A Novel ACKR2-Dependent Role of Fibroblast-Derived CXCL14 in Epithelial-to-MesenchymalTransition and Metastasis of Breast CancerElin Sj€oberg1, Max Meyrath2, Laura Milde3, Mercedes Herrera1, John L€ovrot1,Daniel H€agerstrand1, Oliver Frings1, Margarita Bartish1, Charlotte Rolny1,
Erik Sonnhammer4, Andy Chevign�e2, Martin Augsten1, and Arne €Ostman1
Abstract
Purpose: Fibroblasts expressing the orphan chemokineCXCL14 have been previously shown to associate with poorbreast cancer prognosis andpromote cancer growth. This studyexplores the mechanism underlying the poor survival associa-tions of stromal CXCL14.
ExperimentalDesign:Tumor cell epithelial-to-mesenchymaltransition (EMT), invasion, and metastasis were studied in invitro and in vivo models together with fibroblasts overexpres-sing CXCL14. An approach for CXCL14 receptor identificationincluded loss-of-function studies followed by molecular andfunctional endpoints. The clinical relevance was furtherexplored in publicly available gene expression datasets.
Results: CXCL14 fibroblasts stimulated breast cancer EMT,migration, and invasion in breast cancer cells and in a xeno-
graft model. Furthermore, tumor cells primed by CXCL14fibroblasts displayed enhanced lung colonization after tail-vein injection. By loss-of function experiments, the atypicalG-protein–coupled receptor ACKR2 was identified to mediateCXCL14-stimulated responses. Downregulation of ACKR2, orCXCL14-induced NOS1, attenuated the pro-EMT and migra-tory capacity. CXCL14/ACKR2 expression correlatedwith EMTand survival in gene expression datasets.
Conclusions: Collectively, the findings imply an autocrinefibroblast CXCL14/ACKR2 pathway as a clinically relevant stim-ulator of EMT, tumor cell invasion, and metastasis. The studyalso identifies ACKR2 as a novel mediator for CXCL14 functionand thereby defines a pathway with drug target potential.
See related commentary by Zhang et al., p. 3476
IntroductionDeath of patients with breast cancer is almost exclusively due
to metastatic disease. Metastasis develops through a multistepprocess, involving tissue invasion, intravasation, survival in thebloodstream and lymph system, extravasation, and tissue colo-nization. This study develops recent correlative studies that haveindicated clinical relevance, in breast cancer, of stroma-derivedexpression of the chemokine CXCL14 by demonstrating signifi-
cant and independent associations between high stromalCXCL14expression and shorter survival in a population-based breastcancer cohort. Notably, epithelial expression of CXCL14 did nothave an impact on breast cancer outcome (1).
During the early steps of metastasis development, tumor cellslose cell-to-cell contacts and epithelial characteristics and insteadgain mesenchymal traits that allow them to invade the surround-ing tissue and metastasize; a process termed epithelial-to-mesen-chymal transition (EMT; ref. 2).
EMT is controlledbydistinct transcriptional programsactivatedby specific transcription factors, including Snail, Slug, Twist, Zeb,and Gsc. Activation of these factors ultimately leads to the loss ofepithelial markers including E-cadherin and cytokeratins, and theupregulation of mesenchymal markers, such as vimentin, alpha-smoothmuscle actin (aSMA), andmatrix-degrading enzymes (3).Although the classical paradigm attributing EMT a crucial role inthe process of metastasis has been recently challenged by studiesin genetic mouse models, other recent studies including in vivoimaging approaches demonstrated that cancer cells displaying anEMT phenotype give rise to metastases (4–6).
Induction of EMT can occur in a paracrine manner by secretedfactors from cells of the tumor stroma, as for example, the cancer-associated fibroblasts (CAF; refs. 7, 8). CAFs constitute a hetero-geneous cell population that contributes to cancer growth andspread by secretion of a variety of protumorigenic factors, includ-ing soluble factors. Among these CAF-secreted factors implicatedin EMT andmetastasis are chemokines, proteins of a size between8 and 14 kDa that stimulate directed cell migration by creating agradient along which cell types expressing the corresponding
1Department of Oncology-Pathology, Cancer Center Karolinska (CCK), Karo-linska Institutet, Stockholm, Sweden. 2Department of Infection and Immunity,Immuno-Pharmacology and Interactomics, Luxembourg Institute of Health(LIH), Esch-sur-Alzette, Luxembourg. 3Division for Vascular Oncology andMetastasis, German Cancer Research Center (DKFZ), Heidelberg, Germany.4Stockholm Bioinformatics Center, Department of Biochemistry and Biophysics,Stockholm University, Science for Life Laboratory, Stockholm, Sweden.
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
M. Augsten and A. €Ostman contributed equally to this article.
Current address for M. Augsten: amcure GmbH, Eggenstein-Leopoldshafen,Germany.
Corresponding Author: Arne €Ostman, Karolinska Institutet, Cancer CenterKarolinska, R8:03, Karolinska University Hospital, Stockholm 17176, Sweden.Phone: 468-5177-0232; Fax: 468-339-031; E-mail: [email protected]
receptor travel (7, 9). Chemokines bind to the pertussis-sensitiveGai-subfamily of G-protein–coupled receptors (GPCR) thatengage different signaling pathways including ERK1/2, PI3K/AKT,and calcium signaling (10). Besides the classical chemokinereceptors, there is a subfamily of atypical chemokine receptors(ACKR) that are predominantly involved in sequestration ofchemokines (11).
In cancer, chemokines are involved in the recruitment ofvarious cell types into tumors and thereby affecting inflammation,angiogenesis, tumor growth, invasion, and metastasis (12). Aparacrine chemokine cross-talk between stromal cells and tumorcells, involving effects onEMT, has been demonstrated to enhanceformation of metastases (13–16). Expression of certain chemo-kines in distant tissues has also been reported to determinemetastasis formation in specific organs, a process termed meta-static tropism (17).
The orphan chemokine CXCL14, earlier designated BRAK,MIP-2g , BMAC, or KS1 stimulates migration of various immunecells, including B cells, NK cells, and monocytes, but not T cells(18–21). CXCL14 expression has been shown to be upregulated inCAFs, as compared with normal fibroblasts, in human breast andprostate cancer (22, 23). Experimental studies exploring the func-tion of CXCL14 during tumor progression and metastasis forma-tion have demonstrated context-dependent pro- and antitumoraleffects. The reasons for these effects remain largely unknown andcould possibly dependent on the cell type that express the chemo-kine and on the profile of chemokine receptors and ACKRsexpressed. Some studies with CXCL14 overexpression in tumorcellshavedemonstratedantitumoral effectsof this chemokine (24).In contrast, tissue culture andmouse cancermodel studies of breastand prostate cancer have demonstrated protumoral effects ofCXCL14 expressed by stromal fibroblasts, associated withCXCL14-induced changes in fibroblast secretomes (22, 23, 25).
