bet proteins exhibit transcriptional and functional ...ising results for in vitro and in vivo cancer...

BET Proteins Exhibit Transcriptional andFunctional Opposition in the Epithelial-to-Mesenchymal TransitionGuillaume P. Andrieu1 and Gerald V. Denis1,2

Abstract

Transcriptional programs in embryo-genesis and cancer, such as the epithe-lial-to-mesenchymal transition (EMT),ensure cellular plasticity, an essentialfeature of carcinoma progression. Aseffectors of signal transduction, thebromodomain and extraterminal (BET)proteins are well suited to supportplasticity because they function as co-activators or co-repressors of mammali-an transcriptomes. Here, using bothhormone-sensitive and triple-negativebreast cancer (TNBC) model systems,we systematically altered EMT transcrip-tional profiles by manipulating indi-vidual BET proteins and found thatBRD2 positively regulates EMT, whereasBRD3 and BRD4 repress this program.Knockdown of individual BET proteinsrevealed independent transcriptionalnetworks that differed from each otherand from the small-molecule pan-BETinhibitor JQ1, which previously hadbeen misleadingly asserted to beBRD4-selective. Available small-mole-cule pan-BET inhibitors, proposed asantiproliferative agents in cancer clinicaltrials, obscure these biological differ-ences. Transcriptional profiling revealsthat individual BET proteins, inhibitedseparately, engage in and control EMTthrough unique processes.

Implications: The distinct and opposing functions of BET proteins in the EMT process suggests the need for more member-selectiveepigenetic targeting agents.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/4/580/F1.large.jpg. Mol Cancer Res; 16(4); 580–6. �2018 AACR.

IntroductionThe bromodomain and extraterminal (BET) family of tran-

scriptional regulators includes three somatic members BRD2,

BRD3, BRD4, and testis-specific BRDT. The bromodomain, aprotein motif first described in brahma, binds to e-N-aminoacetylgroups of nucleosomal histone lysine and recruits histone

1Cancer Center, Boston University School of Medicine, Boston, Massachusetts.2Department of Pharmacology and Experimental Therapeutics, BostonUniversity School of Medicine, Boston, Massachusetts.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Gerald V. Denis, Boston University School of Medicine,Room K520, 72 East Concord Street, Boston, Massachusetts. Phone: 617-414-1371: Fax: 617-638-5673; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-17-0568

�2018 American Association for Cancer Research.

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modification enzymes, transcriptional co-activators and co-repressors, and chromatin remodeling activities to gene promo-ters. These epigenetic readers therefore function to upregulate ordownregulate gene expression in response to cellular signals (1).Human BET proteins are homologs of female sterile homeotic, atranscriptional regulator important in Drosophila development, afeature of many proteins involved in human cancer. BET proteinsshare common structural features, with evolutionarily conserved,tandembromodomains that interact with acetylated histones andan extraterminal domain that recruits specific interactors. Thecontribution of BET proteins to cancer progression has largelybeen reported, reinforcing their value as therapeutic targets nota-bly for acute myeloid leukemia, B-cell lymphoma, lung, breast,prostate, pancreatic, and colorectal cancers (1). BET proteins, ascrucial transcriptional regulators, control cancer progression.First-generation small molecule inhibitors have been developedthat 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 prom-ising 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 individualbiological functions of each BET protein and can presentpotential risks for patients (9). Indeed, BET proteins exertspecific, protective roles that are ablated by pan-BET inhibition.One illustration is BET protein-controlled active repression ofHIV-1 transcription; pan-BET inhibitors reactivate latent HIV-1and viral outgrowth in infected human T cells (10, 11). Despitetheir mutual homology, BET proteins have nonidenticalgenome occupancy patterns, are engaged in different regulatorycomplexes, and modulate distinct signaling pathways andbiological functions (12–15). Thus, full comprehension of thecontribution of each BET protein is crucial before envisagingthem durably as therapeutic targets for cancer treatment. Wehave previously reported that depletion of BRD4 inhibitsinvasion and migration in cellular models of triple negativebreast cancer, as a regulator of Jagged1/Notch1 signaling (16).Interestingly, only BRD4 regulates this pathway, not BRD2 orBRD3, revealing that some transcription programs are notregulated by all BET proteins (16). Because the dependence oftarget genes on individual BET proteins in each cancer cell typeis impossible to predict a priori, it is necessary to map thesignaling pathways with an unbiased approach.

