Attenuates STAT1-Mediated IFN Signaling in Breast Cancer

This information is current as Merit L. Goodman, Gloria M. Trinca, Katherine R. Walter, of September 30, 2021. Evangelia K. Papachristou, Clive S. D'Santos, Tianbao Li, Qi Liu, Zhao Lai, Prabhakar Chalise, Rashna Madan, Fang Fan, Mary A. Markiewicz, Victor X. Jin, Jason S. Carroll and Christy R. Hagan

J Immunol 2019; 202:3076-3086; Prepublished online 1 Downloaded from April 2019; doi: 10.4049/jimmunol.1801152 http://www.jimmunol.org/content/202/10/3076 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2019/03/29/jimmunol.180115 Material 2.DCSupplemental References This article cites 54 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/202/10/3076.full#ref-list-1

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Progesterone Receptor Attenuates STAT1-Mediated IFN Signaling in Breast Cancer

Merit L. Goodman,*,†,‡,1 Gloria M. Trinca,*,†,‡,1 Katherine R. Walter,*,†,‡ Evangelia K. Papachristou,x Clive S. D’Santos,x Tianbao Li,{ Qi Liu,{ Zhao Lai,{,‖ Prabhakar Chalise,# Rashna Madan,** Fang Fan,** Mary A. Markiewicz,†† Victor X. Jin,{ Jason S. Carroll,x and Christy R. Hagan*,†,‡

Why some tumors remain indolent and others progress to clinical relevance remains a major unanswered question in cancer biology. IFN signaling in nascent tumors, mediated by STAT1, is a critical step through which the surveilling immune system can recognize and destroy developing tumors. In this study, we have identified an interaction between the (PR) and STAT1

in breast cancer cells. This interaction inhibited efficient IFN-induced STAT1 phosphorylation, as we observed a decrease in Downloaded from phospho-STAT1 in response to IFN treatment in PR-positive breast cancer cell lines. This phenotype was further potentiated in the presence of PR ligand. In human breast cancer samples, PR-positive tumors exhibited lower levels of phospho-STAT1 as compared with their PR-negative counterparts, indicating that this phenotype translates to human tumors. Breast cancer cells lacking PR exhibited higher levels of IFN-stimulated gene (ISG) RNA, the transcriptional end point of IFN activation, indicating that unliganded PR alone could decrease transcription of ISGs. Moreover, the absence of PR led to increased recruitment of STAT1,

STAT2, and IRF9 (key transcription factors necessary for ISG transcription) to ISG promoters. These data indicate that PR, both in http://www.jimmunol.org/ the presence and absence of ligand, attenuates IFN-induced STAT1 signaling, culminating in significantly abrogated activation of genes transcribed in response to IFNs. PR-positive tumors may use downregulation of STAT1-mediated IFN signaling to escape immune surveillance, leading to the development of clinically relevant tumors. Selective immune evasion of PR-positive tumors may be one explanation as to why over 65% of breast cancers are PR positive at the time of diagnosis. The Journal of Immunology, 2019, 202: 3076–3086.

umor escape from the surveilling immune system is a cell surface receptor, allowing the JAK-STAT signaling cascade to critical first step in tumor development and essential initiate. Type I IFNs, such as IFN-a/b, bind their cognate IFN-a/b T for tumor progression (1). The cancer immunoediting receptor complex, which is associated with tyrosine kinases. The by guest on September 30, 2021 hypothesis postulated by Schreiber et al. (2) highlights that the activation of both JAK1 and tyrosine kinase 2 (TYK2) results in innate and adaptive immune responses work together to “flag” the downstream phosphorylation of both STAT1 and STAT2. early neoplastic lesions for immune-mediated elimination. An The phosphorylated STAT1–STAT2 heterodimer associates with a early mediator of this elimination process is activation of type I third , IFN regulatory factor 9 (IRF9), leading IFN signaling (3–5). Thus, suppression of IFN signaling may to the formation of the IFN-stimulated gene (ISG) factor 3 (ISGF3) help developing tumors evade the critical early steps of immune complex. The ISGF3 complex then translocates to the nucleus recognition and clearance. and binds DNA sequences referred to as IFN-stimulated response IFN signaling encompasses the mechanism through which IFNs elements (ISREs) within the ISG promoter regions, leading to ISG (type I–III) are made and released by the host cell in response to a transcription. Collectively, the IFN-induced production of these viral infection. IFNs are cytokines that bind their corresponding ISGs limits the spread of virally infected cells by promoting an

