Tumor-inducedStromalSTAT1AcceleratesBreast Cancer via ... · the protumor effects of the TME in conjunction with traditional cancer chemotherapies (4). ... majority of mammary gland

Oncogenes and Tumor Suppressors

Tumor-induced Stromal STAT1Accelerates BreastCancer via Deregulating Tissue HomeostasisVictoriaR. Zellmer1,2, PatriciaM. Schnepp1,2, SarahL. Fracci1, XuejuanTan1,2, ErinN.Howe1,2,and Siyuan Zhang1,2

Abstract

The tumor microenvironment (TME), the dynamic tissuespace in which the tumor exists, plays a significant role intumor initiation, and is a key contributor in cancer progression;however, little is known about tumor-induced changes in theadjacent tissue stroma. Herein, tumor-induced changes in theTME were explored at the morphologic and molecular level tofurther understand cancer progression. Tumor-adjacent mam-mary glands (TAG) displayed altered branching morphology,expansion of myofibroblasts, and increased mammosphereformation, broadly suggesting a tumor-induced field effect.FACS analysis of TAGs demonstrated an increased number ofLin�CD24þ/CD49þ enriched mammary gland stem cells(MaSC), suggesting deregulated tissue homeostasis in TAGs.Comparative transcriptome analysis of TAGs and contralateralcontrol glands coupled with meta-analysis on differentiallyexpressed genes with two breast cancer stromal patient micro-array datasets identified shared upregulation of STAT1. Knock-

down of STAT1 in cancer-associated fibroblast (CAF) cocul-tured with human breast cancer cells altered cancer cell pro-liferation, indicating a role for STAT1 as a stromal contributorof tumorigenesis. Furthermore, depletion of STAT1 in CAFssignificantly reduced periductal reactive fibrosis and delayedearly breast cancer progression in vivo. Finally, cotreatment withfludarabine, a FDA-approved STAT1 activation inhibitor andDNA synthesis inhibitor, in combination with doxorubicin,showed enhanced therapeutic efficacy in treating mouse mam-mary gland tumors. Taken together, these results demonstratethat stromal STAT1 expression promotes tumor progressionand is a potential therapeutic target for breast cancer.

Implications: Tumors induce stromal STAT1-dependent cyto-kine secretion that promotes tumor cell proliferation andcan be targeted using clinically-approved inhibitors of STAT1.Mol Cancer Res; 15(5); 585–97. �2017 AACR.

IntroductionStepwise accumulation of mutations in key oncogenes and

tumor suppressors in somatic cells typifies the classic tumorprogression model (1). Cancer cells coexist with normal cells inthe same tissue space, communicating with one another andconstituents of their shared tumor microenvironment (TME).Seminal findings over the past decade have established the sig-nificance of the heterogeneous TME in tumor initiation andprogression (2). Multi-omics analyses demonstrated that altera-tions within tumor-associated stroma are predictive indicators ofclinical outcome (3) and targeting tumor-promoting modulatorsof the TME has been perceived as a promising strategy to combatthe protumor effects of the TME in conjunction with traditionalcancer chemotherapies (4).

Breast cancer arises from a dynamic mammary gland ecosys-tem. A diverse array of cell types establish the context in which the

majority of mammary gland development occurs (5–7). Giventheir fundamental role in deposition of the extracellular matrix(ECM), fibroblasts are particularly influential components of themammary milieu (8). Reminiscent of myofibroblasts are carci-noma-associatedfibroblasts (CAFs), whichbearmolecular resem-blance to activated normal fibroblasts during the wound healingprocess (9). Through enhanced ECM production, CAFs have thecapacity to alter the TME through tissue remodeling includingrecruitment of immune cells to TME, perturbing adipocyte dif-ferentiation, disrupting the function of epithelial stem cells, andinstigating other discordant events that promote the hallmarks ofcancer (10, 11).

CAFs also display increased proliferation and secrete adiverse array of cytokines (12). At the core of secretory factorregulation is the Janus kinases (JAK) and STAT pathways (JAK–STAT). Upon phosphorylation-dependent activation, isoformsof STAT proteins function as highly specific transcription factorsindependent of secondary messengers (13). Specifically, STAT1is a central mediator of both type I (alpha and beta) and type II(gamma) IFNs, which play an essential role in cell growth,regulation, and antiviral and immune defense (14). In mousemammary tissue, STAT1 expression is observed in the virginmammary gland and diminishes into gestation, only to reap-pear during involution, potentially expressed in recruitedimmune cells (15). Deregulated STAT1 expression has beenimplicated in breast cancer development with both tumor-suppressing and -promoting roles of STAT1 (16, 17). Despitethe known contributions of various STATs throughout mam-mary gland development, the roles of STAT1 in mammary

1Department of Biological Sciences, College of Science, University of NotreDame, Notre Dame, Indiana. 2Mike and Josie Harper Cancer Research Institute,University of Notre Dame, South Bend, Indiana.

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

Corresponding Author: Siyuan Zhang, Department of Biological Sciences,University of Notre Dame, A130 Harper Hall, Notre Dame, IN 46556. Phone:574-631-4635; Fax: 574-631-2156; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-16-0312

�2017 American Association for Cancer Research.

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tumor progression remain highly controversial and appear tobe tumor stage- and cell type-specific (15).