The tumor-promoting roles of CAF-derived CXCL14 havebeen shown to depend on nitric oxide synthase 1 (NOS1)and involve stimulation of angiogenesis and recruitment ofmacrophages (25).
Continued exploration of the roles of CXCL14 in tumor biol-ogy and possible exploitation of this chemokine as a therapeutictarget depend on the identification of critical mediators ofCXCL14 signaling, including receptors. This study thereforeaimed at providing a better understanding of the molecularmechanismunderlying the documented poor survival associationof stroma-derived CXCL14.
Materials and MethodsCell lines and chemicals
The mouse fibroblast cell line NIH3T3 (and derivatives), thebreast cancer cell lines MCF7, SKBR3 MDA-MB-231, 4T1, and
Hs578t were cultured in DMEM (Hyclone), supplemented with10% FBS (Hyclone), 1% glutamine (Hyclone) and 1% penicillin/streptomycin (Hyclone). DMEM-F12 (Hyclone), supplementedwith horse serum (Biochrom), 1% glutamine, and 1% penicillin/streptomycin was used for culturing the MCF10-DCIS cell line.Starvation was performed in medium without serum. All cellswere maintained at 37�C in humidified air with 5%CO2. NIH-ctrand NIH-CXCL14 fibroblasts have been characterized earlier, andfibroblasts secrete physiologic levels of CXCL14 (22). Cell lineswere purchased from ATCC or received from collaboration part-ners and continually tested for Mycoplasma infection during thestudy. The identity of the cell lines used was confirmed by shorttandem repeat (STR) profiling at Uppsala Genome Center. Allexperiments were performed with cells of passage 3–20.
Western blot analyses used antibodies against p-ERK (#9101),ERK (#9102), E-cadherin (#3195), Snail (#3879), NOS1 (#4234)(Cell Signaling Technology), b-actin (A1978), and a-tubulin(T5201; Sigma-Aldrich). Primary antibodies detecting E-cadherin(#3195), Cytokeratin 8/18 (#4546), and PDGFbR (#3169; CellSignaling Technology) together by fluorescent-linked secondaryantibodies, were used for immunofluorescence staining of xeno-graft tumors.
Pertussis toxin was purchased from Sigma-Aldrich and recom-binant CXCL14 and CCL5 from R&D Systems and PeproTech.
Alexa Fluor 647–coupled chemokines were purchased fromAlmac.
RNA isolation, cDNA synthesis, and qRT-PCR analysisRNA was isolated from xenograft tumors or overnight starved
cells using GeneElute Mammalian Total RNA Miniprep Kit(Sigma-Aldrich). cDNA synthesis was performed with the Super-Script III First-Strand Synthesis System for RT-PCR (Invitrogen),using PolydT primers, in accordance with the manufacturer'sinstructions. The qRT-PCR reaction using SYBR Green UniversalPCR Master Mix (Applied Biosystems) was performed with the7500 Real-Time PCR System (Applied Biosystems). The concen-tration of primers was 200 nmol/L, and expression levelswere normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences forprimers obtained from Sigma-Aldrich are shown in Supplemen-tary Table S1. Other primers were QuantiTect primers obtainedfrom Qiagen.
Immunoblotting, immunofluorescence, and secretomeanalyses
Immunoblotting and immunofluorescence analyses were per-formed as described previously (22). In short, for analysis ofCXCL14-induced p-ERK signaling by immunoblotting, overnightserum-starved cells were stimulated with recombinant CXCL14(R&D Systems or PeproTech) for 7minutes. For experiments withpertussis toxin, cells were treated with the toxin for 1 hour at 37�Cand 5% CO2 prior to CXCL14 stimulation. Cell lysates wereprepared and SDS/PAGE was performed followed by transfer topolyvinylidene difluoride membranes (Millipore). Immunoblot-ting with p-ERK and ERK antibodies (Cell Signaling Technology),diluted 1:1,000, were performed and signals were detected withImageQuant LAS4000 (GE Healthcare) and quantified usingImageJ (http://imagej.nih.gov.proxy.kib.ki.se/ij).
Analysis of EMT markers was performed for 48–72 hours afterstimulation with fibroblast-conditioned medium or coculture ofbreast cancer cells and fibroblasts at a 1:1 ratio. The conditioned
Translational Relevance
Autocrine fibroblast CXCL14/ACKR2 signaling is shown toinduce EMT, migration, and metastasis and to correlate withworse survival in patientswith breast cancer. The identificationof ACKR2 as a novel component in the signaling of the orphanchemokine CXCL14 is relevant for further biomarker studiesand suggests novel targeting opportunities.
Sj€oberg et al.
Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer ResearchOF2
medium was generated by seeding the same number of NIH-ctrand NIH-CXCL14 cells. The next day, medium was changed toDMEM containing low serum (1% FBS) and the conditionedmedium was collected for 48 hours, sterile filtered with a 0.2-mmfilter. Antibodies for immunoblotting, including E-cadherinand Snail, were diluted 1:1,000 and NOS1 1:500. For immuno-fluorescence analysis, E-cadherin, Cytokeratin 8/18, and PDGFbRantibodies were diluted 1:100.
To analyze the secretome from CXCL14 fibroblasts, a proteinarray was performed. A total of 7.0 � 105 NIH-CXCL14 andNIH-ctr cells were seeded in 10-cm dishes. The next day,medium was changed from medium with 10% FBS to lowserum (1% FBS). Conditioned medium was collected after48-hour incubation at 37�C, sterile filtered, and stored at�20�C. Aliquots of conditioned medium (1 mL) fromNIH-CXCL14 and NIH-ctr fibroblasts were subjected to theproteome profiler (mouse angiogenesis array kit ary015, R&DSystems), performed according to the manufacturer's protocol.Array images obtained were analyzed using the ImageJ soft-ware. For all spots, the average background signal from negativecontrol spots was subtracted. The average signal from positivecontrol spots of each membrane was used to normalize the twodifferent types of fibroblast conditions. The relative differencesin protein expression between NIH-CXCL14 and NIH-ctr cellswere expressed as ratio (fold of NIH-ctr).
In vitro growth, migration, and invasion assaysTo study the effect of siRNA and short hairpin RNA (shRNA)-
mediated knockdown of ACKR2 on growth of NIH-ctr and NIH-CXCL14fibroblasts, 2�104 cellswere seededperwell of a 24-wellplate (Sarstedt), in quadruplicates in serum-reduced media. After3days of culture, AlamarBlue (Bio-Rad)wasused todetermine thecell number. A total of 350mLof a 1:10dilutionof AlamarBluedyeinDMEMwas added to eachwell and cells were incubated at 37�Cwith 5%CO2 for 2.5 hours. Tomeasure the conversion of the dye,100 mL was transferred into each well of a white 96-well plate(Costar) and absorbance was measured at a wavelength of570 nm.
To study cell migration of breast cancer cells, a transwellmigration assay was used. A total of 2.5 � 104 breast cancer cellswere seeded in transwell inserts (Corning) with an 8.0-mm pore-sized membrane and placed in a 24-well plate (Corning), induplicates. For analysis of CXCL14 fibroblast–inducedmigration,2.5 � 104 CXCL14- or control fibroblasts were seeded in thebottom chamber.