*Department of Biochemistry and Molecular Biology, University of Kansas Medical Foundation (to V.X.J.), and NCI Cancer Center Support Grant P30 CA168524 Center, Kansas City, KS 66160; †Department of Cancer Biology, University of (to C.R.H.). Kansas Medical Center, Kansas City, KS 66160; ‡University of Kansas Cancer x The sequences presented in this article have been submitted to the Gene Expression Center, Kansas City, KS 66160; Cancer Research UK Cambridge Institute, Univer- { Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126517) under sity of Cambridge, Cambridge CB2 0RE, United Kingdom; Department of Molecular accession number GSE126517. Medicine, University of Texas Health Science Center San Antonio, San Antonio, TX 78229; ‖Greehey Children’s Cancer Research Institute, University of Texas Health Address correspondence and reprint requests to Christy R. Hagan, Department of Bio- Science Center San Antonio, San Antonio, TX 78229; #Department of Biostatistics, chemistry and Molecular Biology, MS3030, University of Kansas Medical Center, 3901 University of Kansas Medical Center, Kansas City, KS 66160; **Department of Rainbow Boulevard, Kansas City, KS 66160. E-mail address: [email protected] Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas The online version of this article contains supplemental material. City, KS 66160; and ††Department of Microbiology, Molecular Genetics, and Immu- nology, University of Kansas Medical Center, Kansas City, KS 66160 Abbreviations used in this article: ChIP, chromatin IP; ER, ; EtOH, ethyl alcohol; FDR, false discovery rate; GSEA, gene set enrichment analysis; IHC, 1M.L.G. and G.M.T. contributed equally to this work. immunohistochemical; IP, immunoprecipitation, immunoprecipitated; IRF9, IFN reg- ORCIDs: 0000-0001-6888-1199 (K.R.W.); 0000-0001-5685-8573 (M.A.M.). ulatory factor 9; ISG, IFN-stimulated gene; ISGF3, IFN-stimulated gene factor 3; ISRE, IFN-stimulated response element; LE, leading edge; MS, mass spectrometry; Received for publication August 21, 2018. Accepted for publication March 11, 2019. MSigDB, Molecular Signatures Database; PR, progesterone receptor; qPCR, quan- This work was supported by the National Cancer Institute (NCI) R00CA166643 titative PCR; RIME, rapid immunoprecipitation mass spectrometry of endogenous (to C.R.H.), Department of Defense Breast Cancer Research Program W81XWH- proteins; RNA-Seq, RNA sequencing; shRNA, short hairpin RNA; TMA, tissue 16-1-0320 (to C.R.H.), Susan G. Komen Foundation CCR16376147 (to C.R.H.), microarray; TYK2, tyrosine kinase 2. V Foundation V2015-025 (to C.R.H.), National Institutes of Health R01GM114142 (to V.X.J.), U54CA217297 (to V.X.J.), and 1S10OD021805-01 (to Z.L.), the Owens Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801152 The Journal of Immunology 3077 antiproliferative and/or proapoptotic response in addition to the Scientific), STAT1 (9172; Cell Signaling), p-STAT1 (7649; Cell Signal- activation of immune clearance mechanisms (reviewed in Ref. 6). ing), p-TYK2 (68790; Cell Signaling), TYK2 (14193; Cell Signaling), IFN responsiveness and signaling play a critical role during viral STAT2 (4594; Cell Signaling), IRF9 (10793; Santa Cruz), IFIT1 (14769; Cell Signaling), IFIT2 (sc-390724; Santa Cruz Biotechnology), infections; however, emerging data suggest an important role for IFIT3 (sc-393512; Santa Cruz Biotechnology), OAS1 (14498; Cell Sig- IFN in tumorigenesis through modulation of immune surveillance. naling), topoisomerase II-a (12286; Cell Signaling), and b-tubulin (2128; Independent of viral infection, both IFNs and ISG expression have Cell Signaling). All Western blotting experiments were performed in been found in human tumors (4, 7, 8). Although the effects of IFN triplicate, and representative experiments are shown. signaling and ISG phenotypes vary in cancer biology, it remains Breast cancer tissue microarray clinically relevant to gain a deeper understanding of IFN signaling Tissue microarrays (TMAs) were constructed from archival formalin-fixed, regulation in this context (4, 9). We have previously shown that paraffin-embedded samples of invasive mammary carcinoma (43 patients) the progesterone receptor (PR), a steroid-activated nuclear re- as well as matched benign breast tissue (35 patients). These samples were ceptor implicated in breast cancer, can transcriptionally repress identified from the pathology departmental archives of the University ISGs in breast cancer via decreased recruitment of ISGF3 com- of Kansas Medical Center from 1997 to 2011. Based on review of the ponents to ISREs in response to progestins (10). In this study, we original pathology reports, the invasive mammary carcinomas were typed as invasive ductal carcinoma (39 patients), metaplastic carcinoma (1 patient), present a mechanism whereby PR, in the absence of its activating invasive mammary carcinoma with ductal and lobular features (1 pa- ligand, can attenuate IFN signaling through decreased STAT1 tient), invasive ductal carcinoma with a minor lobular component (1 patient), activation/phosphorylation. The potent loss in IFN signaling sen- and invasive lobular carcinoma (1 patient). Using the semiautomated sitivity may contribute to the escape of malignant cells from the TMArrayer (Pathology Devices, Westminster, MD), TMA paraffin blocks were assembled with 2.0-mm cores. surveilling immune system. Downloaded from Immunohistochemistry Materials and Methods Phospho-STAT1 (no. 8826; Cell Signaling), STAT1 (no. 14994; Cell Signaling), Cell lines and constructs and PR (no. M3568; Dako) Abs were used for immunohistochemical (IHC) staining according to the following procedure: 4-mm paraffin T47D-co, T47D-Y, and T47D-YB have been previously described and sections were mounted on Fisherbrand Superfrost slides and baked for were a generous gift of Dr. C. Lange (Minneapolis, Minnesota) (11, 12). 60 min at 60˚C then deparaffinized. Epitope retrieval was performed MCF7 (13) and T47D-co short hairpin RNA (shRNA) (10) cells have been in a Biocare Decloaking Chamber (pressure cooker) under pressure http://www.jimmunol.org/ previously described. Cells were treated with the following reagents (when for 5 min, using pH 6 citrate buffer followed by a 10-min cooldown applicable): R5020 (10 nM; Sigma-Aldrich) and human rIFN-a (IFN-a2A, period. Endogenous peroxidase was blocked with 3% H2O2 for 10 min, SPR4594; Sigma-Aldrich). followed by incubation with phospho-STAT1 (1:800), STAT1 (1:3200), or Rapid immunoprecipitation mass spectrometry of PR (1:2000) primary Ab for 30 min (PR) or 45 min (p-STAT1, STAT1), followed by Mach 2 HRP Polymer (Biocare Medical) for 30 min (p-STAT1, endogenous proteins STAT1) or Envision+ Anti-Mouse (Dako) for 30 min (PR) and DAB+ Rapid immunoprecipitation (IP) mass spectrometry (MS) of endogenous chromogen (Dako) for 5 min. IHC staining was performed using the proteins (RIME) experiments were performed as previously described IntelliPATH FLX Automated Stainer at room temperature. A light hema- (14–16) in R5020-treated (10 nM for 60 min) T47D-YB or T47D-Y cell toxylin counterstain was performed, following which, the slides were lines that were cross-linked with 1% formaldehyde for 10 min. IPs were dehydrated, cleared, and mounted using permanent mounting media. A by guest on September 30, 2021 performed using a PR Ab (20 mg, sc-7208; Santa Cruz Biotechnology). The pathologist then scored the slides according to the intensity of staining (0 = peptide samples were analyzed on a Dionex Ultimate 3000 UHPLC system no staining, 1 = mild intensity, 2 = moderate intensity, 3 = strong intensity), coupled with the LTQ Orbitrap Velos mass spectrometer (Thermo Fisher percentage of positive cells, and the subcellular localization of the staining. Scientific). For the separation of the peptides, a multistep gradient elution A second pathologist blindly scored the slides to identify any discordant was used: mobile phase (A) was composed of 2% acetonitrile, 0.1% formic results. For statistical analysis, the Wilcoxon rank-sum test was used to acid, and 5% DMSO, and mobile phase (B) was composed of 80% ace- assess the differences in the phospho-STAT1 and STAT1 intensities be- tonitrile, 0.1% formic acid, and 5% DMSO. The gradient elution method at tween two groups of subjects (PR-positive versus PR-negative tumors). flow rate 300 nl/min was as follows: for 65 min, gradient up to 45% (B); for 10 min, gradient up to 95% (B); for 10 min, isocratic 95% (B); for Subcellular fractionation 5 min, down to 5% (B); and for 10 min, isocratic equilibration 5% (B) at Subcellular fractionation studies were performed as described previously 40˚C. The full scan was performed in the Orbitrap in the range of 400– (17), with modifications noted in the figure legend. 1600 m/z at 60K resolution. The tandem MS scan was performed with collision-induced dissociation energy 30% and exclusion duration 30 s. RNA sequencing The raw data were processed in Proteome Discoverer 1.4 using the Sequest HT search engine. The node for Sequest HT included the following pa- Approximately 500 ng total RNA was used for stranded total RNA se- rameters: precursor mass tolerance, 20 ppm; fragment mass tolerance, quencing (RNA-Seq) library preparation by following the KAPA Stranded 0.5 Da; and dynamic modifications, which were oxidation of M (+15.995 Da) RNA-Seq Kit with RiboErase (HMR) sample preparation guide. The first and deamidation of N and Q (+0.984 Da). Significant peptides were filtered step in the workflow involved the depletion of rRNA by hybridization of at a false discovery rate (FDR) ,1%, and specific interactors were con- cDNA oligonucleotides, followed by treatment with RNase H and DNase sidered if they were identified in both RIME replicate experiments in to remove rRNA duplexed to DNA and original DNA oligonucleotides, T47D-YB cells but not in the T47D-Y (PR-null) cell line. respectively. Following rRNA removal, the RNA was fragmented into small pieces using divalent cautions under elevated temperature and Co-IP magnesium. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers. This was fol- For co-IP experiments, cell lysates were collected in RIPA buffer (sup- lowed by second-strand cDNA synthesis using DNA polymerase I and plemented with protease/phosphatase inhibitors) and incubated on ice for RNase H. Strand specificity was achieved by replacing dTTP with dUTP 30 min. Cell lysates containing equivalent protein concentrations (1000 mg) in the second strand marking mix. The incorporation of dUTP in second- were incubated overnight at 4˚C with 2 mg of appropriate Ab or control IgG. strand synthesis effectively quenches the second strand during amplifica- Protein G agarose (Roche Diagnostics, Indianapolis, IN) was added for the tion because the polymerase used in the assay will not incorporate past this final 2 h of incubation time. Immune complexes were washed three times with nucleotide. These cDNA fragments then went through an end repair pro- supplemented RIPA buffer, resuspended in Laemmli sample buffer containing cess, the addition of a single “A” base, and then ligation of the adapters. 2-ME, boiled for 5 min, and subjected to Western blotting analysis. The products were then purified and enriched with PCR to create the final Immunoblotting RNA-Seq library. RNA-Seq libraries were subjected to quantification process, pooled for cBot amplification, and subsequently sequenced with Immunoblotting/Western blotting was performed as previously described 50-bp single-end sequencing run with an Illumina HiSeq 3000 platform. (10–12). Membranes were probed with primary Abs recognizing total After the sequencing run, demultiplexing with Bcl2fastq2 was employed PR (sc-7208; Santa Cruz Biotechnology or MS-298-P; Thermo Fisher to generate the FASTQ file for each sample. 3078 PR ATTENUATES STAT1 SIGNALING Downloaded from http://www.jimmunol.org/

