2019, 66 (1), 65-74 Original Flightless-I mediates the repression of estrogen receptor α target gene expression by the glucocorticoid receptor in MCF-7 cells Liu Yang and Kwang Won Jeong Gachon Institute of Pharmaceutical Sciences, College of Pharmacy, Gachon University, Incheon 21936, Republic of Korea Abstract. The human homologue of flightless-I (FLII) belong to the gelsolin protein family and contain a gelsolin-like domain at the C-terminus and a leucine-rich repeat (LRR) domain at the N-terminus. FLII regulates estrogen receptor alpha (ERα) and glucocorticoid receptor (GR)-mediated transcription by direct interaction through different domains, suggestive of its potential role in the crosstalk between the ERα and GR signaling pathway. Here, we demonstrate that FLII plays a critical role in GR-mediated repression of ERα target gene expression. In FLII-depleted cells, the reduction in 17-β-estradiol (E2)- induced ERα occupancy following treatment with dexamethasone (Dex) at the estrogen responsive element (ERE) site of the ERα target gene was significantly inhibited. The ERE binding of GR by the cotreatment with E2 and Dex was significantly inhibited by FLII depletion, indicating that FLII is required for the recruitment of GR at the ERE sites of ERα target genes. In addition, the recruitment of ERα-induced FLII to ERE sites was significantly reduced by Dex treatment. In protein binding assays, GR inhibited the E2-induced interaction between ERα and FLII, suggesting that GR interferes with the binding of ERα and FLII at the ERα target genes, resulting in the release of ERα and FLII from EREs. Taken together, our data reveal an unknown mechanism by which the transcription coactivator FLII regulates the GR-mediated repression of ERα target gene expression in MCF-7 cells. Key words: Flightless-I, Estrogen receptor α, Glucocorticoid receptor, Dexamethasone BREAST CANCER remains the leading cause of breast cancer is documented. Glucocorticoids have been cancer-related deaths among females worldwide [1-3]. shown to control apoptosis and proliferation in breast Numerous evidences have suggested that 17β-estradiol cancer models and may enhance the chemo-sensitivity of or estrogen (E2), a steroid hormone that regulates vari‐ breast cancer cells when used in combination [11, 12]. ous human physiological functions and influences However, the molecular mechanism underlying the diverse pathological processes, acts as one of the major effects of glucocorticoids in the therapy of breast cancer risk factors in breast cancer development and progres‐ is yet unclear. Similar to estrogen, glucocorticoid signal‐ sion [4, 5]. The effect of estrogen is mainly mediated via ing is regulated by binding to its cognate intracellular estrogen receptor alpha (ERα), a member of nuclear receptor known as glucocorticoid receptor (GR), a receptor superfamily of proteins that play a critical role member of the nuclear receptor superfamily of ligand- in cellular processes such as cell proliferation, differen‐ dependent transcription factors. Extensive studies have tiation, apoptosis, and migration, all of which influence focused on the crosstalk between GR and ERα to explore the development and progression of cancer [6, 7]. As a the underlying mechanism in breast cancer cells. Some result, antiestrogens that block ERα-mediated transcrip‐ findings have demonstrated that GR inhibits E2- tion and estrogen synthesis inhibitors have been devel‐ stimulated ERα target gene expression. GR occupies oped for the treatment of breast cancer [7-10]. ERα-binding regions (EBRs) through binding to the acti‐ The contribution of glucocorticoids in the treatment of vator protein 1 (AP-1) within EBRs [13, 14]. The recruit‐ ment of GR to EBRs destabilizes ERα and steroid Submitted Aug. 20, 2018; Accepted Oct. 4, 2018 as EJ18-0343 receptor coactivator-3 complex, leading to the repression Released online in J-STAGE as advance publication Oct. 26, 2018 of ERα target gene expression [15]. Furthermore, coacti‐ Correspondence to: Kwang Won Jeong, Gachon Institute of Phar‐ maceutical Sciences, College of Pharmacy, Gachon University, 191 vators such as steroid receptor coactivators (SRC-2 and Hambakmoero, Yeonsu-gu, Incheon 21936, Republic of Korea. SRC-3) and the mediator component MED14 have been E-mail: [email protected] reported to contribute to the interplay between GR and ©The Japan Endocrine Society 66 Yang et al. ERα [16]. GR has been recently shown to bind to ERα- GGUGUUUGACAACGACdTdT-3' (sense) and 5'-GUC occupied enhancers depending on its SUMOylation and GUUGUCAAACACCUGCdTdT-3' (anti-sense); siNS, association with the N-CoR/SMRT-HDAC3 complex. 