Author Manuscript Published OnlineFirst on February 7, 2018; DOI: 10.1158/1541-7786.MCR-17-0568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. BET Proteins Exhibit Transcriptional and Functional Opposition in the Epithelial-to-mesenchymal Transition Guillaume P. Andrieu1, Gerald V. Denis1,2* 1 Cancer Center, Boston University School of Medicine, Boston, Massachusetts, 02118, USA. 2 Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts, 02118, USA. Running title: Distinct and Opposing Functions of BRD2 and BRD4 in EMT Keywords: bromodomain, epigenetics, plasticity, breast cancer, EMT Grant support: This study and all the authors were supported by grants from the National Institutes of Health (DK090455 and U01 CA182898, GVD). Corresponding author: Gerald V. Denis, Cancer Center, Rm K520, Boston University School of Medicine, 72 East Concord Street, Boston, Massachusetts, 02118, USA Phone: 617-414-1371 Fax: 617-638-5673 E-mail: [email protected] Conflicts of interest. The authors state that they have no potential conflicts of interest to disclose. Word count: 2,280 excluding Abstract Abstract word count: 147 Figures: 3 Tables: 0 Supplementary Figures: 2 Supplementary Tables: 2 References: 23 Supplementary information: A file of supplementary figures and data is provided. 1 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 7, 2018; DOI: 10.1158/1541-7786.MCR-17-0568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Transcriptional programs in embryogenesis and cancer, such as the epithelial-to- mesenchymal transition (EMT), ensure cellular plasticity, an essential feature of carcinoma progression. As effectors of signal transduction, the bromodomain and extraterminal (BET) proteins are well suited to support plasticity because they function as co-activators or co-repressors of mammalian transcriptomes. Here, using both hormone-sensitive and triple-negative breast cancer (TNBC) model systems, we systematically altered EMT transcriptional profiles by manipulating individual BET proteins and found that BRD2 positively regulates EMT, whereas BRD3 and BRD4 repress this program. Knockdown of individual BET proteins revealed independent transcriptional networks that differed from each other and from the small-molecule pan- BET inhibitor JQ1, which previously had been misleadingly asserted to be BRD4- selective. Available small-molecule pan-BET inhibitors, proposed as anti-proliferative agents in cancer clinical trials, obscure these biological differences. Transcriptional profiling reveals that individual BET proteins, inhibited separately, engage in and control EMT through unique processes. 2 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 7, 2018; DOI: 10.1158/1541-7786.MCR-17-0568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction The bromodomain and extraterminal (BET) family of transcriptional regulators includes three somatic members BRD2, BRD3, BRD4 and testis-specific BRDT. The bromodomain, a protein motif first described in brahma, binds to ε-N-aminoacetyl groups of nucleosomal histone lysine and recruits histone modification enzymes, transcriptional co-activators and co-repressors, and chromatin remodeling activities to gene promoters. These epigenetic readers therefore function to upregulate or downregulate gene expression in response to cellular signals (1). Human BET proteins are homologs of female sterile homeotic, a transcriptional regulator important in Drosophila development, a feature of many proteins involved in human cancer. BET proteins share common structural features, with evolutionarily conserved, tandem bromodomains that interact with acetylated histones and an extraterminal domain that recruits specific interactors. The contribution of BET proteins to cancer progression has largely been reported, reinforcing their value as therapeutic targets notably for acute myeloid leukemia, B-cell lymphoma, lung, breast, prostate, pancreatic and colorectal cancers (1). BET proteins, as crucial transcriptional regulators, control cancer progression. First generation small molecule inhibitors have been developed that compete with BET bromodomain/acetyl-peptide binding, displacing both BRD2, BRD3, BRD4 and BRDT from chromatin. These compounds, including JQ1 (2) and I-BET (3), show promising results for in vitro and in vivo cancer