Human Cancer Biology Estrogen Receptor A Binds to Peroxisome Proliferator ^ Activated Receptor Response Element and Negatively Interferes with Peroxisome Proliferator ^ Activated Receptor ; Signaling in Breast Cancer Cells Daniela Bonofiglio,1Sabrina Gabriele,1Saveria Aquila,1Stefania Catalano,1Mariaelena Gentile,2 Emilia Middea,1Francesca Giordano,2 and Sebastiano Ando' 2,3 Abstract Purpose: The molecular mechanisms involved in the repressive effects exerted by estrogen receptors (ER) on peroxisome proliferator ^ activated receptor (PPAR) g^ mediated transcriptional activity remain to be elucidated. The aim of the present study was to provide new insight into the crosstalk between ERa and PPARg pathways in breast cancer cells. Experimental Design: Using MCF7 and HeLa cells as model systems, we did transient trans- fections and electrophoretic mobility shift assay and chromatin immunoprecipitation studies to evaluate the ability of ERa to influence PPAR response element ^ mediated transcription. A pos- sible direct interaction between ERa and PPARg was ascertained by coimmunoprecipitation assay, whereas their modulatory role in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway was evaluated by determining PI3K activity and AKT phosphorylation. As a biological counter- part, we investigated the growth response to the cognate ligands of both receptors in hormone-dependent MCF7 breast cancer cells. Results:Our data show for the first time that ERa binds to PPAR response element and represses its transactivation. Moreover, we have documented the physical and functional interactions of ERa and PPARg, which also involve the p85 regulatory subunit of PI3K. Interestingly, ERa and PPARg pathways have an opposite effect on the regulation of the PI3K/AKT transduction cascade, explaining, at least in part, the divergent response exerted by the cognate ligands 17 h-estradiol and BRL49653on MCF7 cell proliferation. Conclusion: ERa physically associates with PPARg and functionally interferes with PPARg signaling. This crosstalk could be taken into account in setting new pharmacologic strategies for breast cancer disease. Peroxisome proliferator–activated receptors (PPAR) are ligand- tiation of malignant breast epithelial cells in vitro and induced activated transcription factors belonging to the nuclear receptor morphologic changes associated with apoptosis and fibrosis in superfamily (1). Activation of PPARs is a multistep process that breast tumor cells injected in mice (15, 16). involves ligand binding, heterodimerization with retinoic X A large body of evidence has shown that estrogen receptor receptor (RXR), interaction with cognate DNA sequences, and (ER) a is involved in the development of breast cancer (17–20). recruitment of coregulatory proteins (1). Three PPAR isoforms, On ligand binding, ERa undergoes a conformational change a, h/y, and g, are expressed in multiple species in a tissue- allowing chromatin interaction and the transcriptional regula- specific manner (2–4). As for PPARg, its involvement has been tion of target genes (21). It has also been reported that ERa reported in several metabolic pathways, in adipocyte differen- binds to the p85 regulatory subunit of phosphatidylinositol tiation, and even in the growth inhibition of different cancer cell 3-kinase (PI3K), leading to the activation of the protein kinase lines (5–14). In addition, PPARg promoted terminal differen- B/AKT pathway, which in turn regulates diverse processes like cell survival and proliferation (22, 23). Recently, an increasing physiologic significance has been Authors’ Affiliations: Departments of 1Pharmaco-Biology and 2Cell Biology, and attributed to the crosstalk among nuclear receptors, which was 3Centro Sanitario, University of Calabria, Cosenza, Italy observed at several levels of the signal transduction cascades Received 11/30/04; revised 6/7/05; accepted 6/15/05. (24–28). As it concerns the interaction between PPAR and ER Grant support: Associazione Italiana per la Ricerca sul Cancro 2003. The costs of publication of this article were defrayed in part by the payment of page pathways, the PPAR/RXR heterodimer has been shown to bind charges. This article must therefore be hereby marked advertisement in accordance to estrogen response element (ERE)–related palindromic with 18 U.S.C. Section 1734 solely to indicate this fact. sequences; however, it cannot transactivate due to a nonper- Requests for reprints: Sebastiano Ando' , Department of Cell Biology, University missive natural promoter structure (26). On the other hand, of Calabria, Cosenza 87036, Italy. Phone: 39-0984-496201; Fax: 39-0984- 496203; E-mail: [email protected]. ERs negatively interfere with PPAR response element (PPRE)– F 2005 American