Interferon-StimulatedGenesAreTranscriptionally Repressed ...mcr.aacrjournals.org/content/molcanres/15/10/1331.full.pdf · Tianbao Li5, Sean M. Holloran1,2,3, Gloria M.Trinca1,2,3,

Chromatin, Epigenetics, and RNA Regulation

Interferon-StimulatedGenesAreTranscriptionallyRepressed by PR in Breast CancerKatherine R.Walter1,2,3, Merit L. Goodman1,2,3, Hari Singhal4, Jade A. Hall1,2,3,Tianbao Li5, Sean M. Holloran1,2,3, Gloria M. Trinca1,2,3, Katelin A. Gibson1,2,3,Victor X. Jin5, Geoffrey L. Greene6, and Christy R. Hagan1,2,3

Abstract

The progesterone receptor (PR) regulates transcriptional pro-grams that drive proliferation, survival, and stem cell pheno-types. Although the role of native progesterone in the develop-ment of breast cancer remains controversial, PR clearly alters thetranscriptome in breast tumors. This study identifies a class ofgenes, Interferon (IFN)-stimulated genes (ISGs), potentlydownregulated by ligand-activated PR which have not beenpreviously shown to be regulated by PR. Progestin-dependenttranscriptional repression of ISGs was observed in breast cancercell line models and human breast tumors. Ligand-independentregulation of ISGs was also observed, as basal transcript levelswere markedly higher in cells with PR knockdown. PR repressedISG transcription in response to IFN treatment, the canonicalmechanism through which these genes are activated. LigandedPR is robustly recruited to enhancer regions of ISGs, and ISGtranscriptional repression is dependent upon PR's ability to

bind DNA. In response to PR activation, key regulatory tran-scription factors that are required for IFN-activated ISG tran-scription, STAT2 and IRF9, exhibit impaired recruitment to ISGpromoter regions, correlating with PR/ligand-dependent ISGtranscriptional repression. IFN activation is a critical early stepin nascent tumor recognition and destruction through immu-nosurveillance. As the large majority of breast tumors are PRpositive at the time of diagnosis, PR-dependent downregulationof IFN signaling may be a mechanism through which early PR-positive breast tumors evade the immune system and developinto clinically relevant tumors.

Implications: This study highlights a novel transcriptionalmechanism through which PR drives breast cancer develop-ment and potentially evades the immune system. Mol Cancer Res;15(10); 1331–40. �2017 AACR.

IntroductionThere is an emerging role for the ovarian steroid hormone,

progesterone, and its receptor, the progesterone receptor (PR), inthe development of breast cancer (1–3). Clinical data have shownincreased breast cancer incidence in women taking postmeno-pausal hormone replacement therapy (HRT) whose combinedregimens included estrogen andprogestins; this increased riskwasnot present in women taking estrogen-only HRT (4). Althoughmuch debate continues regarding the role for native progesteronein breast cancer development, it is clear that PR, alone or in

combination with the estrogen receptor (ER), affects the tran-scriptional landscape of breast cancer (5–8).

PR is a ligand-activated transcription factor that binds DNAeither directly through progesterone response elements (PRE),or indirectly through tethering interactions with other tran-scription factors. These interactions, together with the recruit-ment of transcriptional coregulators, lead to transcriptionalactivation or repression of PR target genes. In the breast, thesePR-dependent gene programs can drive proliferation, cell sur-vival, and mammary stem cell self-renewal (reviewed in ref. 3).The mechanisms for PR-dependent transcriptional activationhave been well studied, while PR-mediated transcriptionalrepression, especially direct repression in response to ligand,remains less understood.

Interferon (IFN) signaling is a critical response of the innateimmune system, which typically occurs following pathogendetection. Following IFN (types I–III) production and bindingto their cognate receptors, a signaling cascade mediated byJAK/STAT is initiated, culminating in a transcriptional responsewhose gene products aid the cell in responding to a pathogenicthreat. The genes activated in response to IFNs are collectivelycalled IFN-stimulated genes (ISG; reviewed in ref. 9). In responseto type I IFNs, such as IFNa, a heterodimeric receptor [IFNAR1(IFNa receptor 1) and IFNAR2] complex is activated/autopho-sphorylated in response to ligand, promoting JAK1/tyrosinekinase 2 (TYK2)-dependent phosphorylation of STAT1 andSTAT2. Phosphorylated STAT1 and STAT2, together with IFN-regulatory factor 9 (IRF9), form a transcriptional complex

1Department of Biochemistry and Molecular Biology, University of KansasMedical Center, Kansas City, Kansas. 2Department of Cancer Biology, Universityof Kansas Medical Center, Kansas City, Kansas. 3University of Kansas CancerCenter, University of Kansas Medical Center, Kansas City, Kansas. 4Departmentof Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School,Boston, Massachusetts. 5Department of Molecular Medicine, University of TexasHealth San Antonio (UTHSA), San Antonio, Texas. 6The BenMayDepartment forCancer Research, The University of Chicago, Chicago, Illinois.

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

K.R. Walter and M.L. Goodman contributed equally to this article.

Corresponding Author: Christy R. Hagan, University of Kansas Medical Center,3901 Rainbow Blvd, Kansas City, KS 66160. Phone: 913-588-0795; Fax: 913-588-9896; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-17-0180

�2017 American Association for Cancer Research.