The epithelial-to-mesenchymal transition (EMT) is a develop-mental program that cancer cells often activate to acquire a highlyplastic phenotype that promotes invasion, metastasis, as well aschemoresistance and cancer stem cell generation (17). Severaltranscription programs triggered by key transcription factorsinduce drastic changes in epithelial cells to confer mesenchymalphenotypes and properties. Here, we investigated patterns oftranscriptional activation and repression of genes important forEMT that are controlled byBETproteins in breast cancermodels toresolve the unique and independent functions of these transcrip-tion regulators.

Materials and MethodsCell culture

Human breast cancer cell lines maintained at the NCI Officeof Physical Sciences-Oncology Centers (PS-OC) Network

Bioresource Core Facility (PBCF) were contractually obtainedthrough the ATCC, under a Material Transfer Agreement. The celllines have been authenticated by the NIH Physical SciencesOncology Consortium. Mycoplasma contamination was pre-vented by treating the cells with Plasmocin (25 mg/mL for2 weeks, Invivogen) following thawing prior the experiments.MDA-MB-231 and MCF-7 were cultured in DMEM. SUM149PTcells were cultured inDMEM/F12þ 5 mg/mL insulin and 0.5 mg/mLhydrocortisone (Sigma). T47D cells were cultured in RPMI. Allculture media were supplemented with 10% FBS. Cells werecultured at 37�C in a humid 5% CO2 atmosphere.

Antibodies and reagentsThe 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) (CellSignaling Technology), anti-Snai3 (Abcam), anti a-tubulin(DM1A), Twist (H-81), and anti-ZEB2 (E-11) (Santa CruzBiotechnology). Fluorochrome-conjugated secondary antibodieswere obtained from The Jackson Laboratory. JQ1 was purchasedfrom Tocris Bioscience.

Plasmids, siRNAs and transfectionPlasmids coding for His-tagged BET proteins or control

vector pReceiver-M01 were purchased from GeneCopoeia.ON-TARGETplus BET proteins siRNAs were obtained fromDharmacon. Cells were transfected with plasmids and siRNAsby Lipofectamine 2000 reagent (Thermo Fisher Scientific) aspreviously validated (16). Efficient depletions or overexpres-sions were obtained 3 days posttransfection.

qRT-PCRTotal RNA was extracted using the RNEasy Kit (Qiagen).

Reverse transcription reactions were performed on 1 mg of RNAwith the QuantiTect Reverse Transcription Kit (Qiagen). Theprimer sequences used for this study are listed in SupplementaryTable S1. PCR amplifications were performed with the MESAGREEN qPCRMasterMix (Eurogentec) on an ABI Prism 7500 FastBlock thermal cycler.

The gene screening was conducted with the RT2 Profiler PCREMT Array (Qiagen). Z scores were calculated and heatmaps weregenerated using MATLAB software (MathWorks).

Immunocytochemistry staining, confocal imaging, andanalysis

Cells were fixed in absolute methanol for 5 min at�20�C thenpermeabilized with 0.2% Triton X-100 in PBS buffer for 10minutes. After saturation in blocking buffer (0.02% Triton X-100, 2% BSA in PBS) for 30 minutes, cells were incubated withprimary antibodies, then fluorochrome-conjugated secondaryantibodies, both diluted in blocking buffer for 1 hour. Finally,coverslips were mounted with ProLong Gold with DAPI (ThermoFisher Scientific). Image acquisition was conducted using a LeicaSP5 confocalmicroscope. For z-stack acquisition, a step of 0.3 mmwas set. Fluorescence intensities were determined using ImageJsoftware (NIH). Intensities were corrected for background thenexpressed as a ratio of mean intensities per cell area beforenormalization.

Distinct and Opposing Functions of BRD2 and BRD4 in EMT

www.aacrjournals.org Mol Cancer Res; 16(4) April 2018 581




Statistical analysesStatistical analyses were either performed with Student t test or

ANOVA according to the datasets by using GraphPad Prism 7software. The following symbols were used to indicate significantdifferences: ns, P > 0.05; ��, P < 0.01; ��, P < 0.001.

All the experiments executed for this study have beenconducted in accordance with the NIH guidelines underthe review of the Boston University Institutional BiosafetyCommittee.