FIGURE 1. PR interacts with STAT1. (A) Search Tool for Retrieval of Interacting Genes/Proteins (STRING) network of the top 30 PR interactors identified in RIME. The size of the node increases proportionally to the number of identified peptides, and thick edges denote high-confidence STRING interactions (0.7–0.99). Asterisk used to denote location of PGR and STAT1 on STRING diagram. (B) Sequence coverage of PR and STAT1 in both replicate RIME experiments. Green highlights high-confidence peptides at FDR ,1%. PR and STAT1 have been identified by 27 and 19 unique peptides, respectively. (C) Left: PR was IP from starved T47D-YB cell lysates (with or without R5020; 60 min), and the resulting associated protein complexes were by guest on September 30, 2021 analyzed by Western blotting. Bottom panels represent total input cell lysates. Species-specific (rabbit or mouse) IgG was used as a control for the IP. Right: STAT1 was IP from T47D-co cell lysates (with or without R5020), and the resulting associated protein complexes were analyzed by Western blotting. Bottom panels represent total input cell lysates. PR-B (upper) and PR-A (lower) isoforms are both recognized by the PR Ab. Species-specific IgG was used as a control for the IP; the band visible in the IgG only lanes represents nonspecific binding between PR/STAT1 and the IgG Ab. These experiments were performed in triplicate, and a representative experiment is shown.

