Bisphenol A Induces Gene Expression Changes and Proliferative Effects through GPER in Breast Cancer Cells and Cancer-Associated Fibroblasts

1Bisphenol A (BPA), used largely in the manufacture of polycarbonate plastics, is the constituent of a wide array of consumer products, including plastic food containers, baby bottles, and the lining of metal food cans. Humans are exposed to BPA mainly at the time of consumption of water and foods through the materials used for containers and packages.

2BPA is able to accelerate growth and puberty, alter the ovarian cycle in females, interfere with embryonic development, and to induce aneuploidy. Moreover, a relationship between BPA blood levels, obesity, polycystic ovary syndrome, repeated miscarriage, and endometrial hyperplasia has been found in women, suggesting that it may act as an endocrine disruptor. Exposure to BPA has also been correlated with the incidence of diverse types of tumors.

3BPA has estrogenic activity both in vivo and in vitro and is thought to be an environmental estrogen. Previous investigations (reviewed by ) have demonstrated that BPA binds to and activates the estrogen receptor (ERα and ERβ), although the affinity of BPA for these receptors was approximately 10,000-fold weaker with respect to estradiol. In recent years, the identification of G protein-coupled receptor (GPER) as a novel estrogen receptor has suggested new possibilities by which estrogenic compounds might cause biological effects in different cell types. In this regard, we reported a characteristic signature elicited by estrogenic GPER signaling in SKBR3 breast cancer cells and we identified a network of transcription factors, such as c-FOS, early growth response protein 1 (EGR-1), and connective tissue growth factor (CTGF), that may be involved in important biological functions.

4BPA is one of several environmental estrogens that have exhibited the ability to bind to GPER and to activate transduction pathways involved in the biological responses of both normal and neoplastic cells. For example, BPA stimulated the proliferation of mouse spermatogonial cells and human seminoma cells and induced chemoresistance in breast cancer cells through activation of GPER.

5The contribution of the stromal microenvironment to the development of a wide variety of tumors has been highlighted by clinical evidence and the use of mouse models. A growing body of data has also suggested that tumor cells actively recruit cancer-associated fibroblasts (CAFs), which remain activated and play a prominent role in cancer progression. In breast carcinoma approximately 80% of stromal fibroblasts may acquire the activated phenotype that promotes the proliferation of cancer cells at metastatic sites, stimulating tumor growth such as for the primary tumor.

6In this study, we demonstrate that BPA exerts a stimulatory action through GPER in breast cancer cells and CAFs.

7Reagents. We purchased bisphenol A (BPA), N-[2-(p-bromocinnamylamino)ethyl]-5-soquinolinesulfonamide dihydrochloride (H89), PD98059 (PD), and arsenic trioxide (As2O3) from Sigma-Aldrich (Milan, Italy); AG1478 (AG) from Biomol Research Laboratories (DBA, Milan, Italy), and 1-(4-(6-bromobenzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)-ethanone (G-1) from Calbiochem (Merck KGaA, Frankfurt, Germany). As2O3 was dissolved in phosphate-buffered saline, and BPA and PD were dissolved in ethanol; AG1478, H89, and G-1 were solubilized in dimethyl sulfoxide (DMSO).

8Cell culture. SKBR3 cells. SKBR3 human breast cancer cells were maintained in phenol red-free RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Cells were changed to medium without serum the day before experiments for immunoblotting and reverse-transcription polymerase chain reaction (RT-PCR).

9CAFs. CAFs were extracted as previously described. Briefly, breast cancer specimens were collected from primary tumors of patients who had undergone surgery. Signed informed consent was obtained from all the patients and from the institutional review board(s) of the Regional Hospital of Cosenza. Tissues from tumors were cut into smaller pieces (1–2 mm diameter), placed in digestion solution (400 IU collagenase, 100 IU hyaluronidase, and 10% serum, containing antibiotic and antimycotic solution), and incubated overnight at 37°C. The cells were then separated by differential centrifugation at 90 × g for 2 min. Supernatant containing fibroblasts was centrifuged at 485 × g for 8 min; the pellet obtained was suspended in fibroblasts growth medium (Medium 199 and Ham’s F12 mixed 1:1 and supplemented with 10% FBS) and cultured at 37°C in 5% CO2. Primary cells cultures of breast fibroblasts were characterized by immunofluorescence. Briefly cells were incubated with human anti-vimentin (V9) and human anti-cytokeratin 14 (LL001), both from Santa Cruz Biotechnology DBA (Milan, Italy). To assess fibroblasts activation, we used anti-fibroblast activated protein α (FAPα) antibody (H-56; Santa Cruz Biotechnology DBA) (data not shown).