ResultsWe sought to determine how individual BET proteins tran-

scriptionally control EMT in breast cancer cells. We performed aPCR array analysis of 84 genes involved in EMT regulation indifferent breast cancer cell lines, specifically depleted for eachBET protein (Fig. 1). Our analysis revealed that individualdepletion of each BET protein produced a unique transcriptionprofile, indicating that BRD2, BRD3, and BRD4 exert indepen-dent control over EMT (Fig. 1A). Under BRD2 depletion, wefound that 34 genes were significantly downregulated andtwo others upregulated in triple-negative MDA-MB-231 cells(Fig. 1B, Z score �2 or ��2, P-value < 0.05). Conversely, BRD3depletion (16 genes upregulated, 3 downregulated) and BRD4depletion (7 genes upregulated, 2 downregulated) are mostlyassociated with gene upregulation, suggesting these BETproteins principally act as repressors. Remarkably, only a fewgenes co-vary in the different BET-depleted signatures, revealingthat BET proteins regulate EMT sometimes in opposition toeach other (Fig. 1A and B). Similar results were obtained intriple-negative breast cancer SUM149PT cells or luminal Abreast cancer MCF-7 cells (Supplementary Fig. S1A and S1B).Functional analysis of the genes deregulated under BET deple-tion, suggests that BRD2 positively regulates EMT, whereasBRD3 and BRD4 repress this program.

We also compared the transcriptional consequences of singleBET silencing with pan-BET inhibition by JQ1 treatment. Apanel of downregulated genes was common to BRD2 depletionor JQ1 treatment, whereas only few co-occurrences wereshared with JQ1 in BRD3 or BRD4 depletion (Fig. 1B). Mostof the commonly regulated genes are EMT transcription factors(Fig. 1B and C and Supplementary Table S2), suggesting thatBET proteins exert a transcriptional control on EMT. This resultstrongly suggests that pan-BET inhibition is most similar toBRD2 depletion, and clearly opposes single BRD3 or BRD4depletion, indicating that any result obtained with pan-BETinhibitors like JQ1 should not be interpreted as a specifictargeting of any BET protein, as misleadingly asserted in severalreports (9).

EMT is driven by multiple transcription programs inducedby several major transcription factors, including the Snailfamily (Snail, Slug, Snai3), Twist and the ZEB family (ZEB1,ZEB2; ref. 17). To confirm that BET proteins regulate EMTthrough different transcription programs, we modulated singleBET protein expression by either specific depletion or over-expression of each and monitored the transcriptional responseof the main EMT transcription factors in multiple breast can-cer cell lines. Triple-negative breast cancer MDA-MB-231 andSUM149PT cell lines exhibit a mesenchymal phenotype. Inthese cell lines, BRD2 depletion induced a significant down-regulation of Snail, Slug, Snai3, but also Twist and ZEB1, ZEB2

(Fig. 2A and B and Supplementary Fig. S2A). Conversely, BRD3and BRD4 depletions significantly increased the expression ofall these major EMT transcription factors. The luminal A breastcancer line MCF-7 presents an epithelial phenotype and barelyexpresses EMT transcription factors under normal conditions(Fig. 2B). Upon BRD2 depletion, we noted a moderate down-regulation of Snail and Twist. However, BRD4 silencing led to astrong upregulation of the Snail and ZEB family members alongwith Twist. Interestingly, BRD3 depletion phenocopies BRD4silencing but fails to upregulate Snai3 or ZEB2 in our models,suggesting independent control of these transcription factors.We then overexpressed each BET protein in these cell lines andmonitored the expression of the major EMT transcriptionfactors (Fig. 2C). We found that BRD2 overexpression inducedexpression of each of these factors, assayed in both cell lines(Fig. 2D and Supplementary Fig. S2B). However, BRD3 andBRD4 overexpression led to downregulation of several EMTfactors, including Twist, ZEB1, and ZEB2. These data supportthe idea that BRD2 positively regulates EMT, whereas BRD3 andBRD4 repress this program. Importantly, the overexpression ofsingle BET proteins also led to individual and distinct tran-scriptional signatures relevant to EMT (Supplementary Fig. 1C).To compare single BET depletion versus pan-BET targeting onEMT transcription factor expression, we then treated breastcancer cell lines with JQ1 and repeated the aforementionedexperiments. We found that JQ1 treatment led to a significantdownregulation of SNAI1, SNAI3, TWIST1, ZEB1, and ZEB2in MDA-MB-231 and MCF-7 cells (Supplementary Fig. S2Cand S2D), as also observed under BRD2 targeting (Fig. 2B andSupplementary Fig. S2B). Collectively, our results demonstratethat BRD2 opposes BRD3 and BRD4 to transcriptionally regu-late EMT. This duality may be explained by divergent regulationof the key EMT transcription factors. Interestingly, BRD3 seemsto exert only moderate control of EMT transcription programscompared to BRD4; BRD3 modulation does not affect all theEMT transcription factors depicted here. Critically, we con-firmed that, in a model of transcription control of EMT, pan-BET inhibition with JQ1 most closely parallels BRD2 silencingand opposes BRD3 or BRD4 targeting.