RNA-Seq data processing was performed as previously described (18). in the figure legends. qPCR was performed using the Faststart Essential DNA Briefly, RNA-Seq FASTQ files were aligned to hg19 using TopHat (version Green Master (Roche) on a Roche LightCycler 96. Relative concentrations 2.0.14), and only uniquely mapped reads were used for further downstream were quantified using the LightCycler 96 (software 1.1, Absolute Quantification analysis (19). Expression levels of genes were measured with normalized Analysis; Roche) using a six-point standard curve. counts of reads by their respective lengths using the Cufflinks 2.0.2 package, followed by their distribution analysis as fragments per kilobase million unit Chromatin IP assays (20). These data have been deposited in the National Center for Biotech- Chromatin IP (ChIP) was performed using the ChIP-IT Express Kit (Active nology Information’s Gene Expression Omnibus (21) and are accessible Motif) according to manufacturer’s instructions, using sonication for chromatin through Gene Expression Omnibus series accession number GSE126517 shearing. Lysates were IP overnight (18 h) with the following Abs: STAT1 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126517). (sc-346; Santa Cruz), STAT2 (sc-476; Santa Cruz), IRF9 (sc-10793; Santa Cruz), or an equal amount of negative control mouse or rabbit IgG. Resulting Gene set enrichment analysis DNA was analyzed using qPCR as described above, and data are represented Gene set enrichment analysis (GSEA) was performed using the javaGSEA asapercentageofinputDNA. desktop software; the c2 Molecular Signatures Database (MSigDB) version 5.2 was queried (22, 23). Dataset files were developed based on normalized Illumina Statistical analysis expression intensities from PR-null and PR-positive T47D cells (as described Statistical significance for all experiments was determined using an RNA sequencing in the section). Specifically, the log2-transformed expression unpaired Student t test, unless otherwise specified. A p value #0.05 is a values were compared for two phenotypes: T47D PR null (IFN- /vehicle) considered statistically significant. The delta method was used to calculate a and T47D PR positive (IFN- /vehicle). GSEA was executed using the default SD for the ratio of two variables using their individual SD, as seen when settings, except the permutation type was set to Gene_set with 1000 permu- plotting fold-relative RNA expression data between two treatment groups tations, and the metric for ranking genes was set to Diff_of_Classes, because and cell lines (24). normalized expression data were log2 transformed. Leading edge (LE) analysis was performed on the 29 gene sets from MSigDB c2 analysis that achieved FDR values #0.05. Results Real-time quantitative PCR PR interacts with STAT1 RNA isolation, cDNA creation, and quantitative PCR (qPCR) were performed Our laboratory has a long-standing interest in defining how as previously described (10–12), with modifications noted in this article and PR affects the activity of other transcription factors, particularly The Journal of Immunology 3079 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 2. PR attenuates IFN-induced STAT1 phosphorylation. (A) T47D cells that are PR negative (T47D-Y) or PR positive (T47D-co) were treated with IFN-a for 0–2 h. Isolated protein lysates were analyzed by Western blotting. Densitometry of the ratio of p-STAT1/total STAT1, as determined using ImageJ analysis, is shown to the right of the immunoblot. (B) T47D-co cells were starved for 18 h in serum-free media, followed by treatment with IFN-a and R5020 or vehicle (EtOH) for 0–2 h. Isolated protein lysates were analyzed by Western blotting. Densitometry of the ratio of p-STAT1/total STAT1, as determined using ImageJ analysis, is shown to the right of the immunoblot. (C) T47D-co cells were treated as in (B). Whole cell lysates were subjected to nuclear/cytoplasmic fractionation, and resulting subcellular lysates were analyzed by Western blotting. b-tubulin (cytoplasmic) and topoisomerase II (topo II; nuclear) are shown as fractionation markers. These experiments were performed in triplicate, and a representative experiment is shown. through protein–protein interactions. To identify novel protein the confounding effects of estrogen. T47D-Y (PR-null) cells binding partners of PR, we used an IP/MS approach called RIME. can be used to determine the effect of PR isoform variants or This technique, pioneered by the Carroll laboratory to identify mutants, such as PR-A (T47D-YA cells) and PR-B (T47D-YB cells). estrogen receptor (ER)–interacting proteins, has been a powerful We have published extensively using these cell line models to tool to study cross-talk between steroid receptors and other nu- define isoform- and phosphorylation-specific PR gene regulation clear proteins (14, 15). PR is an important target gene of ER, and protein–protein interactions (10–12, 28–30), and this cell line and, as such, PR expression is regulated by estrogen in most model remains a powerful and well-established system for tissues (25, 26). To differentiate between the effects of ER/estrogen studying the transcriptional activity of PR (31). and PR/progesterone, our laboratory uses PR-positive (T47D-co) We used T47D-YB (stably expressing the full-length PR-B isoform) and PR-null (T47D-Y) variants of the ER/PR+ breast cancer cell and PR-null (T47D-Y) cells as a model system to study PR–protein line T47D (27). T47D-co cells endogenously express both iso- interactions using RIME. Briefly, T47D-YB and T47D-Y cells were forms of PR, PR-A, and PR-B, without the need for exogenously cross-linked following treatment with a synthetic PR ligand (R5020) added estrogen, allowing us to study the function of PR without or vehicle (ethyl alcohol [EtOH]). PR was IP from isolated nuclei, 3080 PR ATTENUATES STAT1 SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 3. Regulation of p-STAT1/STAT1 is disrupted in PR-positive tumors. (A) TMA analysis was performed using IHC staining with phospho- STAT1 (p-STAT1), STAT1, and PR Abs. Boxplots and stripcharts show the distribution of p-STAT1 (left) and STAT1 (right) staining intensities in PR-negative and PR-positive breast cancer samples. (B) Select PR-negative (left) and PR-positive (right) breast cancer (BrCa) cases stained for PR, p-STAT1, and total STAT1 are shown at original magnification 320. Negative control isotype-only control staining is shown. (C) Scatter plot of pSTAT1 and STAT1 with different colors for PR positive (red) and PR negative (blue), with trend lines for each. The Spearman correlation coefficient and p values for each group are shown on top. Only tumors with .0 total STAT1 staining were included in the analysis. and PR-interacting proteins were identified using MS; the complete PR and STAT1 interact, and this interaction is potentiated when list of PR-interacting proteins identified using RIME is provided as PR is activated by ligand. Supplemental Table I. Among the top 30 identified PR-interacting proteins was STAT1 (Fig. 1A); peptide sequence coverage for PR PR attenuates IFN-induced STAT1 phosphorylation and STAT1 is shown in Fig. 1B. The interaction between PR and STAT1 is a key mediator of the innate immune response, typically STAT1 was validated using co-IP in T47D-YB cells (cell line used in response to viral infection. However, IFN activation in cancer for the RIME experiments): an interaction between PR and STAT1 cells, independent of viral infection, has