of aromatase in breast cancer tissue has been demonstrated by enzyme activity measurement (1 , 2) , immunocytochemistry (3, 4, 5, 6) , and RT-PCR3 analysis (7, 8, 9) . Cell culture (10 , 11) and nude mouse experiments (12) using aromatase-transfected MCF-7 cells demonstrate, in a direct fashion, that aromatase expressed in breast cancer cells can play a role in stimulating the growth of tumors in both an autocrine and a paracrine manner. In addition, Tekmal et al. (13) have demonstrated that overexpression of aromatase in mammary glands of virgin females mice led to the enlargement of 40% of the ducts, of which 65% had hyperplastic lesions, 20% had dysplastic lesions, and 15% had fibroadenoma lesions. Overexpression of aromatase in involuted mammary glands of transgenic females induces hyperplasia in 75–80% of ducts and glands that exhibit a range of morphological abnormalities. Results from aromatase transgenic mouse studies show that early exposure of mammary epithelium to in situ estrogen and continued exposure to in situ estrogen appear to predispose mammary tissue to preneoplastic changes, which may, in turn, increase the risk of developing neoplasia and increase susceptibility to environmental carcinogens. The results further indicate that in situ-produced estrogen plays a more important role than circulating estradiol in breast tumor promotion.

During the last several years, this laboratory and other laboratories have found that nuclear receptors play important roles in modulating aromatase expression in human breast tissue. The expression of aromatase in adipose tissue was found to be stimulated by glucocorticoids (14) , and aromatase mRNA in adipose tissue was found to contain mainly exon I.4. Characterization of the region upstream of exon I.4 revealed the existence of a TATA-less promoter and an upstream GRE sequence that interacted with the glucocorticoid receptor (15) . A regulatory element (S1) is situated between the two promoters (I.3 and II), and the trans-factors that interact with the element have been found to be mainly nuclear receptors (16 , 17) . S1 behaves as an enhancer when ERR{alpha}-1 binds and as a repressor when COUP-TF, EAR-2 or RAR{gamma} binds (17 , 18) . The function of S1 depends on the expression levels of these nuclear receptors in cells. Because EAR-2, COUP-TFI, and RAR{gamma} are expressed at high levels, it is likely that S1 is a negative regulatory element that suppresses aromatase promoters I.3 and II in normal breast tissue. In cancer tissue, S1 may function as a positive element because ERR{alpha}-1 is expressed, whereas EAR-2 and RAR{gamma} are only present in a small number of tumor specimens (18) . A similar mechanism has been suggested to take place in preadipocytes. Liver receptor homologue-1, an orphan receptor, has been shown to bind to S1, leading to an increase of the promoter II activity (19) . When the cultured human preadipocytes were differentiated into mature adipocytes, a time-dependent induction of peroxisome proliferator-activated receptor {gamma} and a rapid loss of the expression of liver receptor homologue 1 and aromatase were observed (19) . Peroxisome proliferator-activated receptor {gamma} has been shown to down-regulate the activity of promoter II, but the mechanism has not yet been determined (20) . In addition, in choriocarcinoma cells (21) as well as in MCF-7 breast cancer cells (22) , another nuclear receptor, RAR, up-regulated aromatase activity by binding to the imperfect palindromic sequence upstream of exon I.1 of the aromatase gene.

E2 has been shown to modulate aromatase expression in other vertebrates (e.g., Ref. 23 ). The effects of estrogens are mainly mediated by their interaction with the ER (24) . Besides ER{alpha}, the classical ER, a novel ER (i.e., ERß) that is highly homologous to ER{alpha} has recently been cloned (25) . To determine whether E2/ER can modulate aromatase expression in breast cancer cells, we have initiated an ER transfection study in SK-BR-3 breast cancer cells, which are ER negative and express aromatase. The study was extended to another breast cancer cell line, MCF-7. Our results indicate that ER{alpha} transfection and E2 treatment greatly enhance the aromatase expression in these cell lines through a nongenomic fashion and cross-talk with growth factor-mediated pathways.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
E2 and TAM were purchased from Sigma Chemical Co. (St. Louis, MO). ICI 182,780 was purchased from TOCRIS (Ballwin, MO). [1ß-3H]Androst-4-ene-3,17-dione was purchased from New England Nuclear (Boston, MA). SK-BR-3 and MCF-7 breast cancer cells were from American Type Culture Collection (Manassas, VA). Her-2-overexpressing MCF-7 cells were kindly provided by Dr. Dihua Yu (The University of Texas M. D. Anderson Cancer Center, Houston, TX). SK-BR-3 cells were maintained in McCoy’s 5A medium containing 15% FBS and glutamine at 37°C and 5% CO2. MCF-7 cells were maintained in Eagle’s MEM with nonessential amino acid, sodium pyruvate, and 10% FBS. To eliminate the influence of steroid hormones in the medium, for the hormone induction experiments, both types of cells were switched to phenol red-free Eagle’s MEM with nonessential amino acid, sodium pyruvate, and 5% charcoal-dextran-treated FBS. Lipofectin, G418, and TRIzol Reagent were purchased from Life Technologies, Inc. (Grand Island, NY). Oligonucleotide primers were synthesized in the DNA/RNA synthesis laboratory at the City of Hope.