models including ‘nuclear protein in testis’ midline carcinoma (2), leukemia (4), lymphoma (5,6), prostate (7) and breast cancer (8). However, pan-BET inhibition strategies obscure the individual biological 3 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 7, 2018; DOI: 10.1158/1541-7786.MCR-17-0568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. functions of each BET protein and can present potential risks for patients (9). Indeed, BET proteins exert specific, protective roles that are ablated by pan-BET inhibition. One illustration is BET protein-controlled active repression of HIV-1 transcription; pan-BET inhibitors reactivate latent HIV-1 and viral outgrowth in infected human T cells (10,11). Despite their mutual homology, BET proteins have non-identical genome occupancy patterns, are engaged in different regulatory complexes, and modulate distinct signaling pathways and biological functions (12-15). Thus, full comprehension of the contribution of each BET protein is crucial before envisaging them durably as therapeutic targets for cancer treatment. We have previously reported that depletion of BRD4 inhibits invasion and migration in cellular models of triple negative breast cancer, as a regulator of Jagged1/Notch1 signaling (16). Interestingly, only BRD4 regulates this pathway, not BRD2 or BRD3, revealing that some transcription programs are not regulated by all BET proteins (16). Because the dependence of target genes on individual BET proteins in each cancer cell type is impossible to predict a priori, it is necessary to map the signaling pathways with an unbiased approach. The epithelial-to-mesenchymal transition (EMT) is a developmental program that cancer cells often activate to acquire a highly plastic phenotype that promotes invasion, metastasis, as well as chemoresistance and cancer stem cell generation (17). Several transcription programs triggered by key transcription factors induce drastic changes in epithelial cells to confer mesenchymal phenotypes and properties. Here, we investigated patterns of transcriptional activation and repression of genes important for EMT that are controlled by BET proteins in breast cancer models to resolve the unique and independent functions of these transcription regulators. 4 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 7, 2018; DOI: 10.1158/1541-7786.MCR-17-0568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods Cell Culture Human breast cancer cell lines maintained at the NCI Office of Physical Sciences- Oncology Centers (PS-OC) Network Bioresource Core Facility (PBCF) were contractually obtained through the American Type Culture Collection, under a Material Transfer Agreement. The cell lines have been authenticated by the NIH Physical Sciences Oncology Consortium. Mycoplasma contamination was prevented by treating the cells with Plasmocin (25 mg/ml for two weeks, Invivogen) following thawing prior the experiments. MDA-MB-231 and MCF-7 were cultured in Dulbecco’s modified Eagle’s medium (DMEM). SUM149PT cells were cultured in DMEM/F12 + 5 μg/μl insulin and 0.5 μg/μl hydrocortisone (Sigma). T47D cells were cultured in Roswell Park Memorial Institute (RPMI). All culture media were supplemented with 10% fetal bovine serum (FBS). Cells were cultured at 37°C in a humid 5% CO2 atmosphere. Antibodies and Reagents The following antibodies were used: anti-BRD2, anti-BRD3 and anti-BRD4 (Bethyl Laboratories), anti-E-cadherin (24E10), anti-N-cadherin (13A9), anti-Snail (C15D3), anti-Slug (C19G7), anti-vimentin (D21H3), anti-ZEB1 (D80D3) (Cell Signaling Technology), anti-Snai3 (Abcam), anti D-tubulin (DM1A), Twist (H-81) and anti-ZEB2 (E- 5 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 7, 2018; DOI: 10.1158/1541-7786.MCR-17-0568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 11) (Santa Cruz Biotechnology). Fluorochrome-conjugated secondary antibodies were obtained from Jackson Laboratories. JQ1 was purchased from Tocris Bioscience. Plasmids, siRNAs and Transfection Plasmids coding for His-tagged BET proteins or control vector pReceiver-M01 were purchased from GeneCopoeia. ON-TARGETplus BET proteins siRNAs were obtained from Dharmacon. Cells were transfected with plasmids and siRNAs by Lipofectamine 2000 reagent (Thermo Fisher Scientific) as previously validated (16). Efficient depletions