In this study, we demonstrated that mammary tumors seemto induce field cancerization on the seemingly normal tissuesof their microenvironment. Using a mouse-in-mouse modelof breast cancer, we first observed that mammary tumorsinduce field cancerization on the adjacent tissues in themammary gland microenvironment, including increasedmammary gland branching and enriched mammary stem cells(MaSC) and CAFs in tumor-adjacent mammary glands. Tran-scriptional profiling identified STAT1 as the central signalingnode in the tumor-adjacent stroma. Ablation of STAT1 inCAFs decreases cancer cell proliferation through STAT1-depen-dent secretion of pentraxin 3 (PTX3). Depletion of STAT1 inCAFs reduces a-SMAþ periductal-reactive fibroblasts and duc-tal carcinoma in situ (DCIS)-like lesions in a mouse model ofearly breast cancer progression. Finally, using an allograftmodel, we demonstrated that combination of a STAT1 inhib-itor (fludarabine) with chemotherapy (doxorubicin) reducedstromal CAFs, STAT1 expression, and exhibited superior ther-apeutic efficacy.

Materials and MethodsCell culture and STAT1 knockdown in CAFs

Murine mammary tumor cell line (PNA.Met1) was derivedfrom a spontaneous mammary tumor from the MMTV-PyMTmodel. DCIS.COM cell line was originally purchased fromAsterand, Inc. MDA-MBA-231 cell line was purchased fromATCC and has been actively passaged less than six months.The caveolin-1–deficient mouse embryonic fibroblasts wereused were gifted by Dr. Zach Schafer (University of NotreDame, Notre Dame, IN; refs. 18, 19). All cells lines weremaintained in DMEM-F12 growth media supplemented with10% FBS and 5% penicillin–streptomycin at 37 �C with 5%CO2. For siRNA knockdown experiments, CAFs were treatedwith either the ON-TARGETplus SMARTpool siRNA targetingmouse Stat1 or ON-TARGETplus nontargeting siRNA pool (GEHealthcare Dharmacon Inc.) for 6 hours.

EdU assayCells were seeded in serum-free media 24 hours prior to

treatment to allow for cell-cycle synchronization. Followingdesignated treatment, cells were pulsed with 5-ethynyl-2-deox-yuridine (Click-iT Plus EdU Alexa Fluor 647 Imaging Kit, LifeTechnologies), for two hours before fixation in 2% parafor-maldehyde and subsequent EdU detection as per the manu-facturer's protocol. Coverslips were mounted on slides andimaged using a Nikon A1R-MP confocal microscope. Quanti-fication of EdUþ tumor cells were carried out using the cellcounter plugin from the ImageJ software (NIH, Bethesda, MD).The percentage of EdUþ for each field of view captured wasrecorded and analyzed.

Cytokine array analysisBriefly, on day 0, 1 � 106 cells (siControl CAF or siStat1 CAF)

were seeded in complete DMEM-F12. On day 3, serum-freeDMEM-F12 was added and conditioned for 48 hours beforecytokine array analysis by RayBiotech using the Mouse CytokineArray Q4000 (QAM-CA-4000). After fluorescent scanning, dataextraction, and data computation using array-specific scanning

devices and data processing tools (RayBiotech). Concentrationlevels, expressed in picograms per milliliter (pg/mL), were calcu-lated against a standard curve set for each biomarker from thepositive and negative controls. Results from each of the 200different cytokine probes were ranked by expression and sub-jected to bioinformatics analysis.

In vivo animal experimentsMice were housed in the Animal Facility of the Freimann Life

SciencesCenter at theUniversity ofNotreDame (NotreDame, IN)in compliance with the Institutional Animal Care and UsageCommittee (IACUC) standards. Female FVBmice (12–16 weeks)were purchased from The Jackson Laboratory.

Estrous cycle monitoring. Murine estrous cycle monitoring wascompleted by vaginal lavage according to published protocol(20). Briefly, the vaginal cavity of each animal was washed withsterile ddH2Obypipette, whichwas thenpipetted onto clean slideglass. Dried slides were stained with 0.1% crystal violet for 1minute before mounting. Ten fields of view were analyzed foreach animal to determine estrous cycle.

Mammary fat pad tumor model. Two million ZsGreen-labeledPNA.Met1 cells in 100 mL of serum-free DMEM-F12 wasinjected into one of the inguinal (fourth) mammary fat pad(MFP) of female FVB mice with control injections of 100 mLof serum-free DMEM-F12 into the contralateral gland. Upontumor formation (approximately 14–20 days postinjection),mammary glands and tumors were collected for furtheranalysis.

DCIS model (intraductal tumor injections). Fifty-thousand cellsin 8-mL Hank balanced salt solution were injected into thenipple of the inguinal mammary glands of mature age-matched, cage-matched mice via intraductal injection using31 gauge RN needle, Hamilton #7803-03). Mammary tissuewas collected 14 days postinjection. For the CAF coinjectionexperiment, 2 � 106 CAFs were first injected to mammary fatpad. Twenty-four hours later, 5 � 104 DCIS.COM cells wereinjected intraductally.

In vivo drug treatment. FollowingMFP injection of PNA.Met1 cellsinto each inguinal fat pad, we measured and recorded the calcu-lated ellipsoidal tumor volume and randomly distributed miceinto 4 treatment groups (vehicle, doxorubicin, fludarabine, dox-orubicinþfludarabine). Doses of both doxorubicin and fludar-abine (Selleck Chemicals) were determined on the basis of thebody surface area (BSA) of each animal based on reported humandoses (21–23). Drug treatments were administered every 3 daysby intraperitoneal injection and tumor volumewas logged. Uponsacrificing animals, tumors and adjacent mammary glands werecollected for formalin fixation and paraffin embedding (FFPE) forsubsequent evaluation.