10Western blotting. SKBR3 cells and CAFs were grown in 10-cm dishes, exposed to treatments or ethanol (or DMSO), which was used as the vehicle, and then lysed as described previously. Protein concentrations were determined using Bradford reagent (Sigma-Aldrich) according to the manufacturer’s recommendations. Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Biosciences, Milan, Italy). Membranes were probed overnight at 4°C with antibodies against c-Fos (H-125), β-actin (C-2), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2; E-4), Egr-1 (588), CTGF (L-20), ERK2 (C-14), ERα (F-10), or GPR30 (N-15), all from Santa Cruz Biotechnology, DBA (Milan, Italy), or ERβ from Serotec (Space Import Export, Milan, Italy). Results of densitometric analyses of Western blots, obtained using ImageJ software, are presented as optical density (OD; expressed in arbitrary units) relative to the control (ERK2 or β-actin).

11Plasmids and luciferase assays. The Ctgf luciferase reporter plasmid p(-1999/+36)-luc, which is based on the backbone of vector pGL3-basic (Promega), was a gift from B. Chaqour (Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY, USA). The luciferase reporter plasmid for c-FOS encoding a –2.2-kb 5´ upstream fragment of human c-FOS was a gift from K. Nose (Department of Microbiology, Showa University School of Pharmaceutical Sciences, Hatanodai, Shinagawa-ku, Tokyo, Japan). The EGR-1 luciferase reporter plasmid pEgr-1A, which contains the –600 to +12 5´-flanking sequence from the human EGR-1 gene was a gift from S. Safe (Department of Veterinary Physiology and Pharmacology, Texas A&M University, Houston, TX, USA). For the luciferase assays, cells were transferred into 24-well plates containing 500 μL of regular growth medium per well the day before transfection. On the day of transfection, SKBR3 cell medium was replaced with RPMI without phenol red and serum, and transfection was performed using Fugene6 Reagent (Roche Molecular Biochemicals, Milan, Italy) and a mixture containing 0.5 μg of each reporter plasmid. Renilla luciferase (pRL-CMV; 1 ng) was used as a transfection control. After 5–6 hr, ligand was added and cells were incubated for 16–18 hr. We measured luciferase activity using the Dual Luciferase Kit (Promega, Milan, Italy) according to the manufacturer’s recommendations. Firefly luciferase values generated by the reporter plasmid were normalized to Renilla luciferase values. Normalized values obtained from cells treated with ethanol vehicle were set as 1-fold induction, and the activity induced by treatments was calculated based on this value.

12RT-PCR and real-time PCR. Total RNA was extracted using Trizol commercial kit (Invitrogen, Milan, Italy) according to the manufacturer’s protocol. RNA was quantified spectrophotometrically, and cDNA was synthesized from the RNA by reverse transcription using murine leukemia virus reverse transcriptase (Invitrogen). We quantified the expression of selected genes by real-time PCR using SYBR Green as the detection method and the Step One sequence detection system (Applied Biosystems Inc., Milan, Italy). Gene-specific primers were designed using Primer Express software (version 2.0; Applied Biosystems Inc.). Assays were performed in triplicate. We used mean values to calculate expression levels by the relative standard curve method. For the sequences of primer used, see Supplemental Material, Table S1(http://dx.doi.org/10.1289/ehp.1104526).

13Gene silencing experiments. Cells were plated onto 10-cm dishes, maintained in serum-free medium for 24 hr, and then transfected for an additional 24 hr before treatments using Fugene6. The short hairpin (sh) RNA constructs to knock down the expression of GPER and CTGF and the unrelated shRNA control construct have been described previously.