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referred to as IFN-stimulated gene factor 3 (ISGF3), whichbinds to DNA sequences within ISG promoter regions, referredto as IFN-stimulated response elements (ISRE). Binding ofISGF3 to ISREs leads to transcription of ISGs, preparing thecell with a diverse response to viral infection. IFN signalingactivation and ISG expression has also been detected in humantumors, independent of viral infection. The role of IFN signal-ing in human tumors is not well known and remains an area ofintense research (10). The cancer immunoediting hypothesispostulates that IFN signaling is an early step required forimmune-recognition and subsequent destruction of nascenttumors by immunomodulatory cells (11). Alterations in immu-nosurveillance, recognition, and destruction may have dramat-ic effects contributing to the development of clinically overttumors. This effect appears to be most prominent in the earlystages of tumor development. The opposite phenotype is seenin advanced, therapy-resistant tumors: evidence of IFN signal-ing (high ISG levels) is associated with a range of tumor types,including breast, that have escaped chemotherapy, radiation, orimmunotherapy (12). Herein, we present evidence of a novelrole for PR in mediating transcriptional repression of ISGs. Thisnewly described activity of PR has significant clinical implica-tions for immune evasion and development of PR-positivehuman breast tumors.

Materials and MethodsCell lines and constructs

T47D-co, T47D-Y, T47D-YB, T47D-YA, and Hela-PR cellshave been described previously (13, 14) and were a generousgift from Dr. Carol Lange (University of Minnesota, Minnea-polis, MN). T47D cells (unmodified) were obtained from ATCCand cultured as recommended. Cell line authentication iscurrently under way. MDA-MB-231 cells were maintained inMinimum Essential Media (MEM; CellGro) supplemented with5% FBS, 1% penicillin/streptomycin, 1% nonessential aminoacids, and 6 ng/mL insulin (cMEM). BT549 cells were main-tained in RPMI1640 Media (CellGro) supplemented with 10%FBS, 1% penicillin/streptomycin, 1% nonessential amino acids,and 6 ng/mL insulin. The PR-mDBD construct was a generousgift from Dr. Kathryn Horwitz (University of Colorado, Denver,CO). Cells were treated with the following reagents (whenapplicable): R5020 (10nmol/L; Sigma), human recombinantIFNa (Sigma-Aldrich, SPR4594).

Gene set enrichment analysisGene set enrichment analysis (GSEA) was performed using the

javaGSEAdesktop software; the c2Molecular SignaturesDatabase(MSigDB) version 5.2 was queried (15, 16). Dataset files (GEOaccession number:GSE46850) were developed on the basis ofnormalized Illumina expression intensities from cells that con-stitutively expresswt PR-B (T47D-YB cells), as published in ref. 13.Specifically, the log2-transformed expression values were com-pared for two phenotypes: T47D-YB EtOH (vehicle) and T47D-YBR5020. GSEA was executed using the default settings, except thepermutation type was set to Gene_set with 1,000 permutations,and the metric for ranking genes was set to Diff_of_Classes,because normalized expression data was log2 transformed. Lead-ing Edge (LE) analysis was performed on the 54 gene sets fromMSigDB c2 analysis (PR-B EtOH vs. R5020) that achieved FDRvalues �0.05.

Tumor explantsTumor explant processing and treatments were done as

described previously (8). Raw FASTQ data was reanalyzed usingthe VIPER RNA sequencing (RNA-seq) pipeline (https://bitbucket.org/cfce/viper/) at Center for Functional Cancer Epige-netics (CFCE) at the Dana-Farber Cancer Institute (Boston, MA).For visualizing gene expression data, we used heatmap.2 functionof gplots package in R programming language.

ImmunoblottingImmunoblotting/Western blotting was performed as described

previously (13, 14). Membranes were probed with primary anti-bodies recognizing total PR (Santa Cruz Biotechnology, sc-7208or Thermo Fisher Scientific, MS-298-P), IRF9 (Cell SignalingTechnology, 76684), IFIT1 (Cell Signaling Technology, 14769),IFIT3 (Santa Cruz Biotechnology, sc-393512), OAS1 (Cell Sig-naling Technology, 14498), and ERK (Cell Signaling Technology,9102). All Western blotting experiments were performed intriplicate, and representative experiments are shown.

siRNA/shRNAON-TARGETplus SMARTpool (a pool of four individual

siRNAs) designed to target human PR was purchased from Dhar-macon (L-003433-00), as were nonsilencing (NS) siRNA controls(D-001810-01-05) and the suggested DharmaFect 1 TransfectionReagent (T-2001-03). For siRNA experiments, the cells wereplated at a density of 3 � 105 cells per well in a 6-well plate.Twenty-four hours following plating, T47D-co cells were trans-fected with 25 nmol/L NS or PR siRNA per the manufacturer'ssiRNA transfection protocol. At 48 hours posttransfection, thecells were starved in serum/phenol red-free IMEM (Gibco,A10488-01). At 72-hour posttransfection, the cells were treatedwith EtOH or 10 nmol/L R5020 for 6 hours. Subsequent RNAisolation and qPCR experiments were carried out and analyzed asdescribed below. PR shRNA knockdown cells were created usingviral particles (GE/Dharmacon) targeting three different regionsof human PR. Viral transduction protocol was followed as perthemanufacturer's instructions. Transduced, stable cell line poolsexpressing NS or PR shRNA were created in T47D-co or T47D-YBcells following 14 days of selection in 2.5 mg/mL puromycin(MP Biomedicals). Target si/shRNA sequences are listed in Sup-plementary Table S1.

