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Transrepression of the Estrogen Receptor Promoter by Calcitriol in Human Breast Cancer Cells Via Two Negative Vitamin D Response Elements

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Transrepression of the Estrogen Receptor Promoter by Calcitriol in Human Breast Cancer Cells Via Two Negative Vitamin D Response Elements S Swami et al. Calcitriol represses ER via 20:4 565–577 Research two nVDREs Transrepression of the estrogen receptor promoter by calcitriol in human breast cancer cells via two negative vitamin D response elements Srilatha Swami, Aruna V Krishnan, Lihong Peng, Johan Lundqvist1 and David Feldman Correspondence Division of Endocrinology, Department of Medicine, Stanford University School of Medicine, 300 Pasteur Drive, should be addressed Room S025, Stanford, California 94305-5103, USA to D Feldman 1Breast Cancer Group, Cell Death and Metabolism, Danish Cancer Society Research Center, Strandboulevarden 49, Email DK-2100 Copenhagen Ø, Denmark [email protected] Abstract Calcitriol (1,25-dihydroxyvitamin D3), the hormonally active metabolite of vitamin D, exerts Key Words its anti-proliferative activity in breast cancer (BCa) cells by multiple mechanisms including " vitamin D the downregulation of the expression of estrogen receptor a (ER). We analyzed an " calcitriol w3.5 kb ER promoter sequence and demonstrated the presence of two potential negative " transrepression vitamin D response elements (nVDREs), a newly identified putative nVDRE upstream at " estrogen receptor K K " Endocrine-Related Cancer 2488 to 2473 bp (distal nVDRE) and a previously published sequence (proximal nVDRE) nVDREs at K94 to K70 bp proximal to the P1 start site. Transactivation analysis using ER promoter " estrogen signaling deletion constructs and heterologous promoter–reporter constructs revealed that both " breast cancer cells nVDREs functioned to mediate calcitriol transrepression. In the electrophoretic mobility shift assay, the vitamin D receptor (VDR) showed strong binding to both nVDREs in the presence of calcitriol, and the chromatin immunoprecipitation assay demonstrated the recruitment of the VDR to the distal nVDRE site. Mutations in the 50 hexameric DNA sequence of the distal nVDRE resulted in the loss of calcitriol-mediated transrepression and the inhibition of protein–DNA complex formation, demonstrating the importance of these nucleotides in VDR DNA binding and transrepression. A putative nuclear factor-Y (NFY) binding site, identified within the distal nVDRE, led to the findings that NFY bound to the distal nVDRE site interfered with the binding of the VDR at the site and reduced calcitriol- mediated transrepression. In conclusion, the ER promoter region contains two negative VDREs that act in concert to bind to the VDR and both nVDREs are required for the maximal inhibition of ER expression by calcitriol. The suppression of ER expression and estrogen-mediated signaling by calcitriol in BCa cells suggests that vitamin D may be useful C in the treatment of ER BCa. Endocrine-Related Cancer (2013) 20, 565–577 http://erc.endocrinology-journals.org q 2013 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/ERC-12-0281 Printed in Great Britain Downloaded from Bioscientifica.com at 10/02/2021 01:17:57AM via free access Research S Swami et al. Calcitriol represses ER via 20:4 566 two nVDREs Introduction Breast cancer (BCa) is the most common cancer in women explores the mechanism of the transcriptional repression in the USA (Siegel et al. 2012). Estrogens drive the of the ER gene by calcitriol. Recent advances in the genome- proliferation of mammary epithelial cells and therefore wide analysis of transcriptional regulation by calcitriol promote the growth of estrogen receptor-positive (ERC) have revealed that the VDREs could be located both BCa cells. Estrogens act by binding to the estrogen proximal and distal to the transcriptional start sites of receptor a (ER) to regulate gene expression. Therapy for target genes and many, in fact, are located in clusters ERC BCa, therefore, includes hormonal treatments such hundreds of kilobases from their target genes (Pike & Meyer as those with aromatase inhibitors that inhibit estrogen 2010, Pike 2011, Pike et al. 2011a). However, our current synthesis and selective ER modulators that block estrogen analysis demonstrates that the functional activities of two action in the breast by binding to the ER and acting as nVDRE sites within an w3.5 kb promoter region of the antagonists (Burstein et al. 2010, Tomao et al. 2011). ER gene recapitulate the magnitude of the downregulation Another approach to inhibit the growth of ERC BCa is to of ER expression observed in BCa cells following calcitriol decrease the levels of ER, the nuclear receptor that treatment. One of these sites, the proximal nVDRE mediates the growth-promoting effects of estrogens in identified previously by Stoica et al. (1999), is an imperfect breast