been widely reported (4). could be detected in vehicle-treated cells, and this interaction was In response to IFN-a stimulation, STAT1 and STAT2 are phosphor- slightly increased in cells treated with R5020 (Fig. 1C, left). The ylated by JAK1 and TYK2. Phosphorylated STAT1/2 form hetero- interaction between PR and STAT1 was also observed in unmodi- dimers that bind with IRF9 to form a transcriptional complex referred fied T47D-co cells, endogenously expressing both isoforms of PR to as ISGF3. This transcriptional complex binds to ISREs throughout (Fig. 1C, right); both PR-A (lower band) and PR-B (upper band) the genome, activating transcription of a cohort of genes essential for appear to interact with STAT1, suggesting that the interaction be- mounting the cellular antiviral response (5). To determine if the in- tween PR and STAT1 is not unique to the full-length PR-B isoform. teraction between PR and STAT1 affects the function of STAT1, we Interestingly, the interaction between STAT1 and PR-B is increased looked at STAT1 phosphorylation in response to IFN-a treatment. following treatment with PR ligand, as compared with the STAT1/ T47D cells that are positive (T47D-co) or negative (T47D-Y) for PR-A interaction. These data suggest that the STAT1/PR-B inter- PR expression were treated for 0–2 h with IFN-a. Cells lacking PR action is primarily regulated by ligand, whereas the STAT1 inter- expression (PR null) phosphorylated STAT1 on an earlier time action with PR-A may be a more basal interaction, unaffected by course and at a greater magnitude in response to IFN-a,ascom- ligand. Moreover, an interaction between PR and STAT1 was also pared with PR-positive cells (Fig. 2A, left and quantification of shown in MCF7 cells, an additional PR-positive breast cancer cell Fig. 2A, right). There are two isoforms of STAT1, STAT1a (larger) line (Supplemental Fig. 1A). Cumulatively, these data show that and STAT1b (smaller); both isoforms appear to be equally affected The Journal of Immunology 3081 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021 FIGURE 4. TYK2 phosphorylation is attenuated by activated PR. (A) T47D-co cells were starved for 18 h in serum-free media, followed by treatment with IFN-a and R5020 or vehicle (EtOH) for 0–30 min. Isolated protein lysates were IP with the total TYK2 Ab, and the resulting protein complexes were blotted with phospho-TYK2 Ab. Species-specific IgG was used as a control for the IP. Densitometry of the ratio of p-TYK2/total TYK2, as determined using ImageJ analysis, is shown to the right of the immunoblot. (B) TYK2 was IP from T47D-co cell lysates (with or without R5020; 60 min), and the resulting associated protein complexes were analyzed by Western blotting. Bottom panels represent total input cell lysates. PR-B (upper) and PR-A (lower) isoforms are both recognizedbythe PR Ab. Species-specific IgG was used as a control for the IP. These experiments were performed in triplicate, and a representative experiment is shown. by the presence/absence of PR. Moreover, treatment of PR-positive previously described, custom-designed breast tumor TMA (13). cells (T47D-co) with PR ligand (R5020) even further attenuated In brief, this breast cancer TMA is composed of specimens phospho-STAT1 in response to IFN-a treatment (Fig. 2B, left and collected from 39 breast cancer patients seen at the University quantification of Fig. 2B, right). Decreased STAT1 phosphorylation of Kansas Medical Center, 21 of which were PR positive and in the presence of activated PR was repeated in MCF7 cells 18 of which were PR negative. We stained this breast TMA (Supplemental Fig. 1B, 1C). These data suggest that both the with Abs that recognize phospho-STAT1 and total STAT1, and presence (PR negative versus PR positive) and activation of PR staining intensities were blindly scored by a clinical pathol- (with or without ligand) decreases the levels of phospho-STAT1 ogist. Interestingly, PR-positive tumors had lower phospho-STAT1 in response to IFN treatment, likely because of the interaction staining intensity when compared with their PR-negative coun- between PR and STAT1 (see Fig. 1C). STAT2 phosphorylation, terparts. Although statistical analysis (Wilcoxon rank-sum test) another downstream effector of IFN-a signaling, was unaffected did not show statistically significant differences in the intensities by the presence or activation of PR following IFN-a treatment (likely because of the limited numbers of samples on the TMA), (data not shown). Finally, to determine if the decrease in IFN- there is a clear trend toward lower staining intensity in PR-positive induced phosho-STAT1 following treatment with PR ligand was tumors (Fig. 3A, left). The trend between lower phospho-STAT1 restricted to a particular cellular compartment, we used subcellular staining in PR-positive tumors appears to be specific for phos- fractionation experiments to assay phospho-STAT1. We observed phorylated STAT1, as no trend or statistical significance exists for similar decreases in phospho-STAT1 following treatment with IFN total STAT1 staining intensity between PR-positive and PR-negative and PR ligand in the nuclear and cytoplasmic fractions, as well as tumors (Fig. 3A, right). Select examples from the TMA are shown the whole cell lysate, following treatment with PR ligand. in Fig. 3B. As STAT1 itself is an ISG regulated by IFN signaling (32), higher activation of STAT1 (measured via phosphorylation) Regulation of p-STAT1/STAT1 is disrupted in PR-positive is normally positively correlated with total STAT1 levels, representing human breast tumors a positive-feedback regulatory loop. A scatter plot (Fig. 3C) of To determine if there is a correlation between PR positivity and p-STAT1 and STAT1 staining intensities for each tumor high- phospho-STAT1 levels in human breast tumors, we analyzed a lights the positive correlation between p-STAT1 and total STAT1 3082 PR ATTENUATES STAT1 SIGNALING in PR-negative tumors (blue line; r = 0.7188, p = 0.0291); this correlation is not present in PR-positive tumors (red line; r = 0.0291, p = 0.9364). These data suggest that PR disrupts the activation of STAT1, as measured via p-STAT1 levels (Fig. 3A), and the correlation between p-STAT1 and total STAT1 (Fig. 3C). Cumulatively, these data suggest that PR positivity affects phospho-STAT1 levels in human breast tumors in addition to human breast cancer cell lines. IFN-induced TYK2 activity is attenuated by activated PR Interestingly, phospho-STAT1 following treatment with IFN-g (IFNg2type II IFN signaling ligand) was unaffected by PR activation (data not shown), suggesting that the effect of PR on STAT1 phosphorylation is exclusive to pathways/proteins in- volved in type I IFN signaling. TYK2 is the primary kinase responsible for phosphorylating STAT1 in response to IFN-a (reviewed in Ref. 6), whereas JAK1 and JAK2 are responsible for STAT1 phosphorylation following IFN-g. Because TYK2 is specific to type I IFN signaling, we sought to determine how Downloaded from TYK2 activation was impacted by PR. We measured phospho- TYK2 (indicative of TYK2 activation) following treatment with IFN-a in the presence/absence of PR activation. In T47D-co cells treated with PR ligand, IFN-a–induced TYK2 phosphorylation was attenuated (Fig. 4A, left and quantification of Fig. 4A, right). FIGURE 5. PR activation disrupts the ISGF3 complex. STAT2 and IRF9 Importantly, JAK1 and JAK2 phosphorylation was unaffected (or a species-specific IgG control) was IP from