Plasmid Preparation.
The genomic DNA from SK-BR-3 cells was prepared using TRIzol Reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. The promoter I.1 constructs were prepared by either PCR or restriction enzyme digestion. The pGL-3 basic reporter vector (pGL3-Basic; Promega, Madison, WI), which is promoterless and enhancerless, served as a negative control. The genomic fragments (-1693/+85 bp, -840/+85 bp, -300/+85 bp, -280/+85 bp, -260/+85 bp, -240/+85 bp, -225/+85 bp, -210/+85 bp, and -190/+85 bp) were subcloned into the pGL-3 basic reporter vector inserted between the KpnI and XhoI sites. All constructs were sequenced to confirm their authenticity. The construct of three tandem copies of ERE was subcloned into the pGL3 promoter vector (Promega), which contains SV40 promoter. The pSV-ß-galactosidase control vector (pSV-ßGal; Promega) was cotransfected as an internal standard for transfection efficiency. The human ER{alpha} expression vector, pCI-ER{alpha}, is a gift from Dr. Richard Pestell (Albert Einstein College of Medicine, Bronx, NY). The pCI-ERß was generated in the following manner. Human ERß 530 cDNA was amplified by PCR from the mRNA of MCF-7 breast cancer cells and subcloned into the XhoI/NotI sites of pCI-neo vector. The pSG-GRIP1 was kindly provided by Dr. M. R. Stallcup (University of Southern California, Los Angeles, CA; Ref. 26 ).

Aromatase Assay.
Aromatase activity was determined using the [3H]H2O release method that was reported by Zhou et al. (27) . In the "In-cell" aromatase assay, the cells grown in 6-well cell culture plates were washed twice with PBS. One ml of serum-free medium containing 100 nM [1ß-3H]androst-4-ene-3,17-dione as substrate (specific activity, 27.5 Ci/mmol) as well as 500 nM progesterone was then added to each well. After a 3-h incubation at 37°C, the reaction mixture was removed and extracted with an equal volume of chloroform to extract unused substrate and further treated with dextran-treated charcoal. After centrifugation, supernatant containing the product, [3H]H2O, was counted in a liquid scintillation counter. The protein concentration was determined after dissolving cells in 0.5 M NaOH by the method of Bradford (28) . Aromatase activity was calculated as pmol/mg protein/h.

RNA Isolation and Semiquantitative RT-PCR Analysis of Aromatase Transcripts.
The transfection experiments were performed 24 h after seeding approximately 5 x 105 cells/25-cm2 tissue culture flask using 1.3 µg of expression vector and 10.4 µl of Lipofectin. After a 5-h incubation, medium containing Lipofectin and DNA were removed, and the cells were cultured in phenol red-free Eagle’s MEM with 5% charcoal-dextran-treated FBS. After a 24-h incubation, the cells were used for total cellular RNA isolation. Total RNA was prepared using TRIzol Reagent according to the manufacturer’s instructions. Integrity of the RNA was assessed by electrophoresis in ethidium bromide-stained 1.2% agarose-Tris-borate EDTA gels. The absorbance ratio A260 nm/A280 nm was greater than 1.8.

Exon I primer-specific RT-PCR analyses were performed to examine the alternative utilization of exons I of the human aromatase gene as described previously (29) . Briefly, RT-PCR was performed using a reverse primer derived from exon II and forward primers in exons I.1, I.3, I.4, I.5, I.6, PII, and II for the amplification of exons I.1, I.3, I.4, I.5, I.6, PII, and II, respectively. Reverse transcription was performed in a final volume of 40 µl. Four µg of total RNA were denatured at 70°C for 10 min and reverse transcribed in the presence of 1.0 mM each of dGTP, dATP, dTTP, and dCTP; 40 units of avian myeloblastosis virus reverse transcriptase (Life Sciences Inc.); 100 ng of random primer (Life Technologies, Inc.); 1 unit/µl RNase inhibitor; and reaction buffer supplied with the reverse transcriptase [50 mM Tris-HCl (pH 8.3), 100 mM KCl, 4 mM DTT, and 10 mM MgCl2] for 60 min at 42°C, followed by 10 min at 70°C for heat inactivation. The 2.0 µl of cDNA were subjected to PCR amplification in a 25-µl reaction containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 0.5 µM of each primer, and 1.0 unit of AmpliTaq (Perkin-Elmer Cetus, Norwalk, CT). PCR was performed for 29 cycles (exons I.6, PII, and II) or 32 cycles (exons I.1, I.3, I.4, and I.5) for semiquantitative analysis using the following temperature profile: 54°C, 1 min (annealing); 72°C, 1 min (extension); and 94°C, 1 min (denaturation). An additional extension cycle was performed for 10 min at 72°C before cooling the reaction mixture to 4°C. Because all aromatase mRNA contains exon II, regardless of which exon I is present, PCR for each exon I was performed with a unique exon I forward primer and the exon II reverse primer. As a control, we performed PCR using exon II forward and reverse primers to amplify the exon II region. Furthermore, we performed PCR using a set of human ß-actin-specific primers [ß-actin 1, 5'-AGGAGCACCCCGTGCTGCTGA-3' (forward); and ß-actin 2, 5'-CTAGAAGCATTTGCGGTGGAC-3' (reverse)] to amplify the human ß-actin gene, which served as an internal control to normalize aromatase mRNA expression under each condition. Each PCR product was electrophoresed on a 1.2% agarose gel and stained with ethidium bromide. For quantification, their integrated density was analyzed on the pictures using GS-710 Calibrated Imaging Densitometer (Bio-Rad).