14Wound-healing assay. CAFs were seeded into 12-well plates in regular growth medium. When at 70% to 80% confluency, the cells were transfected with shGPER using Fugene6 reagent for 24 hr. Transfected cells were washed once, medium was replaced with 2.5% charcoal-stripped FBS, and cells were treated. We then used a p200 pipette tip to scratch the cell monolayer. In experiments performed using conditioned medium, CAFs were plated into 12-well plates and transfected with 500 ng shRNA control plasmid or shGPER or shCTGF plasmids using Fugene6, as recommended by the manufacturer. After 24 hr, CAFs were treated with 1 μM BPA, and the conditioned medium was collected and filtered through a sterile nonpyrogenic 0.2 μm filter. The conditioned medium obtained was added to subconfluent SKBR3 cells, and a series of scratches were made using a p200 pipette tip. We evaluated cell migration in three independent experiments after 48 hr of treatment; data are expressed as a percentage of cells in the wound area upon treatment compared with cells receiving vehicle.

15Proliferation assay. SKBR3 cells and CAFs were seeded in 24-well plates in regular growth medium. After cells attached, they were washed, incubated in medium containing 2.5% charcoal-stripped FBS, and transfected with 500 ng shGPER or control shRNA plasmids using Fugene6 reagent. After 24 hr, cells were treated with 1 μM BPA, and the transfection and treatment were renewed every 2 days. We counted the cells using the COUNTESS automated cell counter (Invitrogen) following the manufacturer’s recommendations.

16Statistical analysis. For statistical analysis, we used analysis of variance followed by Newman-Keuls testing to determine differences in means. p-Values < 0.05 are considered statistically significant.

17BPA induces ERK1/2 activation through GPER. Using SKBR3 cells and CAFs, which both express GPER and lack ERs [see Supplemental Material, Figure S1 (http://dx.doi.org/10.1289/ehp.1104526)], we evaluated ERK1/2 activation by increasing concentrations of BPA and the GPER ligand G-1, as GPER activation leads to ERK1/2 phosphorylation. BPA and G-1 induced ERK1/2 phosphorylation in both cell types in a dose-dependent manner (Figures 1A,B and 2A,B). When the epidermal growth factor receptor (EGFR) inhibitor AG1478 or the mitogen-activated protein kinase kinase MEK inhibitor PD was added, ERK1/2 activation was not evident, but it was present when the protein kinase A (PKA) inhibitor H89 was added (Figure 1C). Interestingly, ERK1/2 phosphorylation by BPA was abolished by silencing GPER expression (Figures 1D, 2C), suggesting that GPER is required for ERK1/2 activation after exposure to BPA. We ascertained the efficacy of GPER silencing using immunoblots in SKBR3 cells and CAFs as shown in Figures 1E and 2D, respectively. Moreover, to demonstrate the specificity of BPA action, we used the environmental contaminant arsenic, which elicits the ability of breast cancer cells to activate ERK1/2. We observed that ERK1/2 phosphorylation induced by 10 μM As2O3 was still present in SKBR3 cells transfected with shGPER (data not shown).

18BPA stimulates the expression of GPER target genes. GPER-mediated signaling regulates the transcription of diverse target genes. In the present study, BPA transactivated the promoter sequence of c-FOS, EGR-1, and CTGF (Figure 3A), and accordingly stimulated mRNA expression of these genes (Figures 3B, 4A). In accordance with these findings, BPA induced the protein levels of c-FOS, EGR-1, and CTGF (Figure 3C). As observed with ERK1/2 activation, the EGFR inhibitor AG1478 and the ERK inhibitor PD, but not the PKA inhibitor H89, repressed the up-regulation of these proteins by BPA (Figure 3C). Notably, the c-FOS, EGR-1, and CTGF protein increases after exposure to BPA were abrogated by silencing GPER in both SKBR3 cells and CAFs (Figures 3D, 4B). The efficacy of GPER silencing was ascertained by immunoblotting experiments in SKBR3 cells and CAFs as shown in Figures 3E and 4C, respectively. Taken together, these results demonstrate that BPA regulates the expression of c-FOS, EGR-1, and CTGF through the GPER/EGFR/ERK transduction pathway.