Luciferase transcription assayspGL4.45[luc2P/ISRE/Hygro] luciferase construct (Promega)

was stably integrated in HeLa and HeLa-PR cells using Hygro-mycin selection. Luciferase assays were performed as describedpreviously (17) using the Dual Luciferase Reporter Assay (Pro-mega). Starved cells were treated for 18 hours with 10 nmol/LR5020 and/or 10,000 IU/mL IFNa.

qPCRRNA isolation, cDNA creation, and qPCR were performed as

described previously (13, 14), with modifications noted here andin the figure legends. qPCR was performed using the FastStartEssential DNA Green Master (Roche) on a Roche LightCycler96.Relative concentrations were quantified using the LightCycler96(Roche, Software 1.1, Absolute Quantification Analysis), using a6-point standard curve. Primer sets are listed in SupplementaryTable S1. Relevant genomic sequence information is based onGR37 hg19.

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Mol Cancer Res; 15(10) October 2017 Molecular Cancer Research1332



https://bitbucket.org/cfce/viper/

https://bitbucket.org/cfce/viper/


ChIP assaysChromatin immunoprecipitation (ChIP) was performed using

the ChIP-IT Express Kit (Active Motif) according to the manu-facturer's instructions using sonication for chromatin shearing.Lysates were immunoprecipitated (IP) overnight (18 hours)with the following antibodies: PR (Santa Cruz Biotechnology,sc-7208), STAT2 (Santa Cruz Biotechnology, sc-476), IRF9(Santa Cruz Biotechnology, sc-10793), H3K27ac1 (Cell Signal-ing Technology, 8173), or an equal amount of negative controlmouse or rabbit IgG. Resulting DNA was analyzed using qPCRas described above, and data are represented as a percentage ofinput DNA. In silico analysis was performed using MatInspector(Genomatix) to identify potential PRE-binding sites. Primersets are listed in Supplementary Table S1.

Statistical analysisStatistical significance for all experiments was determined

using an unpaired Student t test, unless otherwise specified. AP � 0.05 is considered statistically significant. The Delta meth-od was used to calculate standard deviation for the ratio of twovariables using their individual SDs, as seen when plotting foldrelative RNA expression data between two treatment groups/cell lines (18).

ResultsIFN gene sets enriched in PR-regulated transcriptional dataset

GSEA is a powerful computational tool for comparing geneexpression datasets of interest (i.e., genes regulated by PR) topublished gene sets culled from the literature (15, 16). UsingGSEA, we analyzed our previously published microarray datasetfrom ligand (synthetic progestin; R5020)-treated T47D breastcancer cells stably expressing full-length PR (GEO:GSE46850;ref. 13). GSEA revealed that multiple IFN-related gene sets (exam-ples shown in Fig. 1A) were significantly enriched in the absenceof ligand; this enrichment was lost in cells treated with ligand,representing a negative correlation between ligand treatment andenrichment with IFN-related gene sets. Nearly 20% of the topsignificantly regulated gene sets (54 sets with an FDR � 0.05) areIFN-related gene sets (select sets shown in Fig. 1B). As enrichmentin these gene sets is lost in progestin-treated cells, these data implythat IFN gene programs are repressed/lost in response to ligand.LE analysis is a component of GSEA that allows one to identifyindividual genes that are present in multiple highly significantgene sets (i.e., core genes that drive the enrichment of aparticular gene set). LE analysis identified multiple genes thatare transcriptional targets of IFN signaling pathways (i.e., IFITs,IRF7, OAS1/2, STAT1, and MX2) whose regulation is lost in

Figure 1.

IFN signaling enriched in PR dataset. A, GSEA comparing gene expression datasets obtained using the Illumina Microarray dataset published in Haganet al, 2013. Vehicle versus R5020 (synthetic PR ligand) are compared in T47D cells stably expressing full-length PR. Select gene sets are shown from the C2 MSigDB.B, Summary of IFN-related gene sets that are included in the most significant (FDR < 0.05; 54 gene sets in total) gene sets from MSigDB C2 GSEA analysis.C, Top 30 ranking genes as identified using LE analysis on 54 gene sets referred to in B. Shading denotes genes that are IFN-signaling related and/or ISGs.

PR Represses ISG Transcription

www.aacrjournals.org Mol Cancer Res; 15(10) October 2017 1333




ligand-treated cells (Fig. 1C). These genes, classically activatedby IFNs, are collectively referred to as ISG. Cumulatively, thesecomputational data suggest that IFN gene programs are nega-tively regulated by progestins.