Transient Transfection and Reporter Gene Assay.
The transfection experiments were performed 24 h after seeding approximately 2 x 105 cells/35-mm tissue culture dish using 0.5 µg of expression vector and 4 µl of Lipofectin according to the manufacturer’s instructions. After a 5-h incubation, medium containing Lipofectin and DNA was removed, and the cells were cultured in phenol red-free Eagle’s MEM with 5% charcoal-dextran-treated FBS. After a 48-h incubation following the transfection, the cells were used for aromatase assay. To determine the region that is responsible to ER{alpha}/E2 induction, the DNA deletion and cotransfection experiments were performed using 0.1 µg of ER{alpha} expression vector, 0.25 µg of luciferase reporter vectors (containing DNA fragments derived from promoter I.1 region of the human aromatase gene, as described in "Plasmid Preparation"), 0.5 µg of pSV-ßGal vector, and 4 µl of Lipofectin. The transfected cells were maintained for 48 h and then lysed in Reporter Lysis Buffer (Promega) according to the manufacturer’s instructions. Aliquots of the lysate were used for assay of ß-galactosidase activity. Luciferase activity was measured using the luciferase assay system (Promega), read in a TD-20/20 Luminometer (Tuener Designs, Sunnyvale, CA), and expressed in relative light units. The relative luciferase activity was calculated by dividing the light unit of luciferase activity by the ß-galactosidase activity. Each set of experiments was repeated at least three times.

DNA Mobility Shift Analysis.
The double-stranded estrogen response site on promoter I.1 of the human aromatase gene containing oligonucleotide -305/-275 bp, 5'-TGTAGAGGTGCTTTAGGCCTCAGGAAACAG-3', was end-labeled with [{gamma}-32P]ATP and T4 kinase and used as a probe in the mobility shift assay. ER{alpha} protein was synthesized in vitro using the TNT-coupled reticulocyte lysate system (Promega) with T7-RNA polymerase, according to the manufacturer’s instructions, using 1 µg of pCI-ER{alpha} expression vector in 50 µl of total volume and an incubation time of 60 min at 30°C. As a negative control, protein generated using pCI empty vector was synthesized. Nuclear extractions from SK-BR-3 cells were prepared according to Zhou et al., as described previously (30) . Mobility shift assays were done as described by Singh et al. (31) . Briefly, 5 µl of in vitro-synthesized ER{alpha} protein or 5 µg of nuclear extract from SK-BR-3 cells were incubated on ice for 60 min in 30 µl of a mixture containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 4% (v/v) glycerol, 0.75 mg/ml BSA, and 1 µg of poly(deoxyinosinic-deoxycytidylic acid), with or without 100 nM E2 or 600 ng of D-12 ER antibody (Santa Cruz Biotechnology, Inc.), and then incubated with 10,000 cpm of 32P-labeled probe on ice for an additional 60 min. The reaction mixture was electrophoresed on 4% acrylamide:bis-acrylamide (74:1) gel with 0.5x Tris-borate EDTA at 10 V/cm. Gels were dried and autoradiographed.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Kinetic Analysis of Aromatase in SK-BR-3 Cells.
Aromatase activity in SK-BR-3 cells was measured by the "in-cell" [3H]H2O release method. The Km and Vmax values were determined to be 34.2 nM and 0.16 pmol/mg/h, respectively (Fig. 1)Citation . The Km value was very similar to that of the Chinese hamster ovary cells expressing human placental aromatase, i.e., 32 nM (27) . The SK-BR-3 cell line has a significantly higher aromatase activity than other breast cancer cell lines. For example, the aromatase activity in the MCF-7 cell line is approximately one-twentieth that in the SK-BR-3 cell line.



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  		Fig. 1. Kinetic analysis of aromatase activity in SK-BR-3 cells using the "in-cell" method. The assay was performed in triplicate, and the results are shown with SE.

 
Increase of Aromatase Activity in E2-treated and ER{alpha} Transiently Transfected SK-BR-3 Cells.
To evaluate the effect of estrogen on aromatase activity, the SK-BR-3 cell line was selected for this study because this cell line had a higher aromatase activity/expression than other breast cancer cell lines. The transfection experiments were performed using 0.5 µg of each expression vector. The cells were incubated for 24 h in media containing 5% charcoal-dextran-treated serum and different concentrations of E2 after transfection, and then the cells were rinsed twice with PBS and assayed for aromatase activity. The aromatase activity in SK-BR-3 cells was elevated in a dose-dependent manner with E2 when the cells were transfected with expression plasmid pCI-ER{alpha} but not pCI-ERß (Fig. 2A)Citation . The activity in ER{alpha} transiently transfected cells treated with 100 nM E2 was approximately 4 times that of the control (DMSO vehicle). The observed increase in aromatase activity induced by E2 could be suppressed by ER antagonists, TAM and ICI 182,780, at a concentration of 1 µM (Fig. 2B)Citation . The GRIP1, a nuclear receptor coactivator, enhanced E2-induced aromatase activity in ER{alpha}-transfected SK-BR-3 cells (Fig. 3)Citation . These results indicate that estrogen-induced aromatase activity was depended on ER{alpha} in SK-BR-3 cells. Aromatase converts androgen to estrogen. The aromatase activity in SK-BR-3 cells was also elevated by testosterone in a dose-dependent manner when the cells were transfected with the expression plasmid pCI-ER{alpha} (Fig. 4)Citation . The observed increase in aromatase activity induced by testosterone was suppressed by 1 µM TAM and 4-OHA (Fig. 4)Citation . 4-OHA is an aromatase inhibitor that inhibits the conversion of testosterone to E2. These results indicate that by binding to ER{alpha}, estrogen, the product of aromatase, can increase aromatase activity/expression in SK-BR-3 cells via a feed-forward manner.