For migration experiments, lasting for 16–24 hours, the insideof the insert was wiped with cotton swabs, washed with PBS, andthe cells were fixed in ice-cold methanol. The membrane was cutout and placed on Superfrost Plus slides (Menzel-Gl€aser) andstained with Vectashield Mounting Medium with DAPI (VectorLaboratories). Quantification of cell migration was performed bycounting cell nuclei of the migrated cells. The same principleswere used for the invasion assays, with inserts containing athin Matrigel layer (Corning). Invasion was allowed to occur for72 hours.
Chemokine binding assayHEK-293 cells or HEK-293 cells stably expressing ACKR2
(under 200 mg/mL hygromycin selection) were distributedinto 96-well plates (2 � 105 cells per well) and incubatedwith increasing concentrations ranging from 10 pmol/L to
100 nmol/L of Alexa Fluor 647–labeled CCL5 or CXCL14 for90 minutes on ice. After a washing step, Zombie Green FixableViability Kit (BioLegend) was used to gate on living cells only. Theexperiments were performed in PBS containing 1%BSA and 0.1%NaN3 (FACSbuffer). Chemokine bindingwas quantifiedbymeanfluorescence intensity (MFI) of 10,000 gated cells on a BD FACSFortessa Cytometer (BD Biosciences).
b-arrestin recruitment assayChemokine-induced b-arrestin-1 recruitment to ACKR2 was
monitored by Nanoluciferase complementation assay (NanoBiT,Promega) as described previously (26, 27). A total of 5� 106HEKcells were plated in 10-cm culture dishes and 24 hourslater transfected with plasmids containing human b-arrestin-1N-terminally fused to LgBiT and ACKR2 C-terminally fused toSmBiT. Twenty-four hours posttransfection, cells were harvested,incubated 30 minutes at 37�C with 200-fold diluted Nano-Glo Live Cell substrate, and distributed into a white 96-well plate(1 � 105 cells per well). b-arrestin-1 recruitment was measuredover 25 minutes with a Mithras LB940 luminometer (BertholdTechnologies). For each experiment, signal measured with asaturating concentration (300 nmol/L) of the full agonist (i.e.,CCL5) was set as 100%.
Bioinformatic analysesSequence analyses for novel chemokine receptors started from
Pfam family PF00001 (7tm_1), using the 1679 human domainsfrom 1,664 human sequences in the full alignment. A neighbor-joining tree of these was built using scoredist distances withBelvu (28). A subtree of 114 cytokines was cut out. After removingfragment sequences and >99% identical sequences, 32 sequenceswere left. Following exclusion of DUFFY, not being part of the23,760 homologs in the Pfam family, a candidate list of 31candidates was established (Supplementary Fig. S1).
Transfection of siRNA and generation of stable cell linesFor transfection of siRNA, 1.0–2.0 � 105 cells were seeded
in 6-well plates (Sarstedt) and transfected for 48 hours with100 nmol/L siRNA (ACKR2 target sequence; 50-CTCAATTAGCGT-TATTGGCAA-30; Qiagen) using HiPerFect transfection reagent(Qiagen). To determine the efficiency of siRNA-mediated knock-down of genes of interest, RNA extraction, cDNA synthesis, andqRT-PCR analysis was performed.
Fibroblast derivatives with stable knockdown of ACKR2 wereestablished using shRNA procedures, as described previous-ly (25). In brief, phoenix cells were transfected with 2 unique29mer shRNA constructs against ACKR2 (gene ID ¼ 59289;shACKR2:A and shACKR2:B) or nontargeting control shRNA(shCtr) in retroviral vectors (Origene). After 48 hours, thesupernatant was collected, filtered, and added to NIH-ctr andNIH-CXCL14 cells for 5 hours. Cells were subsequently selectedin 30 mg/mL blasticidin for 2 weeks. The knockdown of ACKR2was confirmed by qRT-PCR. Generation of NIH-ctr and NIH-CXCL14 fibroblasts with a stable knockdown of NOS1 havebeen described previously (25).
Animal experimentsThe animal experiments were conducted in accordance with
national guidelines and approvedby the StockholmNorth EthicalCommittee on Animal Experiments. The generation of xenografttumors was performed as described previously (25).
Fibroblast CXCL14/ACKR2/NOS1 Signaling in Breast Cancer
www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 OF3
The analyses of lungmetastasis formationwere performed aftertail-vein injection of 2� 105 breast cancer cells in three groups of8-week-old female SCID mice, without any further randomiza-tion. The sample size of 10mice in each group was determined bythe 3R criteria together with previous experience. After 4 weeks,mice were sacrificed and lungs were collected, washed in PBS(Hyclone), and snap frozen (for qRT-PCR analysis) or embeddedin optimal cutting temperature medium (OCT) and snap frozen(for histologic analysis). No animals were excluded from thestudy.
For RNA extraction, 1 mL of TRIzol (Life Technologies) wasadded to the lung tissue and homogenized with a polytron 3times, each for 10 seconds. A volume of 0.2-mL chloroform wasadded and the samples were shaken for 15 seconds and incubatedfor 2minutes at room temperature. The samples were centrifugedat 12,000 � g for 15 minutes at 4�C and the aqueous phase wasplaced in a new tube. RNAwas precipitated by addition of 0.5mL100% isopropanol (Merck) to the samples and incubation for10 minutes at room temperature. Following centrifugation at12,000 � g for 10 minutes at 4�C, the RNA pellet was washedwith 75% ethanol and air dried before resuspension in nuclease-free water (Ambion). The RNA concentration of each sample wasdetermined using the Nanodrop ND-1000 spectrophotometer(NanoDrop Technologies). cDNA were synthetized and subse-quent qRT-PCR analysis was performed as described previously,with human- and mouse-specific primers. Percentages of humancells in mice lungs were quantified as described in Malek andcolleagues (29).
Ten frozen sections (10 mm) were made from the OCT-embed-ded lungs. Five sectionswere thrown in between each saved frozensection. Stainings were performed with the human-specific anti-body Stem121 (Takara Bio) and the positive cells were countedin each section and results are displayed as average number perlung. The tail-vein injections were performed blinded and theanalyses of lung metastasis were performed unblinded.
Clinical cohortsThe relation between CXCL14 transcript abundance and EMT
as assessed by an EMT gene expression signature was investigatedin clinical breast cancer cohorts with publicly available transcrip-tome data: the Uppsala (30), Stockholm (31), Rotterdam (32),andMETABRIC (33) cohorts, as well as The Cancer Genome Atlas(TCGA; ref. 34). Each study site in METABRIC is treated as aseparate cohort. TCGA gene expression datasets for breast cancer,ovarian cancer, and prostate cancer was used to investigate thelevels of EMT markers and survival associations in CXCL14/ACKR2 subgroups was additionally performed in gene expressiondatasets for bladder cancer, clear cell renal cell carcinoma(ccRCC), colorectal cancer, esophageal cancer, glioblastomamul-tiforme (GBM), low-grade glioma,head andneck cancer (HNCC),lung adenocarcinoma, pancreas cancer, and stomach cancer (34).