ISGs are downregulated by progestins in human breast cancerWe used a human tumor–explant model, previously described

by Singhal and colleagues (8), to determine whether progestin-dependent ISG downregulation occurs in human breast tumors.Sliced portions of estrogen receptor (ER)/PR–positive tumorsfrom 8 patients were obtained during surgical breast cancerresection. These tumor samples were grown in an ex vivo culturesystem in media containing vehicle (EtOH) or R5020 (24–48hours). RNA-seq was performed on cultures of each tumor sam-ple; these data were analyzed for ISG repression following R5020treatment. The heatmap in Fig. 2 shows fold expression (R5020/vehicle), for RNA-seq data obtained from all 8 patients, for selectISGs. There is a clear trend toward transcriptional repression forISGs in patient ex vivo tumor explants. These data indicate thatISGs are regulated by progestins not only in human breast cancercell lines (Fig. 1), but, importantly, in human breast tumors aswell (Fig. 2).

ISG repression is PR dependentTo further characterize the mechanism through which proges-

tins regulate ISG expression, we studied progestin-dependent ISGrepression in multiple breast cancer cell line models. A brief noteabout ourmost frequently usedmodel system: PR is an importanttarget gene of ER and, as such, PR expression is regulated by

estrogen in most tissues (19, 20). To differentiate between theeffects of ER/estrogen and PR/progesterone, our laboratory usesPR-positive (T47D-co) and PR-null (T47D-Y) variants of the ER/PRþ breast cancer cell line, T47D (21). T47D-co cells endoge-nously express both isoforms of PR, PR-A and PR-B, without theneed for exogenously added estrogen, allowing us to studythe function of PR without the confounding effects of estrogen.T47D-Y cells can also be used to reintroduce single isoformvariants or mutants of PR, such as PR-A (T47D-YA cells) andPR-B (T47D-YB cells) or DNA-binding mutant PR (T47D-PRB-mDBD). We have published extensively using these cell linemodels to define isoform- and phosphorylation-specific PR generegulation and protein–protein interactions (13, 14, 22–24), andthis cell line model remains a powerful and well-establishedsystem for studying PR activity (5).

To characterize regulation of ISGs by progestins, we analyzedRNA levels of ISGs selected from the LE gene list in T47D-co breastcancer cells. T47D-co cells were treated for 6 hours with syntheticprogestins (R5020 or medroxyprogesterone acetate), native pro-gesterone, or vehicle (EtOH); isolated RNA was used for qPCRanalysis. For the ISGs we assayed, six of which are shown in Fig.3A, expression was transcriptionally repressed (2- to 10-fold) byall progestins, both synthetic and native. Individual gene valida-tion (such as those shown in Fig. 3A, and data not shown) andmicroarray data mining (13) showed that this transcriptionalrepression is conserved for a large cohort of ISGs. Progestin-dependent ISG transcriptional downregulation can be mediatedvia both isoforms of PR, as T47D-Y cells stably expressing eitherPR-B or PR-A can both repress ISG RNA levels, although PR-Bappears to have greater transcriptional repressor activity on theISGs assayed (Supplementary Fig. S1A). In addition, because theT47D-co cells are unique in that exogenous estrogen is not neededfor PR expression, we verified that ISG transcripts were repressedfollowing progestin treatment in unmodified, parental T47D cellsgrown in an estrogenic environment (T47D-ATCC; Supplemen-tary Fig. S1B). To determine whether repression of ISGs by PR iscell-type specific,we stably expressed PR-B inHeLa cells (normallyPR-null) and observed ISG transcriptional repression in responseto progestin treatment (Supplementary Fig. S1C). Finally, thistranscriptional repression was associated with a concomitantdecrease in ISG protein levels. ISG proteins were assayed fromT47D-co breast cancer cells treatedwith ligand (R5020; 18 hours);decreased protein levels were observed for all ISGs assayed viaWestern blotting (Fig. 3B). Cumulatively, these data suggest thatligand-activated PR promotes the downregulation of ISG RNAand protein levels.

To determine whether ISG transcriptional repression followingprogestin treatment is dependent upon PR, we assayed ISGtranscript levels in cells where PR expression was knocked downusing a pool of four siRNAs. T47D-co cells transiently transfectedwith NS or PR siRNA were treated with vehicle or R5020 for 6hours, and RNA was analyzed using qPCR. ISG transcriptionalrepression in response to ligand was lost in cells with PR knock-down (Fig. 4A; PR knockdown efficiency shown in Fig. 4B). Thesedata were repeated in cells stably expressing PR shRNA (Sup-plementary Fig. S2). Surprisingly, basal levels (vehicle treated,in the absence of progestin) of ISGs were markedly higher incells lacking PR (compare vehicle bars between NS and PRsiRNA for each gene shown in Fig. 4A). Basal levels of selectISGs were increased 1.5- to 3.5-fold when PR expression wasknocked down using siRNA (Fig. 4C). These data indicate that

Figure 2.

Progestin-dependent ISG repression in human breast tumors. Human breasttumors were grown ex vivo for 24 to 48 hours in media containing vehicle or10 nmol/LR5020, followedbyRNA-seq. Heatmap represents fold RNAexpressionvalues for R5020/vehicle for seven ISGs, in each of eight patient samples. Redshading, downregulation of RNA expression in R5020-treated tumors; blue,gene upregulation in response to R5020 treatment; white, no change.