epithelial cells using compounds that act palindromic sequence located at K94 to K70 bp of the as selective ER downregulators such as fulvestrant ER gene with reference to the P1 start site, the major start (McDonnell & Wardell 2010, Nilsson et al. 2011). site in ERC BCa cells (deConinck et al. 1995). Herein, we Calcitriol, the hormonally active form of vitamin D report the identification and characterization of another (1,25-dihydroxyvitamin D3), plays an important role in putative nVDRE (distal nVDRE) further upstream of the calcium homeostasis through its actions (Feldman et al. published proximal site that also contributes to the 2013). In addition to these classical actions, calcitriol also calcitriol-mediated transrepression of ER. exhibits anti-proliferative, pro-differentiating, and anti- inflammatory activities, indicating its potential use in the prevention and treatment of multiple cancers including Materials and methods BCa (Beer & Myrthue 2004, Deeb et al. 2007, Krishnan & Materials Feldman 2011, Pereira et al. 2012, Welsh 2012). Interest- Endocrine-Related Cancer ingly, our studies reveal that in ERC BCa cells calcitriol The human BCa cell lines used in the study were obtained inhibits both estrogen synthesis and signaling, leading to from the American Type Culture Collection (Rockville, a significant attenuation of estrogen stimulation of cell MD, USA). Tissue culture media, supplements, and FCS were obtained from Gibco BRL, Lonza (Walkersville, MD, growth (Krishnan et al. 2010a). We have shown that USA), and Mediatech, Inc. (Herndon, VA, USA). All calcitriol acts as a selective aromatase modulator (SAM) antibodies were obtained from Santa Cruz Biotechnology. and decreases estrogen synthesis in BCa cells and the All other chemicals and reagents were procured from surrounding mammary adipose tissue by suppressing Sigma Chemical Co., unless otherwise indicated. aromatase expression (Krishnan et al. 2010b, Swami et al. 2011). Furthermore, calcitriol and its analogs also down- regulate ER expression in BCa cells (James et al. 1994, Methods Simboli-Campbell et al. 1997, Swami et al. 2000), thereby causing a significant inhibition of estrogenic bioresponses Cell culture The human BCa cell lines MCF-7, ZR75-1, in these cells, including the suppression of estrogen- and T47D were cultured at 37 8Cunder5%CO2 stimulated cell growth (Demirpence et al. 1994, Swami atmosphere using RPMI-1640 medium supplemented et al. 2000). with 10% calf serum, penicillin (100 U/ml), and strepto- Our earlier study (Swami et al. 2000) and that of Stoica mycin (100 mg/ml). et al. (1999) revealed that the negative regulation of ER expression by calcitriol occurred at the transcriptional Plasmids ER promoter–reporter plasmids containing level. This transcriptional repression is probably mediated a series of 50 deletions with reference to the P1 start through the binding of the vitamin D receptor (VDR) to site were a kind gift from Dr Ronald Weigel (formerly one or more negative vitamin D response elements at the Stanford University and currently at the (nVDREs) present in the ER promoter. The current study University of Iowa, Iowa City, IA, USA). The construction http://erc.endocrinology-journals.org q 2013 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/ERC-12-0281 Printed in Great Britain Downloaded from Bioscientifica.com at 10/02/2021 01:17:57AM via free access Research S Swami et al. Calcitriol represses ER via 20:4 567 two nVDREs of these plasmids has been described previously Electrophoretic mobility shift assay Electrophore- (deConinck et al. 1995). The NFYA expression plasmids tic mobility shift assay (EMSA), as described previously were a kind gift from Dr Roberto Mantovani (Universita di (Peng et al. 2004), was used to analyze the binding of Milano, Milano, Italy). the VDR to the putative nVDREs in the ER promoter using complementary oligonucleotides for the distal and Heterologous constructs for VDRE analysis We proximal nVDREs. ZR75-1 cell extracts were incubated refer to the previously described VDRE by Stoica et al. for 15–20 min with 100 nM calcitriol and [32P]-labeled (1999) (K94 to K70) as the proximal nVDRE and the oligonucleotide probes. For competition assays, a 500-fold newly discovered upstream site as the distal nVDRE molar excess of the corresponding unlabeled oligonucleo- (K2488 to K2473). A 230 bp KpnI–HindIII fragment (C tides was added to the reaction mixture before the 230/0) from the plasmid pGL2-ER promoter (K3500 to addition of the labeled probes. In some reactions, 2 mgof C230) was cloned into the KpnI/SmaI sites of the an anti-VDR polyclonal antibody (H-81) or an anti-RXRa heterologous Simian virus 40 (SV40) promoter-driven polyclonal antibody (D20) were added to the reaction luciferase reporter vector pGL3 (Promega) to generate the mixture 40 min prior to the addition of the labeled probe. nVDRE(null) construct. Oligonucleotides enclosing
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