Gene expression data analysisAn EMT gene expression signature score was derived for each
tumor in thepanel of clinical cohorts as describedpreviously (35).In brief, the signature was identified from the changes in geneexpression shared by upregulation of Gsc, Snail, Twist, andTGFb1 and by downregulation of E-cadherin. PAM50 intrinsicsubtype classificationwasperformedasdescribedpreviously (36).Individual patient data meta-analysis (IPDMA) of all cohortswith a linear mixed-effects model was performed using the
R package nlme [R syntax lme(groupedData(ScaledEMTscore� scaledCXCL14 | cohort, data))]. All gene expression dataanalysis was performed in R/Bioconductor and SPSS 21.0. TheEMT score and CXCL14 data are first centered and scaled to unitSD within cohorts to facilitate comparison between cohorts.Hence, the slope of a linear regression in each cohort in Supple-mentary Fig. S2A is mathematically equivalent to the Pearsoncorrelation coefficient. This equivalence does not hold in sub-groups as in Supplementary Fig. S2B.
The TCGA gene expression data (Supplementary Figs. S3 andS4) are displayed as Z-scores obtained from cBioportal. For thebreast cancer gene expressiondataset, CXCL14low andCXCL14high
groups were divided according to the 50 percentile, whereasACKR2low and ACKR2high expression was determined by fittinga mixture of two normal distributions using the R packagemixtools, resulting in a dichotomization below and above the90.7 percentile. For the TCGA ovarian and prostate cancer geneexpressiondatasets, theACKR2low andACKR2high subgroupsweredivided by the 4th quartile.
Statistical analysisStatistical calculations were performed using Excel 2011 for
Mac (Microsoft Office), R/Bioconductor, or the statistical packageSPSS 21.0 (SPSS Inc.). All data are expressed as mean or medianvalues, and error bars represent the SDor SEM.Data that are beingstatistically compared, relevant for the conclusions, exhibit sim-ilar variation. Statistical differences between groups were deter-mined using two-sided, unpaired Student t test orMann–WhitneyU test. Pearson correlation was used to analyze correlationsbetween different parameters. The Kaplan–Meier method andlog-rank test method was performed to estimate overall survival.Cox proportional hazards model was used to compare HRs inboth uni- and multivariate analyses. The multivariate analysisincluded known clinical relevant parameters, including T-stage,N-stage, M-stage, and molecular subtypes of breast cancer.Results are presented in the multivariate analysis as HRsincluding 95% confidence intervals (95% CI). For all analyses,P values below 0.05 were considered significant (�, P < 0.05;��, P < 0.01; ���, P < 0.001). All relevant data are available fromthe authors.
ResultsCXCL14 fibroblasts induce loss of epithelial markers andenhance expression of mesenchymal markers and EMTtranscription factors in breast tumor xenografts
The findings of stromal CXCL14 as a poor prognostic marker inbreast cancer (1), prompted analyses of the potential effects ofCAF-derived CXCL14 on tumor cell invasion and metastasis.
We first studied the expression levels of EMT markers as anindicator of a proinvasive phenotype in xenograft tumors formedfollowing coinjectionof the epithelial breast cancer cell lineMCF7and either control fibroblasts or CXCL14 fibroblasts (25).
Immunofluorescence staining of xenograft tumor sectionsdemonstrated a significant loss of tumor cell E-cadherin andCytokeratin 8/18 in CXCL14 breast tumors, as compared withcontrol tumors (Fig. 1A; Supplementary Fig. S5A; SupplementaryTable S2). NOS1, an oxidative stress–induced enzyme, was pre-viously shown to functionally contribute to the protumorigenicactions of CXCL14-expressing fibroblasts (25). EMT markerswere therefore also analyzed in MCF7/NIH-CXCL14 tumors
Sj€oberg et al.
Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer ResearchOF4
with a stable NOS1 downregulation in the fibroblasts. Impairedexpression of NOS1 was sufficient to reverse the decrease inepithelial markers induced by CXCL14 fibroblasts in vivo(Fig. 1A; Supplementary Fig. S5A; Supplementary Table S2).Furthermore, CXCL14 fibroblasts also reduced cancer cell Cyto-keratin 8/18 levels, in a NOS1-dependent manner, in a xenograft
coinjection model of prostate cancer (Supplementary Fig. S5B;Supplementary Table S2).
Analyses of mRNA levels of EMT markers substantiated thesefindings and uncovered a reduction of epithelial markers includ-ing E-cadherin (CDH1), Cytokeratin 18 (KRT18), andCytokeratin8 (KRT8), and increase in mesenchymal markers including
MCF7 + NIH-ctr/shCtr
MCF7 + NIH-CXCL14
/shCtr
MCF7 + NIH-CXCL14
/shNOS1
A
B
0
10
20
30
40
50
60
CXCL14
KRT18KRT8
CDH1VIM
ACTA2
SNAI2
TWIS
TMMP2
Rel
ativ
eex
pres
sion
(Fol
dof
CXC
L14
inN
IH-c
tr)
MCF7+ NIH-ctr/shCtr
MCF7+NIH-CXCL14/shCtr
MCF7 + NIH-CXCL14/shNOS1
* **
*
*
* *
*
* *
* *
* *
* *
* *
CMCF + NIH-ctr MCF7 + NIH-CXCL14
0 2 4 6 8
10 12
MCF7+NIH
-ctr
MCF7+NIH
-CXCL1
4
Mea
nn r
ofbu
d di n
gce
l ls/v
isio
nfie
ld
***
Figure 1.
CXCL14 affects regulators of EMTand invasion in a xenograft tumormodel of breast cancer. A,Xenograft tumors of MCF7 cellscoinjected with NIH-ctr orNIH-CXCL14 fibroblasts werestained for E-cadherin. Whitearrowheads indicate epithelial cellswith weak E-cadherin expression.Scale bar, 50 mm. B, qRT-PCRanalysis of transcript levels of genesencoding EMT-regulated markers inxenograft tumors. The analysiscomprises epithelial marker(E-cadherin, Cytokeratin 18, andCytokeratin 8), mesenchymalmarker [Vimentin, a-SMA (encodedby ACTA2), and MMP2], and theEMT transcription factors Slug andTwist (n¼ 5). C, Staining ofxenograft tumors formed followingcoinjection of MCF7 cells and NIH-ctr or NIH-CXCL14 fibroblasts withthe human-specific antibodyStem121. Budding cells (cluster of upto three cells) in the border of thetumor were counted in 10 visionfields, and results are shown asmean number (nr) of cells/visionfield (n¼ 5). Scale bar, 100 mm.P values were derived fromunpaired two-sided Student t tests.��� , P < 0.001; �� , P < 0.01; and� , P < 0.05. Error bars, SEM.