Whole mount analysisMammary glands from each animal were aseptically collected,

fixed, and stained according to the established protocol. Briefly,each gland was fixed for two hours in 4%paraformaldehyde priorto overnight staining with carmine alum solution. Glands werewashed in an ethanol gradient (70%, 95%, and 100% for one

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Mol Cancer Res; 15(5) May 2017 Molecular Cancer Research586




hour each) prior to overnight clearing in xylene andmounting onglass for branching analysis in ImageJ.

Propagation of primary mammospheresMammary glands were collected and digested in collagenase/

hyaluronidase (Stemcell Technologies) in EpiCult-B completegrowth medium (Stemcell Technologies). After overnight diges-tion, each sample was washed with 1� PBS and treated withdispase (5 mg/mL) and DNAse (0.1 mg/mL), then filteredthrough a sterile 40-mm mesh filter. An equal number of cellswere plated in EpiCult B complete growth media on CorningUltra-Low attachment 96-well plates (Sigma-Aldrich). Total num-ber of mammospheres per well was determined after 7 days at10� magnification.

FACSIntraductal injections of PNA.Met1 cells labeled with DiR (Life

Technologies) were carried out on age-matched, cage-matchedFVB mice. Cells were stained with the APC-conjugated lineagecocktail (Lin; BD Biosciences) containing CD45, Ter119, andCD31. Cells were also stained with PE-conjugated CD49f andFITC-conjugated CD24 (BD Biosciences).

Hemotoxylin and eosin staining and IHCFFPE sections (5 mm) were deparaffinized using standard

protocol and stained with Harleco Gill Modified Hematoxylin(EMD Millipore) and eosin (Sigma-Aldrich). Slides remained inxylene until mounted with Permount Mounting Media (ElectronMicroscopy Sciences). For IHC staining, 5-mm FFPE sections weresubjected to antigen retrieval in boiling 10mmol/L sodium citrate(pH 6.0) buffer for 10 minutes. After blocking endogenousperoxidase with 3% H2O2 for 15 minutes, slides were washed3 times in 1� PBS prior to blocking for 60 minutes at roomtemperature (Protein Block, Serum-Free; Dako), followed withprimary antibody (STAT1, 1:1000, #14994S, Cell Signaling Tech-nology; a-SMA, 1:500, #ab124964, Abcam; 1:500. Cytokeratin-8,#ab53280, Abcam), and incubated overnight at 4�C. After wash-ing with PBS, the sections were incubated with secondary anti-body for another 60minutes (Polink-2PlusHRPRabbitwithDABKit, GBI Labs). Each slide was counterstained with Gill hematox-ylin. IHC quantifications were carried out using ImageJ (NIH,Bethesda, MD).

RNA-seq, meta-analysis, and network analysisRNA-seq was conducted after SMART-seq2 library preparation

(24). Following RNA-seq, raw reads were analyzed by DESeq2 toexplore transcriptional differences between control and TAGtissue (25). Meta-analysis and network construction were con-ducted through the web-based bioinformatics package Networ-kAnalyst (http://www.networkanalyst.ca) based on the recom-mended protocol (26, 27). Three stromal transcriptome analysesdatasets used in the meta-analysis are: (i) Zhang_RNA-seq.The TAG gene expression signature was derived from comparisonof the TAG with a contralateral control mammary gland usinga syngeneic mouse mammary tumor transplantation model(this study). (ii) GSE14548: a publicly available microarraydataset of 14 patient-matched normal epithelium, normal stro-ma, tumor epithelium, and tumor-associated stroma samples.(iii) GSE26910: a publicly available microarray dataset of6 patient-matched tumor-adjacent stromal samples with 6 nor-mal stromal samples. Prostate samples in this dataset were

excluded from our studies. The top differentially expressed genesfrom three independent datasets (FDR P < 0.05, <0.01, <0.01for Zhang_RNA-seq, GSE14548, GSE26910, respectively) werecurated for meta-analysis. On the basis of statistical heterogeneityestimated using Cochran Q tests, we applied random effectsmodels (REM, P < 0.05) for metagene analysis. Forty-two differ-entially expressed metagenes were selected for NetworkAnalystusing InnateDB interactome database (26). During the networkanalysis, we removed UBC, ACTB, and GRB2, which are com-monly involved in many nonspecific interactions as suggested inNetworkAnalyst protocol.

Statistical analysisAll quantitative data with normal distribution are compared by

Student t test (two-tailed). Whole-mount branch point analysiswas compared by paired t test. For all analyses, a P < 0.05 wasconsidered statistically significant.

ResultsEarly mammary tumor development deregulates the mammarygland microenvironment and alters stromal cells