We then confirmed that BET proteins regulate morphologicaland phenotypical changes relevant to EMT in breast cancercells. We immunostained breast cancer cell lines, to detectepithelial and mesenchymal markers upon BET modulation.Luminal A breast cancer cells, such as MCF-7 or T47D, present acuboidal morphology characterized by strong expression of theepithelial marker E-cadherin at tight junctions, and lack themesenchymal markers N-cadherin or vimentin, as illustratedin control cells treated with scrambled siRNA (Fig. 3A). UponBRD2 depletion, we observed increased expression of E-cad-herin, consistent with repression of EMT. Conversely, BRD3 orBRD4 depletion induced a significant decrease in E-cadherinexpression, and an increase in either N-cadherin or vimentinexpression. Under BRD3 or BRD4 depletion, we observeddisruption of cell morphology, exemplified by cell flatteningand increased cell area. Significantly, opposite results wereobtained by overexpressing BET proteins (Fig. 3B). BRD2 over-expression induced downregulation of E-cadherin, increased N-cadherin, and provoked similar morphological modificationsas observed in BRD3- or BRD4-depleted cells, consistent withinitiation of EMT. Taken together, the results indicate that BETproteins control EMT transcription factors and either engage

Andrieu and Denis

Mol Cancer Res; 16(4) April 2018 Molecular Cancer Research582




Figure 1.

Individual BET proteins control independent EMT transcriptomes. A, Heatmap presenting Z scores of a PCR array of 84 EMT genes expressed in MDA-MB-231cells upon BET protein depletion by siRNA (50 nmol/L for 3 days; n ¼ 3). Independent transcriptional signatures relevant to EMT regulation wereobtained for each BET protein. Pan-BET inhibition using small molecule JQ1 (400 nmol/L for 3 days) obscured these individual profiles. A color code isused to illustrate Z score variations. Normalization is set to scramble. B, Systemic analysis of the EMT signatures. BRD2 depletion exhibit a strong associationwith JQ1 treatment with 25 common genes downregulated. Conversely, BRD3 and BRD4 depletions are mostly associated with a small number of upregulatedgenes that do not overlap. Most of the BET-regulated genes are EMT transcription factors. Graphs plot Z scores from BET depletion or JQ1 datasets.Significantly altered genes are indicated (Z score � 2 or � �2, P-value < 0.05). C, Functional analysis of the EMT signatures. Most of the genes modulated byBET depletion are transcription factors, indicating that BET proteins transcriptionally regulate EMT.






or repress EMT programs, leading to changes in epithelial/mesenchymal marker expression and cell architecture.

DiscussionOur results reveal functional opposition between BRD2,

BRD3, and BRD4. BRD2 is a positive regulator of EMT,whereas BRD3 and BRD3 are repressors of this program.Similarly to previous studies, we conclude that each BETprotein carries its own functions, sometimes overlappingwith another family member's, sometimes distinct or opposite(12–15). Therefore, investigators in the BET protein field riskover-interpreting one particular member's role, like BRD4,based solely on experiments with pan-BET inhibitors. Rather,studies should be conducted by selectively targeting eachmember. We have commented on how misleading interpreta-tions are problematic for our mechanistic understanding ofhow small molecule BET inhibitors work or should best becombined with other modalities (9). Family member-selectivesmall molecules are urgently needed to advance the clinicaltranslational impact of these recent discoveries. Among thenewly reported small molecules targeting the BET proteins,MZ1 shows promise (18). This Proteolysis Targeted Chimera(PROTAC), which combines a BET binder motif based on JQ1structure and a ligand for the E3 ligase VHL, induces BET

protein degradation in a family member-specific manner,depending on the dose.