Fibroblast CXCL14/ACKR2/NOS1 Signaling in Breast Cancer
www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 OF5
Vimentin (VIM), a-SMA (ACTA2), and MMP2 (MMP2), and anincrease in EMT transcription factors including Slug (SNAI2), andTwist (TWIST1) in CXCL14-breast tumors (Fig. 1B). Furthermore,analyses of MCF7/NIH-CXCL14 tumors with a stable NOS1knockdown in fibroblasts demonstrated NOS1 dependency ofthese gene expression changes (Fig. 1B). However, mesenchymalmarkers, including Fibronectin (FN1), FAP (FAP), and MMP9(MMP9) were upregulated in CXCL14 tumors independently ofNOS1 expression (Supplementary Fig. S5C).
Additional analyses were performed to investigate whetherthe CXCL14-dependent EMT phenotype also was associated withamore invasive growth pattern. As shown in Fig. 1C, MCF7/NIH-CXCL14 tumors displayed a more invasive growth pattern with asignificantly higher number of budding cells in the tumor periph-ery, as compared with MCF7/NIH-ctr tumors.
These data collectively demonstrate that cancer cells coinjectedwith CXCL14 fibroblasts exhibit enhanced EMT and invasionin vivo.
CXCL14 fibroblasts stimulate EMT in vitro and induce amesenchymal morphology of breast cancer cells
Next, the direct impact of CXCL14-expressing fibroblastson the modulation of EMT markers in MCF7 breast cancer cellsand MCF10-DCIS (ductal carcinoma in situ, DCIS) cells wasinvestigated under in vitro coculture conditions. Western blotanalysis demonstrated a reduction of E-cadherin in both MCF7-and DCIS cells after direct coculture with CXCL14 fibroblasts thatwas not seenwith controlfibroblasts (Fig. 2A). To confirm that thedownregulation occurred in the breast cancer cells and did notreflect changes in fibroblast properties or abundance, immuno-fluorescence costaining for E-cadherin orCytokeratin 8/18 togeth-er with the fibroblast marker PDGFRb was performed on MCF7fibroblast cocultures. As shown in Fig. 2B, there was a specific lossof E-cadherin (left) and Cytokeratin 8/18 (right) in the breastcancer cells when MCF7 cells were cocultured with CXCL14fibroblasts, but not in the presence of control fibroblasts. Similarfindings of altered EMT markers in MCF7- and DCIS cells weredetected after treatment with conditioned media (CM) fromCXCL14 fibroblasts (Supplementary Fig. S6A and S6B). Anotherbreast cancer cell line, SKBR3, which has lost E-Cadherin, showedincreased levels of the EMT transcription factor Snail when treatedwith CM from NIH-CXCL14 compared with CM from NIH-ctr(Supplementary Fig. S6B).
Furthermore, treatment of MCF7, DCIS, and SKBR3 breastcancer cells with CM from CXCL14 fibroblasts induced changesin cell morphology. The tumor cells formed filopodium-likeprotrusions and obtained amesenchymal-like morphology whencultured in CM from CXCL14 fibroblasts, but not in CM fromcontrol fibroblasts or in standardDMEM (Fig. 2C; SupplementaryFig. S6C). These phenotypes induced by CXCL14 fibroblastswere not seen in the mesenchymal metastatic breast cancer cellline MDA-MB-231, a cell line that already has undergone EMT(Supplementary Fig. S6C).
These results demonstrate the ability of CXCL14 fibroblaststo induce an EMT phenotype in certain breast cancer cells, in aparacrine manner independent of cell-to-cell contact.
CXCL14 fibroblasts enhance migration and invasion of breastcancer cells
The induction of EMT suggested functional effects of CXCL14fibroblasts on breast cancer cells. Thus, we compared the ability of
NIH-ctr and NIH-CXCL14 fibroblasts to stimulate the migrationand invasion of MCF7, DCIS, and SKBR3 cells. Transwell migra-tion assayswere performed to analyzewhether the changes inEMTmarkers and in morphology were accompanied by an increase incell motility. CXCL14 fibroblasts displayed a stronger ability tostimulate the migration of MCF7, DCIS, and SKBR3 cells, ascompared with control fibroblasts (Fig. 3A). We also investigatedwhether CXCL14 fibroblast–induced EMT and migration couldbe observed in breast cancer cell lines representing the basal(triple negative) molecular subgroup of breast cancer, including4T1 cells and Hs578t cells (Supplementary Fig. S7A and S7B).NIH-CXCL14 cells significantly enhanced the migration (Supple-mentary Fig. S7A) and stimulated EMT (Supplementary Fig. S7B)of 4T1 cells, as compared with NIH-ctr cells. There was atrend toward enhanced migration of Hs578t cells, althoughnot significant, possibly explained by the fact that these cellsalready have undergone EMT (Supplementary Fig. S7A).
Furthermore, invasionofMCF7andSKBR3 cells througha layerof Matrigel was enhanced by NIH-CXCL14 fibroblasts, as com-pared with control fibroblasts (Supplementary Fig. S7C).
Together, these results demonstrate that the CXCL14 fibro-blast–induced changes in EMT markers are accompanied by anenhanced capacity of breast cancer cells to migrate and invade invitro.
CXCL14 fibroblasts enhance lung colonization of MCF7 cellsfollowing tail-vein injection
The findings of CXCL14-induced effects on migration, inva-sion, and EMT, together with previous findings revealing a pro-tumorigenic role of CXCL14, prompted in vivo studies to explorethe effects of CXCL14 fibroblasts.
Tail-vein experiments monitor the ability of cancer cells tosurvive in the circulation, extravasate, and colonize metastaticsites. These abilities have previously been linked to EMT (37–39).Therefore, lung colonization of tail-vein–injected MCF7 cells,"primed" in vitro in a coculture format together with CXCL14fibroblasts or control fibroblasts prior to injection, was studied(Fig. 3B).
Abundance of breast cancer cells in the lungs, 4 weeksafter injection, was determined by qRT-PCR analyses withhuman-specific primers as described previously (29). As shownin Fig. 3C, a significantly higher number of MCF7 cells weredetected in the lungs of mice that had been injected with cancercells "primed" with CXCL14 fibroblasts, as compared with miceinjected with control fibroblast-primed cancer cells.
These findings were independently validated by IHC analysesof tissue sections from lungs of mice subjected to tail-vein injec-tion of coculture primed cancer cells. As shown in Fig. 3D, theseanalyses demonstrated a significantly higher number of breastcancer cells in the lungs of mice that had been injected withNIH-CXCL14–primed breast cancer cells.
These experiments thus demonstrate that CXCL14 fibroblasts,as compared with control fibroblasts, more potently stimulatelung colonization of blood-circulating breast cancer cells.
CXCL14-induced molecular signaling and cellular responsesare mediated by the atypical G-protein–coupled receptorACKR2
Next, we aimed at identifying key signaling components medi-ating the cellular and protumorigenic effects of the orphan che-mokine CXCL14.