19BPA induces cell proliferation and migration through GPER. The aforementioned results were recapitulated in the complex physiologic responses such as cell proliferation and migration. The proliferative effects observed in both SKBR3 cells and CAFs after 5-day treatment with BPA were cancelled when GPER expression was silenced by shGPER (Figure 5A,B). Moreover, in wound-healing assays in CAFs, migration induced by BPA was abolished by knocking down GPER expression (Figure 5C). To evaluate whether the treatment of CAFs with BPA could induce the migration of tumor cells through secreted factor(s), we performed wound-healing assays in SKBR3 cells cultured with conditioned medium from CAFs. Interestingly, the migration of SKBR3 cells was not evident after silencing GPER or CTGF expression in CAFs (Figure 5D). Overall, these findings demonstrate that BPA induces stimulatory effects as a GPER agonist in both ER-negative SKBR3 breast cancer cells and CAFs.

20There has been increased interest in understanding the molecular mechanisms involved in the endocrine-disrupting effects of BPA. In this regard, fetal and perinatal exposures to BPA in rodents have been shown to affect the brain, mammary gland, and reproductive tract, as well as to stimulate the development of hormone-dependent tumors. Moreover, the estrogenic actions of BPA, including increased uterine wet weight, luminal epithelial height, and increased expression of the estrogen-inducible protein lactoferrin, were reported in prepubescent CD-1 mice. Analogously, BPA induced the proliferation of uterine and vaginal epithelial cells in ovariectomized rats. In regard to the mechanisms by which BPA can exert estrogen-like effects, it has been reported that BPA’s two benzene rings and two (4,4´)-OH substituents fitting in the ER binding pocket allow the binding to and activation of both ERα and ERβ, which in turn mediate the transcriptional responses to BPA. In addition, rapid nongenomic effects involving diverse transduction pathways were observed upon exposure to BPA in pancreatic islet, endothelial, and hypophysial cells and in breast cancer cells. In this context, the novel estrogen receptor GPER was recently shown to mediate the BPA-dependent rapid activation of intracellular signaling and the proliferation of both human seminoma cells and mouse spermatogonial cells.

21To investigate the potential of GPER to mediate estrogenic signals such as those elicited by BPA, we used SKBR3 breast cancer cells and CAFs, both of which express GPER and lack ERs. Interestingly, we found that in both cell types BPA triggers rapid ERK activation through the GPER/EGFR transduction pathway and induces the expression of genes that characterize estrogenic GPER-mediated signaling. In addition, we determined that BPA stimulates the proliferation and migration of SKBR3 cells and CAFs through GPER. Of note, conditioned medium from BPA-treated CAFs induced the migration of SKBR3 cells, suggesting that BPA may also promote a functional crosstalk between cancer cells and CAFs. These data regarding CAFs are particularly intriguing given that these cells actively contribute to cancer growth and progression even at metastatic sites.

22The present findings are relevant to the results obtained in a previous study in which we found that atrazine, another environmental contaminant, triggered estrogen-like activity through the GPER/EGFR/ERK transduction pathway in hormone-sensitive ovarian cancer cells. Moreover, in that study we observed that atrazine induced functional crosstalk between GPER and ERα in accordance with the results of who demonstrated a similar interaction in mouse spermatogonial cells after exposure to BPA. Overall, these findings, together with results of the present study, contribute to a better understanding on the multifaceted mechanisms by which environmental estrogens may act as endocrine stimulators in hormone-dependent malignancies.

23BPA is consistently detected in almost all individuals in developed nations, suggesting that humans are exposed to BPA continuously. In addition, the rapid metabolic clearance of BPA and its detectable levels in human blood and urine suggest that the intake of BPA may be higher than indicated by diverse studies and that long-term daily intake may lead to its bioaccumulation. In this regard, previous studies have estimated that human exposure ranges from < 1 μg/kg/day to almost 5 μg/kg/day (0.325 mg/day/adult). However, pharmacokinetic modeling data have shown that oral intakes up to 100 mg/day/adult would be required to explain the reported human circulating levels. Hence, future studies should include mathematical models of potential exposures, particularly because many sources of BPA exposure have been identified. These observations suggest that the BPA concentration used in the present study is achievable in humans. In the present study, we found that BPA is able to trigger GPER-mediated signaling in breast cancer cells and CAFs, which contributes to tumor progression. Thus, GPER may a potential mediator of the estrogen-like activity of BPA, as well as a further biological target in estrogen-sensitive tumors.