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  		Fig. 2. A, dose-response studies of E2 on aromatase activity in ER{alpha} or ERß transient transfected SK-BR-3 cells. The SK-BR-3 cells were transfected with 0.5 µg of pCI-vector (vehicle), pCI-ER{alpha}, or pCI-ERß and then treated with increasing amounts of E2. After a 24-h incubation, aromatase activity was measured by [3H]H2O release assay. Values represent the mean ± SE from triplicate assays. B, effect of estrogen and antiestrogen on aromatase activity in ER{alpha} transient transfected SK-BR-3 cells. The SK-BR-3 cells were transfected with 0.5 µg of pCI-vector or pCI-ER{alpha}. Transfected cells were treated with 100 nM E2 and/or 1 µM TAM or ICI 182780 (ICI). After a 24-h incubation, aromatase activity was measured by [3H]H2O release assay. Values represent the mean ± SE from triplicate assays.

 


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  		Fig. 3. Enhancement of E2-induced aromatase activity by GRIP1 in ER{alpha} transient transfected SK-BR-3 cells. The SK-BR-3 cells were transfected with 0.5 µg of pCI-vector or pCI-ER{alpha}. The cells were also transfected with 1.0 µg of pSG5 vector or pSG5-GRIP1. Transfected cells were treated with 100 nM E2. After a 24-h incubation, aromatase activity was measured by [3H]H2O release assay. Values represent the mean ± SE from triplicate assays.

 


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  		Fig. 4. Inductive effects of testosterone on the aromatase activity in ER{alpha} transient transfected SK-BR-3 cells. The SK-BR-3 cells were transfected with 0.5 µg of pCI-vector or pCI-ER{alpha}. The transfected cells were treated with increasing amounts of testosterone or in combination with 100 nM testosterone and 1 µM 4-OHA or TAM. After a 24-h incubation, aromatase activity was measured by [3H]H2O release assay. Values represent the mean ± SE from triplicate assays.

 
Evaluation of the Induction Mechanism of E2 on Aromatase Expression in SK-BR-3 Cells.
To determine whether the induction of aromatase activity by E2 in SK-BR-3 cells was due to an up-regulation of aromatase expression at the transcriptional level and to better understand the ER-mediated regulatory mechanism of aromatase expression, we performed exon I-specific RT-PCR to examine exon I/promoter usage in ER{alpha}-transfected SK-BR-3 cells. Exons I.1, I.3, I.6, and PII have been shown as the major exons I in aromatase transcripts in SK-BR-3 cells (9) . ER{alpha} transfection in SK-BR-3 and treatment with 100 nM E2 increased the level of exon I.1-containing transcript, as indicated by the increases in the levels of both exons I.1 and II RT-PCR products (Fig. 5)Citation . These results suggest that estrogen induces aromatase expression through the up-regulation of promoter I.1 in ER{alpha}-transfected SK-BR-3 cells.



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  		Fig. 5. Exon I-specific RT-PCR analysis of ER{alpha}-transfected SK-BR-3 cells. The SK-BR-3 cells were transfected with 0.5 µg of pCI vector or pCI-ER{alpha} and then treated with 100 nM E2. The cells were incubated for 48 h, and then total RNA was isolated. Four µg of total RNA were used for RT-PCR analysis as described in "Materials and Methods." The ß-actin transcript was amplified as an internal control. The size of the detected PCR products for exons I.1, I.6, I.3, PII, II, and ß-actin are 256, 1037, 333, 234, 169, and 825 bp, respectively.

 
To evaluate how E2 regulates promoter I.1 and to localize sequences of promoter I.1 responsible for the induction of E2 in ER{alpha}-transfected SK-BR-3 cells, a series of 5'-deletion constructs were created in the 5'-flanking region of promoter I.1. These constructs were cotransfected into SK-BR-3 cells with expression plasmid pCI-vector, pCI-ER{alpha} vector, and pSV-ßGal vector in the presence or absence of 100 nM E2. After treatment with 100 nM E2, the -1683/+85-, -840/+85-, and -300/+85-bp constructs showed an increase of transcriptional activity in ER{alpha}-transfected cells (Fig. 6)Citation . However, estrogen induction of luciferase activity was much lower in the cells that were transfected with the -280/+85-bp construct. This result suggests that the sequence between -300 and -280 bp upstream of the transcriptional start site of exon I.1 is required for the response of promoter I.1 to estrogen in ER{alpha} transiently transfected SK-BR-3 cells. In agreement with the results from the aromatase activity measurements, E2 could not induce the transcriptional activity of promoter I.1 in ERß-transfected cells.