We began by exploring whether tumors induce either physicalor biochemical changes on ostensibly healthy, nontransformedmammary tissues. Considering mammary gland tissue homeo-stasis is under the tight regulation of local and systemic signals (e.g., ovarian hormones; ref. 5), we age-matched and cage-matchedexperimentalmice and confirmed the estrous cycle of each animalby vaginal lavage staining, as indicated by the presence of corni-fied squamous epithelial cells (Supplementary Fig. S1A).We thenperformed an acute mammary fat pad (MFP) transplantation ofsyngeneic PNA.Met1 tumor cells, an aggressive breast cancer cellline derived from a mammary tumor collected from the MMTV-PyMT transgenic mouse, to study changes in tumor-adjacenttissues (Supplementary Fig. S1B; ref. 28). We assayed branchingmorphology in both the vehicle-injected control and contralateraltumor-adjacent glands (TAG) by mammary whole mount (29).We observed a marked increase in the number of branch pointsper duct (P < 0.05) in each TAG compared with control glands 21days postinjection (Fig. 1A). To further assess tumor-inducedchanges in the mammary gland, we performed IHC on TAGs andcontrol glands for cytokeratin 8 (K8 - luminal epithelial cells) anda-SMA (myoepithelial and myofibroblasts; Fig. 1B). Quantifica-tion showed an increase in the K8þ luminal epithelial compart-ment of TAG tissue compared with control (K8, Fig. 1C, left;a-SMA, Fig. 1C, right). Compared with vehicle-injected controlglands, TAG tissue, while visibly undisturbed, also exhibited anincrease in the number of a-SMAþ stromal and myoepithelialcells (Fig. 1C). Given prior knowledge that fibroblast-secretedfactors can alter the mammary epithelial hierarchy (30, 31), wenext examined the impact of tumor burden on theMaSCniche viamammosphere formation of cells derived from TAGs. Weobserved a significant increase (P < 0.05) in the numbermammo-spheres derived from TAGs compared with tissue from the con-tralateral control gland, strongly suggesting an increased MaSCpopulation in TAG (Fig. 1D).

We next asked whether the MaSC expansion occurs at a criticaltransition stage of tumor progression, such as the transition fromductal carcinoma in situ (DCIS) to invasive ductal carcinoma(IDC). To faithfully mimic the DCIS to IDC transition, we usedan intraductal injection model that provides an accurate recapit-ulation ofDCIS and its progression to IDC in vivo (Fig. 1E; ref. 32).

Stromal STAT1 Activity and Targeting in Breast Cancer

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Intraductal injection of PNA.Met1 cells gave rise to DCIS-likelesions in approximately 3 weeks (Supplementary Fig. S1C). Welabeled the tumor cells with DiR to facilitate tumor cell trackingand injected paraformaldehyde (PFA)-fixed tumor cells in thecontralateral gland to control the potential impact of the physical

stress imposed to mammary gland tissue during intraductalinjection (Fig. 1E) and characterize the enrichedMaSCpopulationin mammary tissue using FACS (Supplementary Fig. S1D). It hasbeen widely accepted that lineage-negative (Lin�) CD24þ/CD49flow cells are enriched for luminal epithelial cells, and the

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Tumor development deregulates themammary glandmicroenvironment and alters stromal cells.A,Wholemount analysis of control (left) and tumor-adjacent (TAG,right) mammary glands. Arrows indicate branch points. Right, branch point quantification of matched control and tumor-adjacent glands, with analysis frompaired t test.B,Representative images of IHC staining of cytokeratin 8 (K8) anda-smoothmuscle actin (a-SMA) in control (top) and TAG (bottom).C,Quantificationof IHC (shown in B) of percent of K8þ and a-SMAþ cells per 40� region of interest (ROI) in both control and TAG. D, Representative images of both controland TAG mammospheres with corresponding quantifications at day 10 postplating. E, Schematic depicting experimental strategy for intraductal injectionexperiments. F, FACS plot of MaSCs from both control (left) and TAG (right) mammary glands.

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CD24þCD49hi basal epithelial population is enriched for MaSCs(33, 34). Therefore, we examined TAG tissue for CD24þCD49hi

cells after gating out Linþ cells as well as DiRþ cells (tumor cells).Quantification of the Lin�/CD24þ/CD49fhi population in bothtumor and control glands showed that the enriched MaSC pop-ulation comprised 14.9% of the total cell population in the TAGs,compared with 7.58% in the control glands (Fig. 1F). Takentogether, these results suggest that early-stage tumorsmay imposea cancer field effect on adjacent, ostensibly normal tissue bystimulating the stromal myoepithelial and myofibroblast popu-lation and inducing an expansion of MaSC population (35).

STAT1 is a central node of tumor-stimulated stromal signatureTo explore the molecular mechanisms driving both the shift in

tissue morphology as well as the impact on the mammaryepithelial hierarchy, we conducted comparative transcriptomeanalysis (RNA-seq) of control and TAG tissue (Fig. 2A). A diag-nostic MA plot shows evenly distributed reads around zero (zero-fold change) across read count values (Supplementary Fig. S2A).In total, we detected 17,877 differentially expressed genes (DEG)in TAGs after stimulation by the presence of a tumor. Among thetop DEGs (P < 0.05), approximately 80% of the highest rankedDEGs were upregulated in the control glands, whereas approxi-mately 20%were upregulated in the TAGs (Fig. 2B). To reveal thefunctional implications of DEGs, we performed gene set enrich-ment analysis (GSEA) using allDEGs (P<0.05; q<0.25) based oncurated gene sets (C2) from the Molecular Signature Database(MSigDB). Interestingly, our TAG samples showed significantenrichment of gene sets that represent mammary gland/stem celldevelopment, aggressive tumor phenotypes, and immune activa-tion (Fig. 2C; Supplementary Fig. S2C; Supplementary Table S1).To validate the clinical relevance of our findings and identifypotential candidates that drive the phenotype observed in tumor-adjacent tissue, we performed meta-analysis based on a randomeffect model across our RNA-seq dataset as well as two publiclyavailable clinical breast cancer stromal microarray datasets(GSE14548 and GSE26910) using NetworkAnalyst (http://www.networkanalyst.ca; refs. 26, 36). We identified a total of42 DE meta-genes (FDR < 0.05) across our RNA-seq dataset andtwo breast cancer stromal patient microarray datasets (Fig. 2D;Supplementary Table S2). Moreover, of the 42 DE meta-genes,15 of these meta-genes are shared among all three datasets asdepicted in the cord diagram, which we refer to as our 15 meta-gene signature (Fig. 2E). Downregulated genes of interest includ-ed cluster of differentiation 36 (CD36), fatty acid binding protein4 (FABP4), and caveolin-1 (CAV1). Upregulated genes of interestin the tumor-associated stroma included STAT1 and hematolog-ical and neurological expressed 1 (HN1). Next, we created a nodalnetwork from the 15-gene meta-signature to identify functionallyimportant drivers of tumorigenesis. Using the Module Explorerpanel in NetworkAnalyst, we isolated the network into denselyconnectedmodules whose node genes are likely to work toward acommon biological function. A search algorithm is performed oneach seed node that ranks each node gene based on the number ofits first-degree interactions. Interestingly, NetworkAnalyst dis-tinctly revealed 3 top-ranked key nodes: (i) STAT1, an upregulatednode in TAG tissue (centrality degree ¼ 222, betweeness ¼15442.82); (ii) PPARG, a downregulated node (centrality degree¼ 123, betweeness ¼ 7616.63); and (iii) CAV1, a downregulatednode (centrality degree ¼ 117, betweeness ¼ 7629.22; Fig. 2F).The loss of CAV1 expression is a reported feature of CAFs, a major