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PR has ligand-independent functions that appear tomaintain lowlevels of ISG expression. Moreover, when ISG expression wasassayed in two PR-negative (triple negative, lacking ER/PR/HER2)breast cancer cell lines, we saw a similar phenotype: no ISGrepression in response to ligand and high basal levels of ISGs(Supplementary Fig. S3). Together, these data indicate thatPR attenuates ISG expression, even in the absence of ligand,suggesting a concertedbiological programaimedatPR-dependentdownregulation of ISGs.

IFN activation of ISGs is repressed by ligand-activated PRMost typically, ISGs are activated in response to IFNs in spe-

cialized cell types of the innate immune system, such as dendriticcells. However, all cell types have the capacity to produce andrespond to IFNs (including fibroblasts and epithelial cells), aslong as the proper receptors (IFNARs) and transcriptionalmachinery are intact (10). Although our T47D-co cells do notsecrete endogenous IFNs (measured using highly sensitive ELISAassays designed to detect picomolar concentrations; data notshown), they retain their response to exogenous treatment withIFNaby transcriptionally upregulating ISGs (Fig. 5). Interestingly,when we cotreated cells with IFNa and progestin (R5020), wefound that progestin treatment attenuated the IFNa-induced ISGtranscriptional response, reducing it to near baseline levels (Fig.5). We concluded from these data that ligand-activated PR canrepress ISG transcripts in response to IFNa.

PR DNA binding needed for ISG transcriptional repressionThere are many potential mechanisms through which PR can

mediate transcriptional repression of target genes (25). To deter-mine whether DNA binding by PR is required to mediate ISGtranscriptional repression, we employed a well-characterizedDNA-binding domain (DBD) mutant of PR (PR-mDBD). Thismutant contains a single point mutation at Cys587, locatedwithin the first zinc finger of the PR DBD, which abolishes PR'sability to bind DNA (26). Using T47D-PRB-mDBD cells, wemeasured the capacity of mDBD PR to repress ISG transcription.

T47D cells stably expressing wt PR-B (T47D-YB, described above)robustly repress ISG transcription in response to ligand; this effectis lost in T47D-PRB-mDBD cells (Fig. 6A). These data suggest PRbinding to DNA is required for ISG transcriptional repression.IFNa-activated ISG transcription is regulated through ISREs,DNA sequences where the ISGF3 complex (STAT1, STAT2, andIRF9) binds and activates transcription. To test whether PRrepressed transcription via direct or indirect binding to ISREsequences, we used an ISRE-luciferase reporter construct stablyexpressed in Hela or Hela-PR cells. ISRE-linked luciferase activitywas unaffected by the presence of PR or its ligand in either cell line(Fig. 6B); no changes were seen basally or in response to IFNatreatment. These data imply that PR does not repress ISGtranscription by binding to or blocking the ISRE promoter ele-ment directly.

PR is recruited to ISG enhancersTo determine whether PR is directly recruited to promoters/

enhancers that regulate ISG transcription, we usedChIP to detectPR binding at ISG-regulatory regions. Asmentioned above, ISGsare regulated by promoter proximal ISREs, typically within100 bp of ISG transcriptional start sites (TSS; ref. 27). Usingpublished ChIP-seq data for ligand-activated PR binding (6), weidentified that all ISGs we assayed exhibited PR binding in theirpromoter/enhancer regions. It is well documented that nuclearreceptors often bind in intragenic or distal (>40 kb away fromthe TSS) enhancer regions of the genes they regulate (28–32); PRbinding to ISG enhancers falls well within this range (�18 kb toþ5 kb).We identified individual PREs within these binding sitesusing in silico analysis. Figure 7A (left) highlights the position ofthe PREs and ISREs (ISREs defined in ref. 27 or throughENCODE STAT1/2 ChIP-seq binding data; refs. 33–35) in rela-tion to the TSS for select genes assayed via ChIP-qPCR for PR andISGF3 (STAT1/STAT2/IRF9) binding. Using PRChIP-qPCR anal-ysis following R5020 treatment, we showed robust PR recruit-ment to enhancer regions of multiple ISGs (Fig. 7A; Supple-mentary Fig. S4); recruitment of PR to the IFIT3 promoter(Fig. 7A) is shown. All subsequent ChIP-qPCR results will be

Figure 3.

ISGs are repressed by ligand-activated PR. A, T47D PR-positive breast cancer cells (T47D-co) were starved for 18 hours in serum-free media, followed bytreatment with 10 nmol/L R5020, 10 nmol/L medroxyprogesterone acetate (MPA), 100 nmol/L native progesterone, or vehicle for 6 hours. Isolated RNA wasanalyzed for select genes. Gene values were normalized to an internal control (b-actin). Error bars represent SD between biological triplicates. Asterisksrepresent statistical significance between the vehicle-treated groups and all treatment groups (R5020, MPA, and progesterone); P < 0.05, as determined usingan unpaired Student t test. This experiment was performed in triplicate, and a representative experiment is shown here. B, T47D-co cells were starved for18 hours in serum-free media and then treated with 10 nmol/L R5020 or vehicle for 18 hours. Protein lysates were analyzed via Western blotting. ERKrepresents the loading control.






shown for the IFIT3 promoter as a representative example ofprotein recruitment to all ISG enhancer regions assayed andsummarized in Fig. 7A.