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  		Fig. 6. Mapping of E2-responsive element within promoter I.1 region of the human aromatase gene by DNA deletion analysis. A series of the reporter constructs containing promoter I.1 region of the human aromatase gene (0.25 µg) and pSV-ßGal (0.5 µg) were transiently transfected into SK-BR-3 cells together with 0.1 µg of pCI vector or pCI-ER{alpha}. After cells were incubated for 24 h in the absence or presence of 100 nM E2, the luciferase activity was measured. The relative luciferase activity was calculated by dividing the light unit of luciferase activity by the ß-galactosidase activity.

 
To confirm the interaction of ER{alpha} with the region between -300 and -280 bp, DNA mobility shift and antibody supershift experiments were carried out using in vitro-synthesized ER{alpha} protein and double-strand oligonucleotide -305/-275 bp, 5'-TGTAGAGGTGCTTTAGGCCTCAGGAAACAG-3'. Whereas proteins in the nuclear extract from SK-BR-3 cells bound to the -305/-275-bp probe, ER{alpha} binding could not be demonstrated (data not shown). These results suggest that promoter I.1-driven E2-induced aromatase gene expression is not due to a direct interaction of ER{alpha} with the -305/-275-bp region.

ER{alpha}-mediated Induction Pathway on Aromatase Expression Cross-talks to EGF/MAPK Pathways.
SK-BR-3 cells express high levels of Her-2. This protein is a member of the EGF receptor family that has been shown not to require ligand for its activation. In addition, the ER signaling pathway is known to cross-talk with several growth factor pathways (e.g., Ref. 32 ). To understand the role of the ER{alpha} signaling pathway on aromatase expression, the following inhibitors were tested for their effects on aromatase expression: EGF receptor tyrosine kinase inhibitor (PD153035 hydrochloride; Ref. 33 ); platelet-derived growth factor receptor kinase inhibitor (AG1296; Ref. 34 ); insulin-like growth factor I receptor kinase inhibitor (AG538; Ref. 35 ); phosphatidylinositol 3-kinase (wortmannin; Ref. 36 ); mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor (PD98059; Ref. 37 ); and the Src family tyrosine kinase inhibitor (PP2; Ref. 38 ). The transfection experiments were performed using pCI vector or pCI-ER{alpha} expression vector. After a 5-h incubation, the medium was changed to phenol red-free Eagle’s MEM with 5% charcoal-dextran-treated FBS. Cells were treated with these inhibitors for 30 min before additional treatment with 100 nM E2. After a 24-h incubation, the cells were assayed for aromatase activity. It was found that E2 induction on aromatase activity could be suppressed by 1 µM PD153035 hydrochloride or 10 µM PD98059 (Fig. 7)Citation . The other inhibitors could not suppress E2-induced aromatase activity (data not shown). These results suggest that E2 could up-regulate aromatase expression by a nongenomic action of ER{alpha} by means of cross-talk with EGF/Ras-mediated pathways.



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  		Fig. 7. Suppression of the ER{alpha}-mediated induction of aromatase expression in SK-BR-3 cells by PD153035 and PD98059. The SK-BR-3 cells were transfected with 0.5 µg of pCI vector or pCI-ER{alpha}. After a 5-h incubation, the medium was changed to phenol red-free Eagle’s MEM containing 5% charcoal-dextran-treated FBS. Cells were treated with (A) 1 µM PD153035 hydrochloride (PD153035) or 10 µM PD98059, (B) increasing amount of PD153035 hydrochloride, and (C) increasing amount of PD98059 for 30 min before the addition of 100 nM E2. After a 24-h incubation, the cells were assayed for aromatase activity. Values represent the mean ± SE from triplicate assays.

 
The induction was significantly reduced when cells were treated in a serum-free condition rather than in 10% FBS (Fig. 8A)Citation . In addition, EGF addition was found to increase aromatase activity in ER{alpha}-transfected SK-BR-3 cells (Fig. 8B)Citation . These results also support a cross-talk mechanism between ER{alpha} and EGF/Ras-mediated pathways. In contrast to aromatase induction, the E2-induced ERE-mediated reporter activity in ER{alpha}-transfected SK-BR-3 cells was not inhibited by PD153035 and PD98059 (data not shown), confirming that the ER{alpha}-mediated aromatase induction did not follow the typical "genomic" mechanism.



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  		Fig. 8. A, effect of FBS on E2-induced aromatase activity in ER{alpha}-transfected SK-BR-3 cells. The SK-BR-3 cells were transfected with 0.5 µg of pCI vector or pCI-ER{alpha}. After a 5-h incubation, the medium was changed to phenol red-free Eagle’s MEM with different amounts of FBS. Cells were treated with or without 100 nM E2. After a 24-h incubation, the cells were assayed for aromatase activity. Values represent the mean ± SE from triplicate assays. B, the enhancement of E2-induced aromatase activity in ER{alpha}-transfected SK-BR-3 cells by EGF. The SK-BR-3 cells were transfected with 0.5 µg of pCI vector or pCI-ER{alpha}. After a 5-h incubation, the medium was changed to phenol red-free Eagle’s MEM containing 5% charcoal-dextran-treated FBS. Cells were then treated with 100 ng/ml EGF with or without 0.1 nM E2. After a 24-h incubation, the cells were assayed for aromatase activity. To better examine the effect of EGF, results are shown as the ratio of the aromatase activity in the E2-treated sample/DMSO-treated sample.