By accomplishing EMT, cancer cells acquire numerous prop-erties relevant for migration, invasion, and survival in responseto chemotherapy or in a stressful microenvironment, or cellidentity and differentiation (17). Furthermore, reports haveshown that EMT can generate cancer stem-like cells (CSC;refs. 19, 20). Depending on the activated EMT transcriptionfactors, several programs can be triggered, leading to differentEMT-related outcomes. For instance, EMT is not always associ-ated with increased metastatic potential. A recent publicationreported that EMT is dispensable for metastasis but elicitsdevelopment of chemoresistance in a pancreatic cancer model(21). In a previous report, we showed that BRD4 silencingablates breast cancer cell migration and invasion (16). Therefore,we can speculate that EMT triggered by BRD4 loss in breastcancer may not generate highly invasive cells but rather mightelicit subpopulations with higher survival or CSCs properties.BRD4 has been reported to control pluripotency and thereforeembryonic stem cell (ESC) identity (22). In ESCs, BRD4 target-ing induces EMT markers, indicating that the regulation wediscovered may not be limited to cancer but can also occurduring development and physiologic EMT. The role of BETproteins in normal and cancer stem cell functions demandsfurther investigation, considering that BET proteins likely

Figure 2.

BRD2 opposes BRD3 and BRD4 to control key EMT transcription factors. A, Validation of BET depletion by siRNA in MDA-MB-231 and MCF-7 cells (50 nmol/Lfor 3 days). B, Protein expression of key EMT transcription factors upon BET protein depletion in MDA-MB-231 or MCF-7 cells. C, Validation of BEToverexpression in MDA-MB-231 and MCF-7 cells. D, Protein expression of key EMT transcription factors upon BET protein overexpression in MDA-MB-231 orMCF-7 cells. Blots are representative of three independent experiments. Molecular weights are indicated (kDa).


Andrieu and Denis




Figure 3.

BET protein manipulation triggers EMT in epithelial breast cancer cells. A, Representative images of MCF-7 depleted for BET proteins (50 nmol/L for 3 days)and stained for E-cadherin (green), N-cadherin (red), and vimentin (gray). B, Representative images of MCF-7 overexpressing BET proteins and stainedfor E-cadherin (green), N-cadherin (red), and DNA (DAPI, blue). For measurement of relative fluorescence intensities, lines represent means � SEM ofthree independent experiments. Each dot represents a single cell value. For cell area measurement, histograms represent means � SEM of threeindependent experiments. Blots depict protein expression of key EMT markers upon BET protein depletion (A) or overexpression (B) in MCF-7 cells.Molecular weights are indicated (kDa). Statistical analyses were conducted by one-way ANOVA. The following symbols were used to indicate significantdifferences: ns, P > 0.05; �� , P < 0.01; �� , P < 0.001. Images and blots are representative of three independent experiments. Bar scale, 10 mm.






activate trithorax (23), which balances Polycomb groupproteins to regulate proliferation and self-renewal in stem cells.Previously, we reported that BRD2 deficiency in murine ESCsinduces insulin transcription, reinforcing the concept that thesefactors play important roles during the earliest stages of mam-malian development. Understanding the biological functions ofeach individual BET proteins is critical knowledge preliminaryto improved design and development of targeted epigenetictherapeutics for cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: G.P. AndrieuDevelopment of methodology: G.P. AndrieuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G.P. Andrieu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G.P. AndrieuWriting, review, and/or revision of the manuscript: G.P. Andrieu, G.V. DenisStudy supervision: G.V. DenisOther (provided funding): G.V. Denis

AcknowledgmentsThis study and all the authors were supported by grants from the NIH

(DK090455 and U01 CA182898, to G.V. Denis). The funders had no rolein study design, data collection and analysis, decision to publish, orpreparation of the manuscript. This study and all the authors weresupported by grants from the NIH (DK090455 and U01 CA182898, toG.V. Denis).

The authors acknowledge the Boston University Cellular Imaging CoreFacility and its imaging equipment. We thank Drs. Alessio Ciulli and DinahSinger for discussion.

Received October 5, 2017; revised December 23, 2017; accepted January 24,2018; published first February 7, 2018.

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