IFN-a–treated (2 h), starved http://www.jimmunol.org/ T47D-co cell lysates (with or without R5020; 60 min), and the resulting by PR activation (data not shown), thus reinforcing the specificity associated protein complexes were analyzed by Western blotting. This of this effect to type I IFN signaling. As such, like PR and STAT1, experiment was performed in triplicate, and a representative experiment is we identified an interaction between PR and TYK2 that was shown. increased in response to PR ligand (Fig. 4B). Again, similar to PR-STAT1, the PR-TYK2 interaction in response to PR ligand appears to be primarily driven by PR-B (larger isoform, upper transcriptional programs. T47D cells that are positive (T47D-co) band). Together, these data suggest that an interaction between or negative (T47D-Y) for PR expression were treated with PR and TYK2 leads to decreased TYK2 activation, which subse- IFN-a (or vehicle) for 18 h. GSEA revealed that multiple by guest on September 30, 2021 quently translates to decreased STAT1 phosphorylation when PR IFN-associated gene sets were enriched in the PR-null RNA-Seq is present/activated. dataset; this enrichment was lost in cells expressing PR (22, 23). The top significantly regulated gene sets in PR-null cells (as PR activation disrupts the ISGF3 complex compared with PR-positive cells) are shown in Fig. 6A; select Following STAT1 phosphorylation, a transcriptional complex is examples of enrichment are shown in Fig. 6B. LE analysis, a formed containing STAT1, STAT2, and IRF9. Formation of this component of GSEA that allows the identification of core genes complex is key to transcriptional activation of genes turned on that drive the enrichment of a particular gene set, identified mul- in response to IFN treatment, such as ISGs. To determine if PR, tiple genes that are transcriptional targets (ISGs) of IFN signaling because of the interaction between PR and STAT1, affects the pathways (i.e., IFITs, MX1, OASs) whose regulation is enriched in formation or integrity of the ISGF3 complex, we used co-IP PR-null cells but lost in PR-positive cells (Fig. 6C). Using qPCR, assays to interrogate the interactions within the ISGF3 com- we validated a core set of ISGs and confirmed on the individual plex. In T47D-co cells treated with IFN-a, the interaction gene level that IFN activation of IGSs is transcriptionally atten- between STAT2 and STAT1 decreased with the addition of PR uated (ranging from 50 to 85% reduction) in T47D cells ligand (R5020; Fig. 5). A decreased interaction was also ob- expressing PR, as compared with PR-null cells (Fig. 6D). Protein served between IRF9 and STAT2 in the presence of R5020 expression for select ISGs mimics the same phenotype; IFN activa- (Fig. 5). These data suggest that PR activation decreases the tion of ISG protein levels is higher in PR-negative cells as compared integrity of the ISGF3 complex, either through promoting dis- with PR-positive cells (Fig. 6E). Cumulatively, these data suggest assembly of the complex or preventing efficient assembly. Cu- that unliganded PR attenuates the transcriptional response to IFN-a mulatively, these data suggest that the interaction between PR through the downregulation of ISG RNA and protein. and STAT1 disrupts the functionality of STAT1 through decreased ISGF3 recruitment to ISREs is attenuated in PR-positive cells phosphorylation (Figs. 2, 3) and disruption of STAT1-containing protein complexes (Fig. 5). ISGs are transcriptionally activated following ISGF3 (composed of STAT1, STAT2, and IRF9) binding to ISRE sequences in the PR decreases ISG transcriptional response and protein levels proximal promoter regions of these genes. To determine if the The end point of type I IFN signaling is transcriptional activation interaction between PR and STAT1 may affect recruitment of of ISGs, genes that canonically orchestrate the cellular response to the ISGF3 complex to ISREs, we used ChIP assays to measure viral pathogens. As PR presence attenuated STAT1 phosphoryla- STAT1, STAT2, and IRF9 recruitment in T47D-co cells expressing tion in response to IFN treatment and promoted the dissociation nonsilencing or PR shRNA [previously described in (10) and of the ISGF3 complex, we performed RNA-Seq on IFN-treated Supplemental Fig. 2]. In response to IFN-a treatment, ISGF3 PR-positive and PR-negative breast cancer cells to determine components (STAT1, STAT2, and IRF9) were potently recruited to how the presence of unliganded PR affects global IFN-activated ISRE sequences of the IFIT genes (Fig. 7). Significantly, in cells The Journal of Immunology 3083 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 6. PR decreases IFN-induced gene expression. (A) RNA-Seq was performed on RNA isolated from IFN-treated (20 IU/ml IFN-a for 18 h) T47D PR-null or PR-positive cell lines. GSEA was performed using the c2 MSigDB collection, comparing RNA-Seq gene expression datasets obtained from IFN-treated PR-null and PR-positive cells. Shown are the top most-significantly enriched gene sets (FDR , 0.05); select enrichment examples are shown in (B). (C) Top 20 ranking genes as identified using LE analysis on 29 gene sets referred to in (A). (D) T47D PR-null or PR-positive cell lines were treated as in (A). Isolated RNA was analyzed for multiple ISGs using qPCR. Gene values were normalized to an internal control (b-actin). Error bars represent SD between biological triplicates. Asterisks (*) represent statistical significance between groups; p , 0.01, as determined using an unpaired Student t test. (E) Cells were treated with 20 IU/ml IFN-a for 6 h, and isolated protein was analyzed via Western blotting with respective Abs. The experiments presented in (D) and (E) were performed in triplicate, and a representative experiment is shown. lacking PR expression (PR shRNA), ISGF3 components exhibited The inhibitory effects of PR on IFN signaling are seen in the absence of more robust recruitment to ISREs, compared with cells expressing PR ligand (ligand independent) and are further potentiated when PR PR (nonsilencing shRNA). These data indicate that the interac- ligand is added. These observations are clinically relevant, as we see tion between PR and STAT1 decreases DNA recruitment of the decreased phospho-STAT1 in PR-positive breast tumors compared transcriptional complex required for ISG transcription, thereby with their PR-negative counterparts. These data regarding the actions leading to a decrease in ISG RNA levels. of unliganded (ligand independent) PR, together with our recently published data showing PR ligand-dependent transcriptional re- Discussion pression of ISGs (10), suggest a novel, multifactorial role for PR in The data presented in this study support a model whereby PR posi- attenuation of IFN signaling. tivity can influence the potency of type I IFN signaling through an IFN signaling has been classically defined as a critical part of interaction with STAT1. We show that PR positivity leads to a decrease the innate immune system’s response to viral infection. However, in IFN-stimulated STAT1 phosphorylation, a collapse of the ISGF3 there is an emerging role for IFNs in aiding the immune system transcriptional complex, decreased recruitment of STAT1 to ISG in detection and clearance of early malignancies. The cancer immu- enhancers, and, subsequently, lower levels of ISG RNA and protein. noediting hypothesis, first put forth by Robert Schreiber, highlights 3084 PR ATTENUATES STAT1 SIGNALING Downloaded from http://www.jimmunol.org/