Sj€oberg et al.
Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer ResearchOF6
CXCL14 fibroblasts induce loss of epithelial marker in breast cancer cells. A, Protein levels of E-cadherin (E-cad) in MCF7 and DCIS cells upon coculture with NIH-ctr or NIH-CXCL14 fibroblasts were detected byWestern blot analysis. Representative blots are shown in the top, and quantifications of three independentexperiments are shown in the bottom. B, Immunofluorescence of MCF7 cells (green) cocultured with NIH-ctr or NIH-CXCL14 fibroblasts (red) for the markersdepicted in the figure. Arrowheads mark sites of loss of E-cadherin (E-cad) or Cytokeratin 8/18. Scale bar, 50 mm. C, Light microscopy pictures of MCF7 cellsexposed for 48 hours to conditioned medium (CM) collected from NIH-ctr or NIH-CXCL14 cells (10�magnification). The number of cells with protrusions wascounted in five vision fields in three independent experiments. Results are shown as fold of untreated MCF7 cells. unstim, unstimulated. P values were derivedfrom unpaired two-sided Student t tests. ��� , P < 0.001; �� , P < 0.01; and � , P < 0.05. Error bars, SD.
Fibroblast CXCL14/ACKR2/NOS1 Signaling in Breast Cancer
www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 OF7
We have previously found that CXCL14 enhances MAPK sig-naling in certain cancer cells and defined them as CXCL14-responsive cell lines (22). Initial experiments, analyzing ERKphosphorylation subsequent to stimulation with recombinantCXCL14, identified a panel of CXCL14-responsive cell lines(MCF7, DCIS, SKBR3, NIH-3T3) and nonresponsive cell lines(MDA-MB-231 and LNCaP) (Supplementary Fig. S8; ref. 22).Treatment of the CXCL14-responsive cell lines MCF7 and
NIH-3T3 with pertussis toxin, which specifically inhibits the Gai
subfamily of GPCRs, blocked CXCL14-induced ERK signaling inboth cell types (Supplementary Fig. S9). This finding suggests thatCXCL14, as other chemokines, signals through the Gai subfamilyof GPCRs.
Next, a sequence alignment approach was initiated toidentify GPCRs that exhibit similarities with known chemo-kine receptors (see "Materials and Methods" for details;
A
B
Cancercells
Fibroblasts
0
1
2
3
Mig
ratio
n
(fold
of c
ontr
ol)
MCF7 NIH-ctrNIH-CXCL14
+−−
++−
−
+ +0
1
2
Mig
ratio
n (f
old
of c
ontr
ol)
DCIS NIH-ctrNIH-CXCL14
+−−
++−
−
+ +0
10
20
30
Mig
ratio
n
(fold
of c
ontr
ol)
SKBR3 NIH-ctrNIH-CXCL14
+−−
++−
−
+ +
− NIH-ctr NIH-CXCL14
MCF7
SKBR3
DCIS
******
***
***
MCF7 NIH-ctr + MCF7 NIH-CXCL14 + MCF7D
05
10 15 20 25 30
MCF7
MCF7 (NIH
-ctr)
MCF7 (NIH
-CXCL1
4)
M
ean
nr o
f
MC
F7 c
ells
/lung
******
0
0.1
0.2
0.3
0.4
% H
uman
cel
ls
in m
ice
lung
s
MCF7
MCF7 + NIH-ct
r
MCF7 + NIH-C
XCL14
**
*****
Fibroblasts
Cancer cells
C
Figure 3.
CXCL14-expressing fibroblastsenhancemigration and stimulatelung colonization of breast cancercells. A,MCF7, DCIS, and SKBR3cells were allowed to migratetoward NIH-ctr or NIH-CXCL14fibroblasts in a transwell migrationassay for 24 hours. Migration wasdetermined by counting DAPI-stained cells that had movedthrough an 8-mmpore sizemembrane of the transwell (seedetails in "Materials andMethods"). Results are derivedfrom three independentexperiments and are presented asfold of MCF7, DCIS, or SKBR3 cellsalone. B,MCF7 cells primed for72 hours in a transwell cocultureassay with NIH-ctr, or NIH-CXCL14fibroblasts were injected into thetail-vein of 8-week-old SCID mice(n¼ 10). C, Lungs were harvested4 weeks after injection of thecancer cells. The number of MCF7cells (human origin) in mouselungs was semiquantitativelyassessed by qRT-PCR usinghuman- and mouse-specificprimers (see details in "Materialsand Methods").D, Lung sectionsfrom the tail-vein experiment werestained for the human-specificmarker Stem121. The number ofMCF7 cells in the lung was countedin 10 sections/lung and is depictedas average (n¼ 10). Arrowheadsindicate tumor cells. Scale bar,100 mm. nr, number. P values werederived from unpaired two-sidedStudent t tests. ��� , P < 0.001;�� , P < 0.01; and � , P < 0.05.Error bars, SEM.
Sj€oberg et al.
Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer ResearchOF8
Supplementary Fig. S1). CXCL14 is a highly evolutionaryconserved chemokine and the absence of chemokine receptororthologs in species expressing CXCL14 limited the number ofpotential candidates (40). Furthermore, CXCL14 is a highlyselective chemokine for trophoblasts of the placenta, imma-ture dendritic cells, B cells, and NK cells, and does not inducechemotaxis of T lymphocytes. Therefore, mediators of CXCL14signaling are likely present on trophoblasts, and on certainimmune cells, but not expressed on T cells (18–21, 41). Theseconsiderations led to the selection for continued studies of 11candidate proteins from the original list. Expression levels ofthese receptors were tested in CXCL14-responsive and nonre-sponsive cell lines (Supplementary Table S3). These resultsreduced the candidate set to six candidates, which wereanalyzed in preliminary siRNA experiments that led to con-tinued studies on ACKR2, CXCR4, GPR25, and GPR182.
Initial experiments, using CXCL14-induced ERK phosphoryla-tion as an endpoint, were performed in the CXCL14-responsiveMCF7 cells. As shown in Supplementary Fig. S10A and S10B,downregulationof ACKR2 significantly reducedCXCL14-inducedERK phosphorylation. In contrast, downregulation of CXCR4,GPR25, and GPR182 did not affect CXCL14-induced ERK phos-phorylation (Supplementary Fig. S10A and S10C). ACKR2-down-regulated cells maintained ERK responses after stimulation withCXCL12, indicating specificity of the effects of ACKR2 down-regulation on CXCL14 signaling (Supplementary Fig. S10D).
These initial findings were extended in two other CXCL14-responsive cell lines with the same endpoint. As shownin Supplementary Fig. S11A–S11E, CXCL14-induced ERKphosphorylation in NIH-3T3 and SKBR3 cells was also atten-uated after siRNA-mediated downregulation of ACKR2. More-over, the enhanced growth of CXCL14 fibroblasts was signif-icantly reduced after downregulation of ACKR2 (Supplemen-tary Fig. S11F). In contrast, no effect on cell growth wasobserved after downregulation of CXCR4, GPR182, and GPR25(Supplementary Fig. S11F).