 
Induction of Aromatase Activity in MCF-7 Cells.
Whereas the aromatase gene is amplified in the MCF-7 breast cancer cell line, very low aromatase activity is detected in this cell line (39) . The suppressive mechanism of aromatase expression in MCF-7 cells is not well understood. We have found that aromatase activity was greatly enhanced after MCF-7 cells were transfected with pCI-ER{alpha} and treated with E2 (Fig. 9A)Citation . To determine whether the ER-mediated induction of aromatase in MCF-7 cells cross-talked with the EGF-mediated pathway, we compared aromatase activity in MCF-7 and Her-2-overexpressing MCF-7 cells after ER{alpha} transfection and E2 treatment. As shown in Fig. 9ACitation , E2-induced aromatase activity in Her-2-overexpressing MCF-7 cells was significantly higher than that obtained in normal MCF-7 cells. It was found that E2 induction of aromatase activity in ER{alpha} transiently transfected MCF-7 cells could also be suppressed by 1 µM PD153035 hydrochloride (Fig. 9B)Citation . These results again suggested that in MCF-7 cells, E2 could up-regulate aromatase expression via cross-talk between the ER{alpha} and the EGF-mediated pathway. It was also found that E2 induction of aromatase activity in ER{alpha}-transfected MCF-7 cells could be suppressed by 1 µM ICI 182,780. However, TAM was found to act as an agonist that induced the expression of aromatase in the transfected cells (Fig. 9B)Citation . As mentioned above, TAM acted as an antagonist in ER{alpha}-transfected SK-BR-3 cells (see Fig. 2BCitation ). The possible explanation for the observed difference in TAM action in MCF-7 cells and SK-BR-3 cells will be discussed in the "Discussion."



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  		Fig. 9. A, induction of aromatase activity in ER{alpha}-transfected MCF-7 cells and ER{alpha}-transfected Her-2-overexpressing MCF-7 cells. The MCF-7 cells and Her-2-overexpressing MCF-7 cells (Her2 MCF7 cells) were transfected with 0.5 µg of pCI vector or pCI-ER{alpha}. Transfected cells were treated with 0.1 nM E2. After a 24-h incubation, aromatase activity was measured by [3H]H2O release assay. Values represent the mean ± SE from triplicate assays. B, suppression of E2/ER{alpha}-induced aromatase expression in MCF-7 cells by PD153035. The MCF-7 cells were transfected with 0.5 µg of pCI-ER{alpha}. After a 5-h incubation, the medium was changed to phenol red-free Eagle’s MEM containing 5% charcoal-dextran-treated FBS. Cells were treated with 1 µM PD153035, 1 µM TAM, or 1 µM ICI 182780 (ICI) for 30 min before the addition of 0.1 nM E2. After a 24-h incubation, the cells were assayed for aromatase activity. Values represent the mean ± SE from triplicate assays.

 
Because MCF-7 cells are ER{alpha} positive, we have also examined the E2-induced ERE-mediated reporter activity in both vector-transfected MCF-7 cells and ER{alpha}-transfected MCF-7 cells. The ERE-mediated reporter activities in two types of transfected cells were found to be similar (data not shown), suggesting that transient transfection of the ER{alpha} expression plasmid did not significantly increase the functionally active ER{alpha} when compared with the level found in the vector-transfected cells. These results would indicate again that ER{alpha}-mediated aromatase induction in MCF-7 cells did not follow the typical "genomic" mechanism (discussed further in "Discussion").


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have found that E2 could induce aromatase expression in the ER{alpha}-overexpressing SK-BR-3 human breast cancer cell line. Our results indicate that E2 up-regulates promoter I.1 of the aromatase gene, and DNA deletion experiments have revealed that the section between -300 and -280 bp upstream from exon I.1 is important for mediating E2 induction. Several investigators have characterized the promoter I.1 region of the human aromatase gene. Toda et al. (40) identified a distal element between -2141 and -2115 bp upstream of exon I.1, which binds nuclear factor interleukin 6, a member of the CCAAT/enhancer-binding protein family, and a cell type-specific enhancer element located between -242 and -166, which consists of two subelements, element I (located between -238 and -200 bp) and element II (located between -196 and -176 bp; Ref. 41 ). Yamada et al. (42) have identified two elements within -300 bp upstream of exon I.1, which recognized the same transacting factor that binds to the trophoblast-specific element identified previously in the enhancer region of human glycoprotein hormone {alpha}-subunit gene. Sun et al. (21) described that an imperfect palindromic sequence between -183 and -172 bp upstream of exon I.1 was modulated by retinoids through retinoid X receptor and RAR. There is no classic ERE with the consensus sequence, i.e., 5'-GGTCANNNTGACC-3', in the promoter I.1 region. In addition to binding to ERE, both ER{alpha} and ERß are known to be capable of stimulating gene expression via the AP-1 site that binds members of the Jun/Fos family of transcription factors (43) . Two putative AP-1 sites were found between -70 bp and the exon I.1 start site. However, the region between -300 and -280 bp, 5'-AGGTGCTTTAGGCCTCAGGAA-3', does not contain an AP-1 site that can be recognized by ER{alpha}. Recently, other mechanisms by which the ERs modify the expression of target genes have been reported. ER{alpha} was shown to stimulate gene expression by binding to the Sp1 factor (44) . The AF-1 domain of ER{alpha} was responsible for activation at a Sp1 element (45) . The sequence between -300 and -280 bp of promoter I.1 region is GC-rich, i.e., a potential binding site for Sp1. However, this possibility is ruled out because the ER{alpha}-mediated induction of aromatase expression is enhanced by cotransfection with the coactivator GRIP1 and suppressed by antiestrogens such as TAM and ICI 182,780. Saville et al. (45) reported that TAM- and ICI 182,780 induced Sp1-mediated activity and p160 coactivators such as GRIP1 could not enhance ER{alpha}/Sp1 action. Thus, the induction of aromatase expression by ER{alpha} via the region between -300 and -280 bp is through an uncharacterized mechanism.