5'-UUCUCCGAACGUGUCACGUdTdT-3' (sense) and This complex may block the recruitment and the Mega‐ 5'-ACGUGACACGUUCGGAGAAdTdT-3' (anti-sense). Trans complex to repress ERα directed transcriptional Total RNA was isolated from MCF-7 cells with Trizol program [17]. (Invitrogen, Carlsbad, USA) after treatment with 10 nM Previous studies have demonstrated that flightless-I E2 or 100 nM dexamethasone (Dex; Sigma-Aldrich, (FLII) functions as a coactivator for the ERα-mediated Louis, USA) for 24 h alone or in combination. RNA was transcription [18, 19]. FLII comprises an N-terminal subjected to reverse transcription by iScript cDNA syn‐ leucine-rich repeat (LRR) and a C-terminal gelsolin-like thesis kit (Bio-Rad Laboratories, Hercules, CA, USA) in domain containing two large repeats (GelA and GelB) a total volume of 20 μL. A total of 2 μL of product was [20, 21]. The coactivator function of FLII in ERα signal‐ used for qPCR performed on a LightCycler 480II ing pathway mainly depends on the recruitment of the (Roche, Indianapolis, IN, USA) with the following SWI/SNF chromatin remodeling complex to promoters. primers: TFF1, 5'-GAACAAGGTGATCTGCG-3' To facilitate this process, FLII binds to ERα and BAF53, (forward) and 5'-TGGTATTAGGATAGAAGCACCA-3' an actin-related protein of the SWI/SNF complex, via C- (reverse); GREB1, 5'-CAAAGAATAACCTGTTGGCC terminal gelsolin-like domain in MCF-7 cells [18]. Fur‐ CTGC-3' (forward) and 5'-GACATGCCTGCGCTCTC thermore, FLII regulates GR-mediated transcription by ATACTTA-3' (reverse); CyclinD1, 5'-AAGCTCAAGTG direct binding to GR via the N-terminal LRR domain in GAACCT-3' (forward) and 5'-AGGAAGTTGTT A549 cells [22]. These findings suggest that FLII may GGGGC-3' (reverse); PgR, 5'-GTGCCTATCCTGCC play an important role in the crosstalk between ERα and TCTCAATC-3' (forward) and 5'-CCCGCCGTCGTAA GR. CTTTCG-3' (reverse); 18S, 5'-GAGGATGAGGTGG In the current study, we determined the function of AACGTGT-3' (forward) and 5'-TCTTCAGTCGCTCC FLII in the repression of ERα target gene expression by AGGTCT-3' (reverse). Results shown are mean and GR signaling in MCF-7 cells. In an attempt to elucidate range of variation of duplicate PCR reactions performed the underlying mechanism, we evaluated the role of FLII with the same cDNA sample. Relative expression levels in the regulation of ERα and GR recruitment to ERα tar‐ were normalized to the expression levels of 18S rRNA. get genes. Furthermore, we also observed the binding activity among ERα, GR, and FLII. Together, our data Chromatin immunoprecipitation (ChIP) assay describe the important role of FLII in the crosstalk We performed the ChIP assays according to previously between ERα and GR in the process of GR-mediated described protocols [18]. Briefly, MCF-7 cells were repression of ERα target gene expression. transfected with siRNAs and cultured for 3 days in hormone-free media. At approximately 90% confluency, Materials and Methods cells were treated with 100 nM E2 or 100 nM Dex alone or in combination for 60 min. After crosslinking with Plasmids and cell culture formaldehyde, the cell extracts were prepared from The following plasmids used were previously descri‐ control and hormone-treated MCF-7 cells. Sheared chro‐ bed [18, 23]: pGEX-ERα (LBD), pCDNA3.1-ERα, matin fragments were prepared by sonication. Immuno‐ pCDNA-hGR, and pTriEX-FLII. MCF-7 cells were cul‐ precipitation of sonicated chromatin solutions was tured in Dulbecco’s modified Eagle’s medium (HyClone, conducted by overnight incubation at 4°C with anti-ERα, South Logan, Utah) supplemented with 4 mM L- anti-GR, or anti-FLII antibodies (Santa Cruz Biotech‐ glutamine, 4,500 mg/L glucose, sodium pyruvate, and nology, Carlsbad, USA). Crosslinking was reversed by 10% fetal bovine serum (Gibco, Grand Island, USA) at heating overnight at 65°C, and protein-associated DNA 37°C and in an atmosphere containing 5% CO2. sequence was purified by phenol-chloroform extraction and ethanol precipitation. The purified DNA sequence RNA interference and quantitative reverse was dissolved in 100 μL of nuclease free water and ana‐ transcription PCR (RT-qPCR) lyzed by real-time quantitative PCR using LightCycler Small-interfering RNA experiments followed by RT- 480II system with SYBR Green I Master (Roche, Indian‐ qPCR were performed according to a previously pub‐ apolis, IN, USA). Results shown are mean and range of lished method [18, 24]. Transfection of MCF-7 cells was variation of duplicate PCR reactions from a single performed with Oligofectamine (Invitrogen, Carlsbad, experiment which is representative of at least three inde‐ USA) according to the manufacturer’s protocol. The pendent experiments. Results were expressed as percent‐ sequences of siRNAs were as follows: siFLII, 5'-GCA age of input chromatin (before immunoprecipitation). FLII in GR-mediated gene repression 67 The primers used were as follows: TFF1(ERE1), 5'-CCG