constituent of the tumor stroma (8, 37). Most importantly, thehighest degree of centrality in our 15 meta-gene signature isSTAT1, which indicates that the presence of a tumor in the sametissue space promotes upregulation of STAT1 in tumor-adjacenttissue, which could play a central role in the deregulated TAG. Asthe mammary gland is highly heterogeneous and compartmen-talized, we sought to confirm the expression of STAT1 in tumor-adjacent tissue. We confirmed our RNA-seq findings by stainingfor STAT1 by IHC and found that expression is negligible in thecontrol gland, while more prominently expressed in tumor-adja-cent mammary tissue, primarily in stromal fibroblasts (Fig. 2G).

Fibroblast STAT1-mediated pentraxin 3 secretion promotestumor cell proliferation

Our IHC analyses indicate that the upregulation of STAT1occurs predominantly in the stromal fibroblast compartment ofthe tumor-adjacent mammary glands, not the epithelial compart-ment (Fig. 2G). Therefore, to examine the functional importanceof fibroblast-derived STAT1 in tumor-adjacent mammary tissueon tumor proliferation, we cocultured tumor cells with CAV1-deficient MEFs (herein referred to as CAFs), a well-establishedmodel cell line with similar transcriptome and secretome profileas humanCAFs (18, 19). Transient depletion of STAT1 in CAFs bysiRNA resulted in a decrease of proliferation as evidenced by lessEdU incorporation (Supplementary Fig. S3A and S3B). We cocul-tured the same number of CAFs, transfected with either controlsiRNA or STAT1 siRNA, with MDA-MB-231 breast cancer cells.Interestingly, coculture with siSTAT1 CAFs resulted in decreasedproliferation as evidenced by significantly less EdU incorporationin the tdTomato-red–positiveMDA-MB-231 cancer cells (Fig. 3A).Similar resultswere observed fromCAFs coculturedwithZsGreen-labeled PNA.Met1 breast cancer cells (Fig. 3B) orDCIS.COMcells,a well-characterized early-stage human breast cancer line (Fig.3C). To exclude the possible contact-dependent role of CAFs, wetreated MDA-MB-231 tumor cells with conditioned media col-lected from either siSTAT1 CAFs or nonsilencing control CAFs for48hours. Treatment of conditionedmedia collected fromsiSTAT1CAFs led to significantly weaker EdU fluorescent intensity intumor cells, which is a direct result of decrease of EdU incorpo-ration (Fig. 3D).

Pentraxin 3 is secreted by STAT1-expressing CAFs and promotestumor cell viability and proliferation

To further elucidate the STAT1-downstream secretory factorsreleased in CAF-conditioned media, we performed unbiasedcytokine array analysis to probe for 200 different secretory pro-teins in the conditioned media. Principal component analysis of68 total detectable cytokines showed a 39% total variance of thefirst component (PC1) and 22% total variance of the secondcomponent (PC2), suggesting differentially regulated cytokineprofiles between the si.Control and si.STAT1 group (Supplemen-tary Fig. S3C). As shown in the heatmap of hierarchical clustering,there was a consistent pattern of downregulation of pentraxin 3(PTX3) in siSTAT1-derived conditioned media compared with si.Control group (Supplementary Fig. S3D). Further analysis usingMA plot identified PTX3 as the only abundant cytokine(log[expression] > 2.5) significantly downregulated in the mediafrom siSTAT CAFs (Fig. 4A; Supplementary Fig. S3D). To furtherexplore the role of CAF-derived PTX3 in tumor progression, wetreated PNA.Met1 tumor cells with purified PTX3 protein. Treat-ing PNA.Met1 tumor cells with PTX3 showed a dose-dependent








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Figure 2.

STAT1 is a central node of tumor stimulated stromal signature.A, Bright-field (left) and fluorescent (right) images of ZsGreen PNA.Met1 xenograft tumor and tumor-associated gland (TAG). Dashed line indicates tumor-adjacent tissue extracted for RNA-sequencing. B, Clustering analysis showing the top DE genes incontrol and TAG groups. C, Cleveland plot of gene set enrichment analysis (GSEA) from all DEGs (P < 0.05, q < 0.25) with curated gene sets (C2) from the MolecularSignature Database (MSigDB). D, Heatmap of top 42 DEGs (FDR P < 0.05) from meta-analysis of Zhang_RNA-seq with GSE14548 and GSE26910. E, Cord diagramof 15 meta-genes (red line) shared among all three datasets (Zhang_RNAseq, GSE14548, and GSE26910). F, Nodal network generated from the 15 meta-genesignature showing the top three ranked nodes. G, Representative images of IHC staining for STAT1 in control (left) and TAG (right).