FIGURE 7. PR decreases ISGF3 recruitment to ISG promoters. T47D-co nonsilencing (NS) and PR shRNA cells were serum starved for 18 h and then treated with 1000 IU/ml IFN-a (or vehicle) for 4 h. Fixed lysates were subjected to ChIP with Abs against STAT1, STAT2, IRF9, or a species- specific IgG (control; data not shown), and qPCR was performed on the isolated DNA, using primers designed to amplify select ISG promoters. A

percentage of ChIP DNA over input DNA is shown. All ChIP experiments were performed in triplicate; a representative experiment is shown. Fold by guest on September 30, 2021 recruitment in IFN-a2treated conditions, as compared with vehicle treatment, is displayed above each bar. Error bars represent SD of technical replicates. that immunoediting can both prevent and promote cancer progression, with human luminal A/B tumors, which are predominantly ER/PR- clearing early tumors but helping shape the immunogenicity of more- positive tumors. Further, dysregulation of the JAK2/STAT1 signal- developed tumors (2, 33). The three stages of cancer immunoediting ing axis leads to the survival and proliferation of luminal progenitor are elimination, equilibrium, and escape. The elimination phase cells, the precursors to ER/PR-positive tumors, in the murine involves recognition and destruction of nascent tumors before they mammary gland (39). These data implicate STAT1 loss as an early can advance to tumors of biological relevance. There is a clear event in the development of mammary gland tumors. Our data role for type I IFNs in this process [reviewed in Ref. (34)]. Both suggest that one putative mechanism for STAT1 loss (i.e., loss tumor cells and host immune cells can produce and are responsive of function) is through the PR–STAT1 interaction and subsequent to IFNs, and this plays a key role in tumor cell elimination. As such, decreased STAT1 signaling, presented in this study. Cumulatively, alterations in IFN production, as well as the cellular response to our data, together with previously published data, suggest PR–STAT1 IFNs, can have dramatic effects on recognition and clearance of cross-talk is critical to the development of ER/PR-positive tumors. developing tumors. Multiple studies suggest this is the case, showing These data discussed thus far present a role for STAT1 as a tumor increased tumor formation in mice lacking key components of the suppressor in multiple tumor tissues. Conversely, work from A. Minn, IFN response pathway, such as IFN receptors (IFNΑR and IFGAR) R. Weichselbaum, and others has shown that expression of a core set and key IFN signaling molecules (JAK2 and STAT1) [these data are of genes (mostly ISGs) canonically regulated by STAT1 is correlated thoroughly reviewed in Ref. (34)]. Situations in which the cellular with therapy resistance in multiple tumors types, including breast. response to IFN signaling is decreased or abrogated, such as that This gene set, referred to as the IFN-related DNA damage resistance seen in the PR-positive breast cell line models shown in this study, signature (IRDS), is upregulated in tumors that develop resistance to may have a significant impact on the efficacy of immune-mediated chemotherapy, endocrine therapy, radiation, and immunotherapy and tumor elimination. Decreased IFN/STAT1-signaling, as mediated by can be used to predict poor prognosis (35, 40–44). Moreover, recent PR, may aid early PR-positive tumors in evading immune surveil- work from the Peter Laboratory suggests that CD95-mediated acti- lance, allowing for the development of clinically relevant tumors. vation of IFN signaling via STAT1 promotes a cancer stemness There is a growing body of evidence to suggest that STAT1 is phenotype in breast cancer cells (45). Together, these data highlight involved in breast tumorigenesis, and this has been extensively putative differences for the role of IFN/STAT1-signaling in early reviewed [Refs. (35–37)]. Significantly, STAT1 knockout mice (tumor initiating/immune evasion) versus late (resistance to therapy) develop mammary gland adenocarcinomas, 90% of which are tumor events and underscore the complexity of events that culminate ER/PR positive (38). The molecular signatures of these tumors overlap in tumor formation and, ultimately, progression. The Journal of Immunology 3085