Although STAT1/STAT2/IRF9 historically comprise the ISGF3transcription factor complex, IRF9 and STAT2 complexed together(in the absence of STAT1) can recapitulate ISRE-mediated tran-scription (36). We therefore focused on how PR activation altersSTAT2/IRF9 recruitment to ISG promoter regions. We observedrobust recruitment of STAT2 and IRF9 to ISREs following treat-ment with IFNa (Fig. 7B). Interestingly, IFN-stimulated STAT2/IRF9 recruitmentwas decreased in the presence of PR ligand. BasalSTAT2/IRF9 occupancy in the absence of IFN is too low (at/aboveIgG levels) to generate a consistently robust signal from ChIP-qPCR. However, a trend exists toward STAT2/IRF9 loss whencomparing vehicle and R5020 in the absence of IFNa (Fig. 7B– compare vehicle and R5020 bars). This observation (loss ofSTAT2/IRF9) is potentiated when IFNa treatment, in combina-tion with PR ligand, is used. Moreover, we see a potent increase inH3K27ac1, a histone mark indicative of activated transcriptionand open chromatin, following treatment with IFN. Treatmentwith PR ligand decreases the levels of H3K27ac1, suggesting thatrecruitment of PR (and loss of STAT2/IRF9) leads to activerepression of these enhancers. Finally, in T47D-co cells where PRexpression was knocked down using shRNA directed against PR(or a nonsilencing control; NS), recruitment of STAT2/IRF9 waspotently enhanced in response to IFNa, indicating that the

Figure 5.

IFN-activated ISG expression is repressed by ligand-activated PR. Followingan 18-hour starvation in serum-free media, cells were treated for 18 hourswith IFNa (20 IU/mL) or vehicle (water) for 18 hours, followed by R5020(10 nmol/L) or vehicle (EtOH) for 6 hours. Isolated RNA was analyzed forexpression of IFIT1, IFIT2, and IFIT3. Gene expression values were normalized toan internal control (b-actin). Error bars represent SD between biologicaltriplicates. Asterisks represent statistical significance between the respectivevehicle and R5020-treated groups; P < 0.05, as determined using anunpaired Student t test. This experiment was performed in triplicate, and arepresentative experiment is shown here.

Figure 4.

ISG repression is PR dependent. A, T47D-co cells were transfected with nonsilencing (NS) or a pool of four PR siRNAs. Forty-eight hours following siRNAtransfection, cells were starved for 18 hours, followed by 10 nmol/L R5020 or vehicle for 6 hours. Isolated RNA was analyzed for select genes. Gene values werenormalized to an internal control (b-actin). Error bars represent SD between biological triplicates. Asterisks represent statistical significance between thevehicle and R5020-treated groups; P < 0.05, as determined using an unpaired Student t test. This experiment was performed in triplicate, and a representativeexperiment is shown here. B, PR knockdown efficiency by siRNA was determined using relative PR levels between the NS and PR siRNA transfected cells.Asterisks represent statistical significance; P < 0.0001. C, Fold increases in basal (no ligand) ISG expression between PR and NS siRNA transfected cells.Error bars represent the SD for the ratio of two variables using their individual SDs, calculated using the Delta method.

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presence of PR (ligand-independent) decreases ISG promoteroccupancy of ISGF3 components (Fig. 7C). Cumulatively, thesedata suggest that PRblocks/decreases efficient STAT2/IRF9 recruit-ment to ISRE regulatory regions, thereby decreasing transcriptionof ISGs. The ligand-dependent loss of STAT2/IFR9 occurs whenthe PRE and ISRE sequences are both located in the proximalpromoter regions (as is the case with IFIT3), as well as when thePR-binding sites occur upstream of the ISRE (data not shown).The details regarding how PR recruitment destabilizes STAT2/IRF9 DNA binding/recruitment are the subject of our ongoingexperiments.

DiscussionHerein,wedemonstrate that PR transcriptionally represses ISGs

in human breast cancer. This represents a novel class of genespreviously unknown to be regulated by PR. Although regulationof ISGs has been well defined in response to IFNs, PR-dependentrepression contributes a novel layer of ISG regulation. We showPR-mediated ISG repression in breast cancer cell lines, aswell as intumor explants from breast cancer patients. In addition, ISGrepression exists in response to PR ligand, as well as in unliganded(basal) conditions. These data indicate a concerted PR-dependenteffort aimed at maintaining low ISG levels in breast cancer.

The cancer immunoediting hypothesis highlights that theinnate and adaptive immune responses work together to flagearly neoplastic lesions for immunomediated elimination (11).An earlymediator of this elimination process is activation of type I

IFN signaling (10). Thus, suppression of type I IFN signaling mayhelp developing tumors evade the critical early steps of immunerecognition and subsequent clearance, allowing nascent tumorsto progress. In vivo studies support this notion. For example,female STAT1 knockout mice, lacking a key upstream activatorof type I IFN signaling, develop mammary gland adenocarcino-mas (37). We show that PR represses the end product of IFNsignaling, ISG transcription, suggesting that progesterone, work-ing through PR, may be a key player in tumor immune evasion. Anumber of previous observations support the importance of themechanismwedescribed, in thedevelopment of breast cancer. Forexample, 70% of breast cancers are ER/PR positive at the time ofdiagnosis, and recent data have suggested a positive correlationbetween PR positivity in benign, normal breast tissue and risk forthe development of breast cancer (38). Moreover, >90% of themammary gland tumors that form in the STAT1-deficientmice areER/PR positive and have gene expression profiles that mimic theluminal ER/PR–positive molecular subtype of breast cancer (37).These data suggest there is protumorigenic cross-talk between PRand IFN signaling pathways.