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increase of cell proliferation compared with vehicle-treated con-trol cells (Fig. 4B). Moreover, coculture of MDA-MB-231 breasttumor cells with PTX3-depleted CAFs led to a significant decreaseof EdU incorporation in tumor cells (23.25% in siPTX3 group vs.50.3% in siNS group; Fig. 4C; Supplementary Fig. S3E). Similardecrease of tumor cell proliferation was observed when tumorcells were cocultured with siPTX3 CAFs (Fig. 4D). In addition,overnight treatment of siPTX3 CAFs with 1 mg purified PTX3restored CAF cell proliferation (23.34% PTX3 treated vs.12.24% EdU incorporation in vehicle-treated group; Fig. 4E),suggesting a self-sustained proliferation of CAF via PTX3 auto-crine. Collectively, our data demonstrated that STAT1-dependentsecretion of PTX3 fromCAFs is one of the facilitators of tumor cellproliferation.

Stromal STAT1 influences progression of early-stage breastcancer in vivo

To assess the importance of stromal STAT1 in tumor progres-sion, we first manipulated the mammary gland microenviron-ment by mammary fat pad injection of either STAT1-depleted

CAFs (sh.STAT1) or control CAFs (sh.Control) in the contralateralsite. We then performed intraductal injections of DCIS.COM cellsto model the clinical progression of DCIS (Fig. 5A; ref. 32). Overthe 4 weeks of DCIS formation, we noticed a palpable increase inmammary gland stiffness in the control glands compared with sh.STAT1 glands. The significant increase of periductal fibrosis in thecontrol glands was evidenced in hematoxylin and eosin (H&Estaining; Fig. 5B). Importantly, on average, 25.53%of eachfield ofview was composed of a DCIS or IDC-like lesion in the controlglands, compared with only 7.65% in the sh.STAT1-injectedglands (Fig. 5C), suggesting a less aggressive mammary tumordevelopment conferred by deprivation of STAT1 in CAFs. Toreveal the cellular composition of the abnormalmammary tissue,we stainedK8 for epithelial cells anda-SMA formyoepithelial andreactive fibroblasts. In sh.Control CAF-injected gland, weobserved K8þ DCIS-like and IDC-like tumors, whereas onlyADH/DCIS-like lesions were observed in the sh.STAT1.CAF-injected glands (Fig. 5D). Moreover, we observed a significantdecrease of a-SMAþ cells surrounding mammary ducts in the sh.STAT1.CAF–injected glands (Fig. 5D and E), suggesting a

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Fibroblast STAT1-mediated PTX3 secretion promotes tumor cell proliferation. A, Representative images of EdU assay from tumor cells grown in coculturewith CAFs (siSTAT1; left). Right, quantification of Percent EdUþ cells. B, Representative images of EdU assay ZsGreen PNA.Met1 tumor cells incoculture with CAFs as mentioned in A (left). Right, quantification of Percent EdUþ cells; pink, EdUþ cells in merged image. C, Representative imagesof EdU assay from DCIS.COM tumor cells cocultured with either shPLKO.1 (control) CAFs or with CAFs containing either construct of shSTAT1 (23.1 or 26.1).Right, quantification of percent EdUþ cells. D, Representative merged images of DAPI-stained DCIS.COM cells after treatment for 48 hours withconditioned media collected from siSTAT1 CAFs and nonsilencing control (siNS) CAFs (left). Right, quantification represents log[EdU intensity] ofPNA.Met1 cells.






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PTX3 is secreted by STAT1-expressing CAFs and promotes tumor cell viability and proliferation. A, MA plot showing the cytokine array analysis. Logfold change is plotted as a function of log mean expression. Each circle represents one probe. B, Proliferation index of PNA.Met1 tumor cells and CAFs24 hours after treatment with recombinant human PTX3 in serum-free DMEM-F12. C, Representative images of nonsilencing control (siNS) CAFs (top)or PTX3-depleted CAFs (bottom; left). Right, quantification of percent EdUþ cells. D, Representative images of MDA-MB-231 cells cocultured withnonsilencing control (siNS) CAFs (top) or PTX3-depleted CAFs (bottom; left). Right, Quantification of percent EdUþ cells. E, Quantification of percentEdUþ cells of non-silencing control (siNS) or PTX3-depleted (siPTX3) CAFs treated with vehicle or 1 mg PTX3 overnight.

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reduction in tumor-induced myoepithelial cells as well asdecreased periductal fibrosis (38).