Association for Cancer Research. mediated transcriptional activity (29); however, the molecular doi:10.1158/1078-0432.CCR-04-2453 mechanisms involved still remain to be elucidated. www.aacrjournals.org 6139 Clin Cancer Res 2005;11(17) September 1,2005 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Human Cancer Biology Herein, we have shown for the first time, to our knowledge, MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, 0.2 mmol/L phenyl- that ERa binds to PPRE even in the context of the endogenous methylsulfonyl fluoride, 1 mmol/L leupeptin] by flicking the tube. phosphatase and tensin homologue deleted on chromosome The cells were allowed to swell on ice for 10 minutes and then vortexed 10 (PTEN) promoter sequence, physically interacts with PPARg, for 10 seconds. Samples were then centrifuged for 10 seconds and the supernatant fraction discarded. The pellet was resuspended in 50 AL and generates a ternary complex involving the p85 regulatory of cold buffer B [20 mmol/L HEPES-KOH (pH 7.9), 25% glycerol, subunit of PI3K. Moreover, ERa and PPARg induce opposite 1.5 mmol/L MgCl2, 420 mmol/L NaCl, 0.2 mmol/L EDTA, 0.5 mmol/L effects on the regulation on the PI3K/AKT pathway eliciting DTT, 0.2 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L leupeptin] consequently divergent growth responses on treatment with the and incubated on ice for 20 minutes for high-salt extraction. Cellular respective cognate ligands 17h-estradiol and rosiglitazone debris were removed by centrifugation for 2 minutes at 4jC and the (BRL49653) in hormone-dependent MCF7 breast cancer cells. supernatant fraction (containing DNA binding proteins) was stored at À70jC. In vitro transcribed and translated PPARg and ERa were Materials and Methods synthesized using the T7 polymerase in the rabbit reticulocyte lysate system as directed by the manufacturer (Promega). The probe was Reagents. BRL49653 was a kind gift from GlaxoSmithKline (West generated by annealing single-stranded oligonucleotides and labeled 32 Sussex, United Kingdom). The irreversible PPARg antagonist GW9662, with [g- P]ATP (Amersham Pharmacia, Buckinghamshire, United 17h-estradiol, and hydroxytamoxifen were purchased by Sigma (Milan, Kingdom) and T4 polynucleotide kinase (Promega), and then purified Italy). ICI182780 was generously provided by Zeneca Pharmaceuticals using Sephadex G50 spin columns (Amersham Pharmacia). (Cheshire, United Kingdom). All compounds were solubilized in A double-stranded PPRE was prepared by annealing the following DMSO or in ethanol (Sigma). sense and antisense-oligonucleotides: 5V-GGGACCAGGACAAAGGT- V V V Plasmids. The pGL3 vector containing three copies of a PPRE CACGTT-3 and 5-GGGAACGTGACCTTTGTCCTGGTC-3 (Sigma sequence upstream of the minimal thymidine kinase promoter ligated Genosys, Cambridge, United Kingdom). As control for nonspecific to a luciferase reporter gene (3XPPRE-TK-pGL3)andthePPARg binding, a cold PPRE competitor was included. The protein binding expression plasmid were a gift from Dr. R. Evans (The Salk Institute, reactions were carried out in 20 AL of buffer [20 mmol/L HEPES (pH 8), San Diego, CA). The expression vector of ERa and androgen receptor 1 mmol/L EDTA, 50 mmol/L KCl, 10 mmol/L DTT, 10% glycerol, were previously described (30, 31). The ERh expression vector was 1 mg/mL bovine serum albumin, 50 Ag/mL poly(deoxyinosinic- provided by Dr. J.A. Gustafsson (Karoliska Institute, Stockholm, deoxycytidylic acid)] with 50,000 cpm of labeled probe, 5 Agof Sweden). The constitutively active myristilated AKT mutant (myr-AKT) MCF7 and HeLa nuclear protein, or 2 ALofin vitro transcribed and was kindly provided by Dr. T. Simoncini (University of Pisa, Pisa, Italy). translated PPARg and ERa proteins, and 5 Ag of poly(deoxyinosinic- Cell cultures. Wild-type human breast cancer ERa-positive MCF7 deoxycytidylic acid). The mixtures were incubated at room temperature cells (a gift from E. Surmacz, Sbarro Institute for Cancer Research for 20 minutes in the presence or absence of unlabeled competitor and Molecular Medicine, Philadelphia, PA) were grown in DMEM-F12 oligonucleotides or in vitro transcribed and translated PPARg and ERa containing 10% FCS, 1% L-glutamine, 1% Eagle’s nonessential amino proteins. For the experiments involving PPARg and ERa (F-10 and acids, and 1 mg/mL penicillin-streptomycin. The ER-negative HeLa D-12) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), the cells were maintained with DMEM supplemented with 10% FCS, 1% reaction mixture was incubated with these antibodies at 4jC for 30 L-glutamine, and 1 mg/mL penicillin-streptomycin. minutes before addition of labeled probe. Under the conditions Transfection assay. Transient transfection experiments were done employed, cross-reactivity