Downregulation of multiple components of IFN signaling hasbeen correlated with increased tumor incidence and metastasis.Mice lacking IFNAR, the IFNa-binding receptor that initiatesJAK/STAT signaling, which culminates in ISG transcription, haveenhanced tumor development, impaired ability to reject synge-neic/allogenic tumors, and accelerated metastasis in a spontane-ous mouse mammary gland tumor model (39, 40). A recentreport shows that IFNAR inactivation in tumor-associated stroma

Figure 6.

PR DNA-binding required for ISGtranscriptional repression.A, Followingan 18-hour starvation in serum-freemedia, T47D-YB or T47D-PRB-mDBDcells were treated for 6 hours withvehicle or 10 nmol/L R5020. IsolatedRNA was analyzed for select genes.Gene values were normalized to aninternal control (b-actin). Error barsrepresent SD between biologicaltriplicates. Asterisks representstatistical significance; P < 0.05, asdetermined using an unpaired Studentt test. B, HeLa and HeLa-PR cells thatwere stably transfected with an ISRE-luciferase were starved for 24 hours,followed by an 18-hour cotreatmentwith IFNa (or water) or 10 nmol/LR5020 (or EtOH). Luciferase assayswere performed as described in the"Materials and Methods" section. Errorbars represent SD of biologicalreplicates. The experiments in A and Bwere performed in triplicate, andrepresentative experiments are shownhere.






leads to an immune-privileged niche for developing colon cancers(41). Type I IFNs, in particular, and dendritic cells have beenshown to be critical to tumor cell rejection, and therefore immu-nosurveillance (42).Moreover, IFN-regulatory factor 7 (IRF7) andits downstream targets (many of which are ISGs shown herein tobe repressed by PR), are suppressed in the aforementioned mam-mary gland tumormetastasis model; restoration of IRF7 signalingsuppressed bone metastasis (39). Finally, expression of an IFNgene signature in a cohort of breast cancer patients was correlatedto a lower frequency of breast cancer metastasis (43). Cumula-tively, these data suggest mounting evidence that IFN signaling iscorrelated with tumor development and metastasis. Conversely,high/increased ISG expression has been linked to poor prognosisinmore advanced tumors. An IFN-relatedDNAdamage resistancesignature has been defined by Weichselbaum and Minn that isassociated with chemotherapy, radiation, and most recently,immunotherapy resistance in multiple tumor types, including

breast (12, 44–46). Therefore, ISG expression, and subsequentlythe effect of ISGs on tumor growth,may vary by tumor stage (earlyvs. late).

Other nuclear receptors,most notably the glucocorticoid recep-tor (GR), have been implicated in modulating IFN and inflam-matory responses. Inhibitory interactions between GR andAP1/NF-kB have been shown to inhibit a broad range of inflam-matory functions, largely through a mechanism termed transre-pression. In thismechanism, GR tethers to DNA-bound transcrip-tion factors/regulatory proteins and affects cofactor recruitmentand subsequent gene regulation (reviewed in ref. 47). Of note, GRhas been shown to repress ISG transcription in a cell-type–specificmanner in macrophages by squelching away an ISGF3 cofactor,GRIP1 (glutamate receptor interacting protein 1), needed for IFN-dependent activation of ISGs in macrophages (48). In thisinstance, the repression was independent of GR binding to DNA,although ligand-activated GR effectively repressed ISRE-luciferase

Figure 7.

PR diminishes recruitment of STAT2/IRF9 to ISG promoters. A, Left, table summary of ISG promoters/enhancers assayed by ChIP-qPCR for PR, STAT2,and IRF9 recruitment. Location of IFN-stimulated response elements (ISRE) and progesterone receptor response elements (PRE) from the transcriptional start site(TSS) of each gene is shown in kilobases (kb). Right, T47D-co cells were serum starved for 18 hours. Cells were then treated with 10 nmol/L R5020 or vehiclefor 30 minutes. Fixed lysates were subjected to ChIP with antibodies against PR or a species-specific IgG (control; not shown), and qPCR was performedon the isolatedDNAusingprimers designed to amplify the IFIT3 promoter. Apercentage of ChIP'dDNAover inputDNA is shown.B,T47D-co cellswere serumstarvedfor 18 hours. Cells were then treated with 10 nmol/L R5020, 1,000 IU/mL IFNa, or a cotreatment of both (or appropriate vehicle controls). Fixed lysates weresubjected to ChIP with antibodies against STAT2, IRF9, H3K27ac1, or a species-specific IgG (control; not shown), and qPCR was performed on the isolatedDNA using primers designed to amplify the IFIT3 promoter. A percentage of ChIP'd DNA over input DNA is shown. C, ChIP experiments were performed as inB, but T47D-co NS and PR shRNA cells were used, with only IFNa treatment. All ChIP experiments were performed in triplicate; a representative experiment isshown here. Fold recruitment in treated conditions (R5020, IFNa, or combination), as compared with vehicle treatment, is displayed above each bar.Error bars represent SD of technical replicates.