Inhibition of stromal STAT1 enhances chemotherapeuticefficacy

Given the prominent role of stromal STAT1 in facilitating breastcancer progression, we hypothesized that combined treatment ofSTAT1-inhibiting agents with doxorubicin, a conventional che-motherapeutic agent used to treat breast cancer, could enhancechemotherapeutic efficacy in breast cancer treatment. Fludarabineis a clinically approved chemotherapy that inhibits cytokine-induced activation of STAT1 and is often used for the treatmentof chronic lymphocytic leukemia and other hematologic malig-nancies (22). In this proof-of-concept study, we used our mouse-in-mouse allograft model to evaluate the therapeutic efficacy offludarabine in combination with doxorubicin. Combinationtreatment of fludarabine and doxorubicin further decreased

tumor volume (P < 0.01) compared with single-treatment alone(Fig. 6A). Consistent with reduction of tumor size, we observeddecreased proliferation (Ki67 IHC staining) and decreased necro-sis in residual tumors either single-drug treatment or combinationtreatment (Fig. 6B and C). Interestingly, single treatment offludarabine showed evidence of increased fibrosis within theresidual tumor mass (Fig. 6C, bottom). In addition, fludarabinetreatment normalized the TAG tissue structure. In both the single-treatment fludarabine and combination-treatment group, TAGglands exhibited regular adipocytemorphology and,most impor-tantly, an evident decrease of resident fibroblasts (Fig. 6C, top),suggesting fludarabine inhibits the abnormal expansion of CAFs.Doxorubicin-treated tumor-adjacent tissue showed an increasein STAT1 expression, likely due to drug-induced immune cellinfiltration, whereas either fludarabine- or combination-treatedtumor-adjacent tissue showed a complete inhibition of STAT1expression (Fig. 6D; ref. 11). Consistently, fludarabine or

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Stromal STAT1 influences progression of early-stage breast cancer lesions in vivo.A, Schematic depicting experimental strategy for intraductal injection experiments.Control CAFs were injected into one mammary fat pad (n¼ 5), while STAT1-depleted CAFs (shSTAT1) were injected into the contralateral fat pad (n¼ 5). After 24hours, DCIS.COM cells were injected into the nipple of each mammary gland. B, Representative H&E images of tumor progress in glands injected with control CAFs(left) and shSTAT1 CAFs (right) at both 10� (top) and 40� (bottom).C,Mean percent of DCIS/IDC-like lesion per field of view in glands injectedwith control CAFs (n¼ 5) comparedwith glands injectedwith shSTAT1 CAFs (n¼ 5).D,Representative 40� images showing tumor lesions in glands injected with control CAFs (left) andshSTAT1 CAFs (right) stainedwith either human K8 (top row) ora-SMA (bottom row). DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; ADH, advancedductal hyperplasia. E,Quantification of percenta-SMAþ cells per 40� field of view in 5-mmsections frommammary glands injectedwith sh.Control CAFs (left) or sh.STAT1 CAFs (right).






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Inhibition of stromal STAT1 enhances chemotherapeutic efficacy. A, Percent change of tumor volume at end point compared with day zero for each tumor in eachtreatment group: vehicle control (n¼ 6), doxorubicin (n¼ 10), fludarabine (n¼ 10), or combination of doxorubicin and fludarabine (n¼ 10).B,Representative imageshowing IHC for Ki67þ tumor cells in control treatment group (left). Right, mean number of Ki67þ cells per 40� field of view in each group. C,Hematoxylin and eosin(H&E) staining of tumors from each treatment group. Left, representative images showing the tissue morphology after the treatments. Right, bar graph showing thepercent of necrosis in each 10� field of view.D, IHC analysis of STAT1 in TAG. Left, representative images of IHC STAT1; right, STAT1þ cells per 40� field of view.E, IHCanalysis of STAT1 in TAG a-SMA. Left, representative images of IHC a-SMA; right, a-SMAþ cells per 40� field of view.

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combination treatment significantly suppressed STAT1 expres-sion in residual tumor tissues while doxorubicin treatmentshowed a moderate effect on STAT1 expression in tumor tissues(Supplementary Fig. S4). Finally, all treatment groups showed amild decrease ofa-SMA expression in tumor-adjacent glands (Fig.6E). Taken together, these findings suggest that targeting theSTAT1 pathway could further improve the therapeutic efficacy ofconventional chemotherapy.

DiscussionBreast cancer development is an evolutionary process with

extensive interplay between tumor cells and their stroma. It isindisputable that a "tumor-centric" approach limits our under-standing of the tumor as a component of a dynamic ecosystem.While previous studies primarily focus on the pathologic con-sequences of tumor-associated stroma on tumorigenesis, ourdata collectively demonstrated a field cancerization effect insti-gated by early tumor development on normal adjacent tissue,which facilitates a vicious cycle of the tumor development(Supplementary Fig. S5).

The first focal point of our study is the tissue niche surroundingthe developing tumor. As a consequence of tumor initiation, acancer field effect is induced, reflected in altered branching mor-phogenesis, an increased capacity for mammosphere formation,and an increasing MaSC population (Fig. 1). Mechanistically,analysis of tumor-associated stroma using a systems biologyapproach revealed upregulation of STAT1 specific to CAFs (Fig.2). Transient depletion of STAT1 in CAFs suggested a tumor-promoting role for STAT1mediated by secretion of PTX3 (Fig. 4).In addition, in vivo depletion of STAT1 in CAFs reduced periductala-SMAþ myoepithelial/myofibroblasts and delayed mammarytumor progression, suggesting a stroma-specific role for STAT1in promoting invasion of early-stage lesions (Fig. 5). Finally, weshowed that treatment with the STAT1 inhibitor fludarabine incombination with doxorubicin significantly reduced tumor bur-den compared with mice treated with doxorubicin alone (Fig. 6).