One major unanswered question in breast cancer biology is why 13. Trinca, G. M., M. L. Goodman, E. K. Papachristou, C. S. D’Santos, P. Chalise, R. Madan, C. Slawson, and C. R. Hagan. 2018. O-GlcNAc-dependent regulation the overwhelming majority of breast cancers (over 65%) are ER/PR of progesterone receptor function in breast cancer. Horm. Cancer 9: 12–21. positive at the time of diagnosis (46, 47). Breast cancer is a hor- 14. Mohammed, H., C. D’Santos, A. A. Serandour, H. R. Ali, G. D. Brown, monally driven disease, largely driven by exposure to estrogens A. Atkins, O. M. Rueda, K. A. Holmes, V. Theodorou, J. L. Robinson, et al. 2013. Endogenous purification reveals GREB1 as a key estrogen receptor reg- (48). Estrogen, when binding to the ER, drives proliferative and ulatory factor. Cell Rep. 3: 342–349. survival gene programs that promote oncogenesis (49). However, 15. Mohammed, H., I. A. Russell, R. Stark, O. M. Rueda, T. E. Hickey, G. A. Tarulli, extensive animal model and clinical data suggest that both estro- A. A. Serandour, S. N. Birrell, A. Bruna, A. Saadi, et al. 2015. Progesterone receptor modulates ERa action in breast cancer. [Published erratum appears in gen and progesterone are needed for breast tumor development 2015 Nature 526: 144.] Nature 523: 313–317. (50–52). Despite these data, the role for progesterone and PR in 16.Mohammed,H.,C.Taylor,G.D.Brown,E.K.Papachristou,J.S.Carroll,and breast cancer remains largely unanswered (31). Although ER and C. S. D’Santos. 2016. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes. Nat. Protoc. 11: 316–326. PR likely provide a proliferative advantage (affording one expla- 17. de Queiroz, R. M., R. Madan, J. Chien, W. B. Dias, and C. Slawson. 2016. nation for this preponderance of ER/PR-positive tumors), another Changes in O-linked N-Acetylglucosamine (O-GlcNAc) homeostasis activate nonmutually exclusive explanation is that these tumors have an the pathway in ovarian cancer cells. J. Biol. Chem. 291: 18897–18914. 18. Liu, Q., R. Bonneville, T. Li, and V. X. Jin. 2017. Transcription factor-associated immune-privileged phenotype, allowing for their escape from the combinatorial epigenetic pattern reveals higher transcriptional activity of surveilling immune system. PR-positive tumors, because of their TCF7L2-regulated intragenic enhancers. BMC Genomics 18: 375. ability to downregulate IFN signaling under liganded (10) and 19. Kim, D., G. Pertea, C. Trapnell, H. Pimentel, R. Kelley, and S. L. Salzberg. 2013. TopHat2: accurate alignment of transcriptomes in the presence of insertions, unliganded conditions [data presented in this study and (10)], may deletions and gene fusions. Genome Biol. 14: R36. evade the critical first step of elimination via immune clearance. 20. Trapnell, C., A. Roberts, L. Goff, G. Pertea, D. Kim, D. R. Kelley, H. Pimentel,

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