Walter et al.





activity. This is in contrast to our findings (Fig. 6B) where PRligand treatment was not able to repress an ISRE-luciferase con-struct, implying that PR-dependent ISG repression occurs via amechanism independent of cofactor squelching. Our data suggestthat PR-mediated repression of ISGs in breast cancer cells occursthrough a different mechanism than that proposed for GR-medi-ated ISG repression in macrophages, perhaps indicating cell type-and nuclear receptor–specific regulation. Recent work, however,suggests that select GR-dependent gene repression events mayrequire direct GR binding to DNA through GR response elements(GRE). These GREs are in close proximity to AP1 and NF-kB–binding sites. GRE-mediated binding of GR leads to recruitmentof GRIP1, the aforementioned ISGF3 cofactor and known GRcorepressor, leading to repressive changes in the chromatin andsubsequent gene repression (49). Thismechanismhas similaritiesto the mechanism we propose herein for PR-mediated, and PRE-dependent, ISG transcriptional repression. Because we observedecreased recruitment of ISGF3 components (STAT2 and IRF9) toISRE promoter sequences following PR activation and recruit-ment of PR to PREs, we favor amodel where protein displacementor steric competition occurs between PR and STAT2/IRF9. Werecognize that transcriptional repression is complex, and a com-bination of mechanisms may contribute to PR-dependent ISGrepression. Our future work is focused on detailing this mecha-nism(s).

Although PR gene activation has been well characterized, themechanisms through which PR represses transcription remainpoorly understood. Various mechanisms have been put forth forPR-mediated transcriptional repression, and are similar to whathas beenwell characterized for GR-mediated repression (squelch-ing of cofactors, recruitment of corepressors, chromatin remodel-ing; reviewed in ref. 25). A recent study details ligand-dependentrecruitment of PR, an HP1g–LSD1 repressive complex, and BRG1to repressed target gene promoters/enhancers. This repressivecomplex leads to ligand-dependent changes in chromatin archi-tecture that result in transcriptional repression (50). Although weobserved a decrease in activating histone marks at ISG enhancersfollowing treatment with PR ligand (Fig. 7B), preliminary datashow that knockdown of BRG1, LSD1, or HP1g had no effect onPR-dependent transcriptional repression of ISGs (not shown),suggesting an alternative (non- HP1g/LSD1-dependent) mecha-nism for PR-mediated ISG transcriptional repression. Experi-

ments to investigate PR-dependent recruitment of other chroma-tin modifiers to ISG enhancers are currently under way.

In summary, our results show novel PR-dependent repressionof ISG transcription. These data have significant implications forthe regulation of IFN signaling in breast cancer and provide aputative mechanism through which nascent breast cancers mayavoid immunosurveillance. Future directions will be aimed atunderstanding what effect PR-dependent downregulation of ISGshas on breast cancer development and progression.

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

Authors' ContributionsConception and design: K.R. Walter, M.L. Goodman, C.R. HaganDevelopment of methodology: K.R. Walter, M.L. Goodman, C.R. HaganAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.R. Walter, M.L. Goodman, H. Singhal, J.A. Hall,S.M. Holloran, G.M. Trinca, G.L. Greene, C.R. HaganAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.R. Walter, M.L. Goodman, H. Singhal, J.A. Hall,T. Li, G.M. Trinca, V.X. Jin, G.L. Greene, C.R. HaganWriting, review, and/or revision of the manuscript: K.R. Walter,M.L. Goodman, H. Singhal, J.A. Hall, G.M. Trinca, K.A. Gibson, G.L. Greene,C.R. HaganAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.R. HaganStudy supervision: C.R. Hagan

AcknowledgmentsThe Center for Functional Cancer Epigenetics (CFCE) at the Dana-Farber

Cancer Institute provided the computing infrastructure for RNA-seq analysis.We thank Drs. Joseph Fontes (KUMC), Chad Slawson (KUMC), and ToddKnutson (Minnesota) for critical scientific input.

Grant SupportThisworkwas supported byNCIR00CA166643 (toC.R.Hagan),DODBCRP

W81XWH-16-1-0320 (to C.R. Hagan), Susan G Komen FoundationCCR16376147 (to C.R. Hagan), V Foundation V2015-025 (to C.R. Hagan),and the NCI Cancer Center Support Grant P30 CA168524 (to C.R. Hagan).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 5, 2017; revised June 20, 2017; accepted July 3, 2017;published OnlineFirst July 6, 2017.

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Walter et al.




2017;15:1331-1340. Published OnlineFirst July 6, 2017.Mol Cancer Res Katherine R. Walter, Merit L. Goodman, Hari Singhal, et al. in Breast CancerInterferon-Stimulated Genes Are Transcriptionally Repressed by PR

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Interferon-StimulatedGenesAreTranscriptionally Repressed ...mcr.aacrjournals.org/content/molcanres/15/10/1331.full.pdf · Tianbao Li5, Sean M. Holloran1,2,3, Gloria M.Trinca1,2,3,