The action of estrogen is typically mediated through ER. After estrogen binding, ER enters the nucleus and facilitates transcription by interacting with enhancer elements such as ERE. This is now referred to as a genomic action of estrogen. Recently, the nongenomic actions of estrogen have been reported, that is, the estrogen binding to membrane or cytosol ER that cross-talks with the signal transduction pathways. In nongenomic mechanisms, estrogen activates diverse growth factor/mitogen-like signaling, including the Src/Ras/MAPK and cAMP pathway (32 , 46, 47, 48, 49, 50) . Our preliminary results from the immunocytochemical analysis showed that ER{alpha} was present in the cytosol of a significant number of ER{alpha}-transfected cells, and this could be responsible for the induction of aromatase expression by E2.4 EGF receptor tyrosine kinase inhibitor (PD153035 hydrochloride) and mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor (PD98059) were found to inhibit E2-induced aromatase activity. These results support the hypothesis that the induction of aromatase activity in SK-BR-3 and MCF-7 cells by E2 is through a nongenomic mechanism, that is, an ER{alpha} cross-talks with the EGF-MAPK pathway. Such cross-talk mechanisms in MCF-7 cells have been reported by other investigators. For example, Duan et al. (51) have demonstrated an ER-mediated activation of the serum response element in MCF-7 cells through MAPK-dependent phosphorylation of Elk-1. Song et al. (52) have reported that E2, through ER{alpha}, can activate MAPK and induce morphological changes in MCF-7 cells. SK-BR-3 cells express a high level of Her-2, a member of the EGF receptor family that is constitutively active without need of the ligand. To evaluate the role of Her-2 on aromatase expression, we examined the aromatase activity in wild-type MCF-7 cells and in Her-2-overexpressing MCF-7 cells. MCF-7 cells are ER{alpha} positive. Whereas we have found that the aromatase activity in the pCI vector-transfected cells was very low and was not induced by E2, aromatase activity in ER{alpha} transiently transfected MCF-7 cells was greatly enhanced. Furthermore, the aromatase activity in ER{alpha}-transfected Her-2-overexpressing MCF-7 cells was significantly higher than that in the ER{alpha}-transfected wild-type MCF-7 cells. These results would support a cross-talk mechanism between ER{alpha} and Her-2. In addition, our results from MCF-7 cell transfection experiments suggest that the distribution of newly expressed ER is different from that of the endogenously expressed ER, i.e., cytosolic/membrane versus nuclear distribution. Our hypothesis is supported by a recent study by Zhang et al. (53) that uses an ER{alpha} construct with a membrane localization signal. These authors reported that membrane-associated ER{alpha} is critical in E2-mediated MAPK activation. Our studies also reveal that aromatase induction in SK-BR-3 and MCF-7 cells is ER{alpha} specific. ERß could not induce aromatase expression and could not compete with ER{alpha}, supporting a nongenomic action of this ER{alpha} isoform. The exact nature of an ER{alpha}-selective inductive mechanism is not yet understood.

In a recent Phase III randomized trial, the aromatase inhibitor letrozole was found to be a more effective neoadjuvant endocrine therapy than TAM for ErbB-1- and/or ErbB-2-positive and ER-positive breast cancer (54) . This finding may be explained by the nongenomic mechanism of ER{alpha} described in this paper (see Scheme 1). In ErbB-1/ErbB-2-positive and ER-positive breast cancer cells, aromatase activity may be induced following the nongenomic mechanism. Therefore, by removing estrogen, aromatase inhibitor treatment would be an effective way to suppress the cancer growth. Ellis et al. (54) have suggested that overexpression of ErbB-1 or ErbB-2 may activate ER by phosphorylation and turn TAM into an agonist. To support this hypothesis, we have found that TAM acts as an agonist to stimulate aromatase activity in ER-transfected MCF-7 cells (Fig. 9B)Citation . However, TAM was found to act as an antagonist in ER-transfected SK-BR-3 cells (see Fig. 2BCitation ). The differential effect of TAM in the two cell lines is not yet understood but may be due to a higher level of coactivators [such as SRC-1 or AIB1 (SRC-3)] in the MCF-7 cell line than in the SK-BR-3 cell line. Glaeser et al. (55) have examined AIB1 gene amplification and expression in different breast cancer cell lines. These authors found that AIB1 gene amplification and overexpression were found in MCF-7 cells, but not in SK-BR-3 cells. Furthermore, Osborne et al. (56) have found that the failure of the antitumor activity of TAM in patients with breast cancer is determined by both the levels of AIB1 and Her-2 and the interaction between AIB1 and Her-2. For patients who received TAM therapy, high AIB1 expression was associated with worse disease-free survival, which is thought to be indicative of TAM resistance. In addition, in a recent paper by Shang and Brown (57) , the estrogen-like activity of TAM in the uterus was attributed to a high level of SRC-1 expression. Whereas our proposed mechanism can explain our results from the current study and the findings from the recent Phase III randomized trial on aromatase inhibitor and TAM, the molecular detail has yet to be determined. For example, it is not clear whether ER{alpha} interacts with members of growth factor-mediated pathways in a direct or indirect manner. In addition, the protein(s), which is phosphorylated and binds to the region between -300 and -280 bp upstream from exon I.1, has not yet been identified.