Our results demonstrated a context- and tissue compart-ment-specific role of STAT1 in breast cancer progression. STAT1activity in tumor cells is often ascribed a tumor suppressorfunction; however, in the TME context, recent evidence hasemerged suggesting the transcription factor may also play atumor-promoting role, particularly in a breast cancer. Hix andcolleagues used a syngeneic mouse injection model to dem-onstrate a protumor role for STAT1 in tumor cells due toexpression of STAT1 and recruitment of CD33þ myeloid cells.Similarly, Tymoszuk and colleagues found that high levels ofSTAT1 mRNA and increased levels of STAT1 target genes intumor cells are associated with macrophage infiltration andpoor prognosis (39). These findings imply that, in addition toits cell-autonomous role in controlling tumor cell proliferation,tumor cell–derived STAT1 might also play a pivotal role in TMEimmune recruitment. Distinct from the aforementioned tumorcell–focused studies, in our study, we provide compellingevidence for the role of STAT1 within the mammary TME.Specifically, tumor cell–instigated upregulation of STAT1 inCAFs alters the proliferation of tumor cells through paracrineand autocrine signaling of PTX3 (Figs. 3 and 4). We furtherdemonstrated that CAF-secreted PTX3 promotes tumor cellviability and proliferation. Bonavita and colleagues publishedevidence suggesting the opposite, showing increased suscepti-

bility for epithelial and mesenchymal tumorigenesis and Trp53mutations in PTX3�/� mice (40). Despite compelling analysessuggesting PTX3 promoter methylation in certain subtypesof cancer, we strongly believe that the role of PTX3 in regulatingthe tumor niche is complex and entirely context dependent. Forexample, Chi and colleagues determined that PTX3 is a CCAAT/enhancer binding protein delta (CEBPD)-responsive geneand displays a protumor phenotype (41), including acquireddrug resistance, enhanced migration, and stem-like character-istics. Given these recent findings, we believe our results furthersupport a protumor role for PTX3 in a tissue niche withactivated CAFs.

Furthermore, our novel observation underscores the impor-tance of studying cancer progression as cohesive and evolvingecosystem (42), which will provide mechanistic insight ofperplexing clinical observation of field cancerization in mam-mary tissue (35). Although we have proposed that tumorsimpose field cancerization on their niche constituents resultingin upregulation of STAT1, the means by which tumors initiallyactivate fibroblasts is multilayered and complex. One potentialexplanation is that tumor cells secrete proinflammatory factorsthat promote resident fibroblast activation and maintain fibro-blasts in this reactive state (38, 43). In addition, evidencesuggests that bone marrow–derived circulating myofibroblastshome to tumors by a systemic response reminiscent of woundhealing (44, 45). This phenomenon could possibly occur inparallel with the activation of tissue-resident fibroblasts, pro-ducing a rapid supply of myofibroblasts in the tumormicroenvironment.

Finally, from a translational perspective, identifying molec-ular targets in the tumor-associated stroma provides new ther-apeutic opportunities to "normalize" the tumor-promotingTME. It is increasingly acknowledged that targeting tumorstroma as a neoadjuvant therapy or in combination withchemotherapy is a promising new avenue to enhance thera-peutic efficacy and attenuate drug resistance (46). While aclinically applicable target of STAT1 for breast cancer is cur-rently unavailable, we conducted a proof-of-concept study ofchemotherapy with fludarabine, a FDA-approved chemothera-py that has been show to significantly inhibit STAT1 signaling(24). Our results demonstrated that combinatorial treatment offludarabine with doxorubicin enhanced the overall therapeuticefficacy and reduced stroma abnormalities (Fig. 6). Furtherdevelopment of a STAT1-specific agent could ultimately facil-itate the clinical proposition of cotargeting the tumor andstroma as a strategy to reduce tumor burden.

In summary, our study highlighted the tumor-imposed insultson the microenvironment and mammary gland homeostasis andsubsequent induction of a molecular clutch (STAT1) driving avicious cycle of tumor progression and shed light on future designof rationalized combinatorial therapy targeting both cancer cell aswell as its coevolved tumor microenvironment.

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

Authors' ContributionsConception and design: V.R. Zellmer, S. ZhangDevelopment of methodology: V.R. Zellmer, X. Tan, E.N. Howe, S. ZhangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V.R. Zellmer, S.L. Fracci, X. Tan, S. Zhang






Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V.R. Zellmer, P.M. Schnepp, S.L. Fracci, E.N. Howe,S. ZhangWriting, review, and/or revision of the manuscript: V.R. Zellmer, S. ZhangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): V.R. ZellmerStudy supervision: V.R. Zellmer, S. Zhang

AcknowledgmentsThe authors would like to thank Dr. Zonggao Shi, HCRI Biorepository

Director, Dr. Charles Tessier, and the Notre Dame Genome Core Facility fortheir technical support.

Grant SupportThis work is partially supported by Department of Defense W81XWH-15-1-

0021 (to S. Zhang), NIH 1R01CA194697-01 (to S. Zhang), Walther CancerFoundation Advancing Basic Cancer Research Grant II (to S. Zhang), IndianaCTSI core pilot fund (to S. Zhang), HCRI Notre Dame Day Pilot Fund(to S. Zhang).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 15, 2016; revised December 13, 2016; accepted January3, 2017; published OnlineFirst January 20, 2017.

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2017;15:585-597. Published OnlineFirst January 20, 2017.Mol Cancer Res Victoria R. Zellmer, Patricia M. Schnepp, Sarah L. Fracci, et al. Deregulating Tissue HomeostasisTumor-induced Stromal STAT1 Accelerates Breast Cancer via

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Tumor-inducedStromalSTAT1AcceleratesBreast Cancer via ... · the protumor effects of the TME in conjunction with traditional cancer chemotherapies (4). ... majority of mammary gland