These findings prompted generation of derivatives of NIH-ctrand NIH-CXCL14 cells with stable ACKR2 downregulation withtwo different ACKR2 shRNAs (Fig. 4A). In agreement with find-ings above, CXCL14-induced ERK phosphorylation was signifi-cantly attenuated in NIH-3T3 cells with stable downregulation ofACKR2 (Fig. 4B and C). As an additional endpoint, CXCL14-induced upregulation of NOS1 was studied. As shown in Fig. 4Dand E, stable ACKR2 downregulation reduced NOS1 protein andmRNA levels in NIH-3T3 cells, but not in control cells. Finally,CXCL14-induced proliferation of NIH-3T3 cells was analyzedwith regard to ACKR2 dependency. Notably, downregulation ofACKR2 reduced the growth capacity of CXCL14 fibroblasts,whereas no effect of ACKR2 downregulation was detected inNIH-ctr cells (Fig. 4F).
The above data prompted binding studies to analyze whetherCXCL14 directly interacts with ACKR2. However, contrarily toCCL5, a high-affinity ligand of ACKR2, binding of CXCL14 wasonly weakly detectable at high concentrations on cells overex-pressing ACKR2 and was not different compared with cellsthat lack ACKR2 (Supplementary Fig. S12A). Furthermore, in ab-arrestin1 recruitment assay, a dose-dependent recruitment ofb-arrestin1 towardACKR2 could only bedetected uponCCL5, butnot upon CXCL14 stimulation (Supplementary Fig. S12B).
In summary, these results derived fromanalyses of different celltypes and using multiple endpoints identify ACKR2 as a critical
mediator of CXCL14-induced signaling, although no direct inter-action between CXCL14 and ACKR2 could be found.
ACKR2 and CXCL14 expression correlates with an EMT geneexpression signature and poor prognosis in clinical datasets ofbreast cancer
To explore the clinical relevance of the experimental findingsof pro-EMT effects of CXCL14 fibroblasts, correlative analyseswere performed in publicly available gene expression datasetsof breast cancer to investigate potential associations betweenCXCL14/ACKR2 expression and clinical features (35).
The analyses revealed significant positive correlations betweenexpression of CXCL14 and the EMT gene expression signature ina meta-analysis of nine breast cancer cohorts (SupplementaryFig. S2A). Analysis of the CXCL14:EMT correlation in intrinsicsubtypes of breast cancer across all cohorts revealed no majordifference among the molecular subgroups of breast cancer, but aslightly stronger correlation in the Basal subgroup (Supplemen-tary Fig. S2B). Correlations between CXCL14 and EMT werenot affected by the amount of tumor stroma (SupplementaryFig. S13). This indicates that the CXCL14:EMT correlation trulyis driven by cancer cell EMT rather than reflecting stromaabundance.
To extend these studies, additional analyses were performedthat focused on relationships between CXCL14 expression andindividual EMT-related genes. As shown inSupplementary Fig. S3,CXCL14-high breast cancer displayed, in general, an EMT profilecharacterized by, for example, reduced expression of E-cadherinand increased expression of EMT transcription factors includingSNAI2, TWIST1, and ZEB1 and mesenchymal markers includingVIM, ACTA2, FN1, and collagens. On the basis of earlier studiesimplying a tumor-promoting function of CXCL14 in prostate andovarian cancer (22, 25, 42), analyses were also performed on theovarian and prostate cancer TCGA datasets. These demonstratedresults similar to those seen in the breast cancer analyses (Sup-plementary Fig. S3).
On the basis of the experimental studies, these associationswere also analyzed in subsets defined by their combined CXCL14and ACKR2 status. In agreement with a functional link betweenCXCL14 and ACKR2, the association with the EMT profilewas most prominent in the CXCL14high/ACKR2high subgroup(Fig. 5A). This pattern was also seen in prostate and ovariancancer datasets (Supplementary Fig. S4).
Survival data in the TCGA datasets was also used to exploresurvival associations of the four CXCL14/ACKR2-defined sub-groups. Initial analyses with all four groups in the breast cancerdataset indicated a particularly poor prognosis of the group withhigh expressionofCXCL14andACKR2 (Supplementary Fig. S14).Notably, a significant poor survival association was seen forthe combined CXCL14high/ACKR2high group, when contrastedwith the rest of the TCGA population (P ¼ 0.01; Fig. 5B). A Coxproportional hazard model revealed an increased riskof death for patients in the CXCL14high/ACKR2high subgroup(HR ¼ 2.494; 95% CI ¼ 1.218–5.104). This poor prognosisassociation of the CXCL14high/ACKR2high subgroup in breastcancer also remained significant in multivariate analyses withclinicopathologic characteristics, including breast cancer molec-ular subsets (Supplementary Table S4). Survival correlations ofthe CXCL14high/ACKR2high subgroup were also explored inpublicly available datasets, representing 12 other tumor types(Supplementary Table S5). Besides breast cancer, the CXCL14high/
Fibroblast CXCL14/ACKR2/NOS1 Signaling in Breast Cancer
www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 OF9
ACKR2 mediates CXCL14-stimulated signaling in fibroblasts. A, The suppression of ACKR2 expression following introduction of two different ACKR2-targetingshRNA (A and B) in NIH-ctr and NIH-CXCL14 fibroblasts was analyzed by qRT-PCR. Results are obtained from three independent experiments. B, The activationof ERK1/2 signaling following CXCL14 stimulation was monitored byWestern blot analysis in NIH-3T3 fibroblasts with and without stable downregulation ofACKR2. C,Quantifications of three independent experiments from B. unstim, unstimulated. Analysis of Nos1 transcript (qRT-PCR; D) and Nos1 protein (Westernblot) levels (E) in NIH-ctr and NIH-CXCL14 derivatives with or without stable downregulation of ACKR2. E,One representative blot together with thequantification of three independent experiments. F, The growth of NIH-ctr and NIH-CXCL14 fibroblasts with or without stable downregulation of ACKR2 wasevaluated by the AlamarBlue assay (see details in "Materials and Methods") after culture for 3 days in serum-reduced medium. The results of three independentexperiments are summarized in the figure. P values were derived from unpaired two-sided Student t tests. ��� , P < 0.001; �� , P < 0.01; and �, P < 0.05. Error bars,SD or SEM.
Sj€oberg et al.
Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer ResearchOF10
Patients with breast cancer expressing high levels of CXCL14 and ACKR2 show enhanced EMT and adverse overall survival. A, Z-scores of EMT genes in the TCGAbreast cancer gene expression dataset, in patients divided in different subgroups with high or low expression levels of CXCL14 and ACKR2. B, Kaplan–Meieranalysis of the CXCL14high/ACKR2high subgroup compared with the rest of the population (n¼ 1,100 patients). P value was derived from log-rank test, and HRsincluding confidence intervals (CI) were derived from univariate Cox regression analyses.