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  		Scheme. 1. The nongenomic mechanism of ER{alpha} on aromatase expression.

 
Kirma et al. (58) have found that the level of ER{alpha} is elevated in aromatase-overexpressing mammary gland. Our results indicate that overexpression of ER{alpha} can enhance the expression of aromatase. Therefore, ER{alpha} and aromatase may up-regulate each other’s expression. However, a positive correlation between ER and aromatase expression in breast cancer tissue has not yet been reported. This would suggest that a complex mechanism is involved in regulating aromatase expression in breast cancer tissue. In fact, estrogen is known to have more than one way to modulate aromatase expression. In addition to the up-regulation of aromatase expression through promoter I.1, we have also found that E2 can down-regulate the promoter I.3 through an ER-mediated mechanism (18) . Therefore, different promoter usage has an impact on the effect of estrogen on aromatase expression.

We have proposed that environmental chemicals can modulate aromatase expression (59) . A number of environmental chemicals have been shown to have estrogen-like activity. The following compounds were tested for their effects on aromatase activity in ER{alpha}-transfected SK-BR-3 cells: DES; TAM; 1,1,1-trichloro-2,2-bis(p-methoxyphenyl)ethane (methoxychlor); decachloro-ocyahydro-1,3,4-metheno-2H-cyclobuta(cd)pentalene (kepone); nonylphenol; 1-chloro-2-(2,2,2-trichloro-1-(4-chlorophenyl)ethyl)benzen(o,p'-DTT); 1,4,5,6,7,7-hexachloro-5-norbornene-2,3-dimethanol cyclic sulfite (endosulfan); 2-(2,4-dihydroxyphenyl)-6-hydroxy-3-benzofurancarboxylic acid lactone (coumestrol); 4',5,7-tryhydroxyflavanone (naringenin); 4',5,7-trihydroxyisoflavone (genistein); 2,2-bis(4-hydroxy-phenyl)propane (bisphenol-A); phenol red; dieldrin; chlordane; toxaphene; and CdCl2. SK-BR-3 cells were transfected with 0.5 µg of each expression vector. The cells were treated for 24 h in media containing 5% charcoal-dextran-treated serum and 100 nM E2 or 1 µM of each compound, and then the cells were assessed for aromatase activity. As shown in Fig. 10Citation , at 1 µM, DES, coumestrol, and genistein increased aromatase activity to the same extent as 100 nM E2. Bisphenol-A showed a weak agonistic effect on aromatase activity. Our results were similar to a previous report showing that these compounds stimulated the ERE-transcriptional activity for ER{alpha} (60) .



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  		Fig. 10. Induction of aromatase activity in ER{alpha}-transfected SK-BR-3 cells by environmental estrogens. The SK-BR-3 cells were transfected with 0.5 µg of each expression vector. The cells were treated for 24 h in media containing 5% charcoal-dextran-treated serum and 100 nM E2 or 1 µM of each environmental estrogen, and then the cells were assayed for aromatase activity. Values represent the mean ± SE from triplicate assays.

 
For the first time, results (Fig. 10)Citation are presented to show that chemicals with estrogen-like activity can up-regulate aromatase expression through a specific mechanism. Specifically, DES, coumestrol, genistein, and bisphenol-A were found to be capable of up-regulating aromatase expression. It is speculated that exposure to these chemicals could have an impact on the growth of ErbB-1/ErbB2-positive and ER-positive breast cancer.

In summary, a novel ER{alpha}-mediated nongenomic regulatory mechanism of aromatase expression is identified. This may explain why ErbB-1/ErbB-2-positive and ER-positive breast cancer responds well to aromatase inhibitor treatment. In this mechanism, the activity of promoter I.1 of aromatase is up-regulated. Whereas the molecular details of this nongenomic mechanism have yet to be characterized, our results suggest that this may be another mechanism to modulate aromatase expression in breast cancer tissue.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants CA44735 and ES08258. Back

2 To whom requests for reprints should be addressed, at Beckman Research Institute, City of Hope Medical Center, Department of Surgical Research, 1450 East Duarte Road, Duarte, CA 91010-0269. Phone: (626) 359-8111, ext. 63454; Fax: (626) 301-8972; E-mail: schen{at}coh.org Back

3 The abbreviations used are: RT-PCR, reverse transcription-PCR; DES, diethylstilbestrol; E2, 17ß-estradiol; EGF, epidermal growth factor; ER, estrogen receptor; ERE, estrogen response element; MAPK, mitogen-activated protein kinase; 4-OHA, 4-hydroxyandrostenedione; SRC, steroid receptor coactivator; TAM, tamoxifen; RAR, retinoic acid receptor; FBS, fetal bovine serum; AP-1, activator protein 1; EAR-2, v-erbA-related protein-2. Back

4 Unpublished results. Back

Received 11/25/02. Accepted 4/25/02.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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