Fibroblast CXCL14/ACKR2/NOS1 Signaling in Breast Cancer
www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 OF11
Together, these correlative analyses support the notion that thepreviously observed poor prognosis association of stroma-derivedCXCL14 in breast cancer is related to amolecular pathwaythat also involves ACKR2.
Paracrine effects of CXCL14 fibroblasts depend on autocrineCXCL14 signaling
Data presented above do not clearly resolve whether the poorprognosis–associated CXCL14/ACKR2 pathway reflects autocrineCXCL14/ACKR2 signaling, supporting EMT and metastasis in aparacrine manner, or rather reflects paracrine actions of CXCL14-activated fibroblasts that involves ACKR2 in breast cancer cells.
As shown in Fig. 6A and B, CXCL14 fibroblast–induced cancercell migration and E-Cadherin downregulation was significantlyinhibited by knockdownof ACKR2 infibroblasts. Of note, ACKR2downregulation in control fibroblasts did not affect migration orE-Cadherin levels of cocultured MCF7 cells.
Further evidence supporting autocrine CXCL14 signaling as thedriver of the prometastatic effects was provided by analyses of theeffects of downregulation of NOS1; an earlier identified down-streamcomponent ofCXCL14fibroblast signaling (25). As shownin Fig. 6C, downregulation of NOS1 significantly reduced theability of CXCL14 fibroblasts to stimulate migration of MCF7cells. Notably, NOS1 downregulation did not affect cancer cellmigration induced by control fibroblasts (Fig. 6C). The reductionof NOS1 signaling also attenuated the CXCL14 fibroblast–induced downregulation of E-cadherin and upregulation of Snailin CM-treated MCF7 cells (Fig. 6D and E).
The reduced paracrine effects of CXCL14 fibroblasts follow-ing downregulation of ACKR2 or NOS1 suggest that CXCL14itself is not promoting EMT, but rather stimulates the expres-sion of EMT regulators in fibroblasts in an ACKR2-/ NOS1-dependent manner. To identify such putative EMT-regulatingsoluble factors derived from CXCL14 fibroblasts, we used aprotein profiler and compared the secretome of NIH-CXCL14and NIH-ctr fibroblasts. Among the factors that are moreabundantly expressed by CXCL14 fibroblasts (SupplementaryFig. S15) are proangiogenic factors (e.g., FGF-2, Angiogenin,VEGF-A), supporting the previous notion that NIH-CXCL14cells stimulate angiogenesis (22), molecules involved in matrixremodeling such as Adamts1, MMP8, TIMP-1, as well as indu-cers and effectors of EMT including CXCL1, CX3CL1, TIMP-1,FGF2, HGF, and tissue factor.
These data, together with the findings of Fig. 1, whichshow reduced EMT in the tumors formed after coinjection withNOS1-downregulated CXCL14 fibroblasts, indicate that the pro-migratory and EMT-modulatory effects of CXCL14 fibroblastsdepend on autocrine CXCL14/ACKR2/NOS1 signaling.
DiscussionThis study extends earlier findings that have identified stroma-
derived CXCL14 as a poor prognosis factor in breast cancer.Mechanistic and correlative studies together suggest a novelprometastatic pathway composed of autocrine CXCL14/ACKR2/NOS1 signaling in fibroblasts that generates a fibroblastphenotype that supports cancer cell migration, invasion, EMT,and metastasis (Supplementary Fig. S16).
The finding of prometastatic effects of CXCL14 adds to earlierliterature indicating the involvement of chemokines inmetastasisdevelopment. CCL5 secreted from bone marrow–derived mesen-chymal cells, recruited to the breast tumor stroma,was identified akey player in promoting tumor cell invasion and development ofmetastasis in SCID mice (13). In another study, paracrine cross-talk between tumor cells, myeloid cells, and endothelial cells,involving CXCL1 and CXCL2 signaling, was shown to drivemetastasis and chemoresistance in theMMTV-PyMTmousemod-el of breast cancer (14). In addition, in experimental breasttumors, CXCL12 secreted from CAFs was shown to select forclones of cancer cells with a high Src activity, and the ability tospecifically form metastasis in bone with a CXCL12-richmicroenvironment (15).
Earlier studies have also linked chemokines and their receptorsspecifically to EMT. A constitutively active form of CXCR4, thereceptor for CXCL12, has been shown to be involved in modu-lationof breast tumor cell EMTmarkers and to enhance formationof lymph node metastasis in mice (43). CCR7, the receptor forCCL21, and CXCR5 and its ligand CXCL13 have been shown tosignificantly correlate with EMT markers and enhanced lymphnode metastasis of human breast tumors (44, 45). Moreover, aGM-CSF-CCL18–positive feedback loop have been implied inEMT and breast tumor metastasis in mice and associated withworse outcome in patients with breast cancer (16).
The comparison of proteins secreted by NIH-CXCL14 andNIH-ctr cells provided new insight in CXCL14 signaling infibroblasts and revealed a set of candidates that mediate EMTstimulated by CXCL14-expressing fibroblasts either individuallyand/ or in combination. For example, CXCL1, CX3CL1, HGF, andTIMP-1 and tissue factor have previously been demonstrated toaffect EMT and metastasis of breast cancer cells (39, 46–48).Recently, Wang and colleagues identified CCL17 derived fromCXCL14-activated fibroblasts as another mediator of CXCL14-stimulated breast cancer EMT and metastasis (49). Furthermore,CXCL14 was shown to act as a chemoattractant for M2 macro-phages that are known to promote EMT (16, 50, 51). Together,these findings suggest that CXCL14 operates different axis ofstromal signaling shifting the phenotype of stromal cells towardtumor progression and metastasis.
A key finding of this study is the demonstration that CXCL14-induced molecular signaling and cellular responses depend onACKR2, classified as an atypical chemokine receptor. ACKR2-dependent CXCL14 signaling was shown in cell types of differentorigin (breast cancer cells and fibroblasts) and the downregula-tion of ACKR2 almost completely abolished CXCL14-inducedeffects. These data suggest that ACKR2 is a required component ofCXCL14 signaling. Findings of ACKR2 expression on some breastcancer cells (Supplementary Table S3) also suggest the possibilitythat CXCL14, in certain settings, might be prometastatic orEMT-stimulatory through direct effects on malignant cells. Thistopic should be further explored in future studies. Continuedmechanistic analyses are also warranted regarding the roles ofCXCL14/ACKR2 on other steps of the metastatic process thanthose covered by the analyses of the invasive border (Fig. 1) andthe "tail-vein" experiment (Fig. 3).
Studies have suggested the chemokine receptors CXCR4 andGPR85 to directly bind CXCL14 and modulating signaling(49, 52). However, Otte and colleagues demonstrated thatCXCL14 does not bind and impact on CXCR4 signaling (53).In line with these data, we found that siRNA-mediated
Sj€oberg et al.
Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer ResearchOF12
Paracrine effects of fibroblast-derived CXCL14 depend on NOS1 and ACKR2. A,Migration of MCF7 cells for 24 hours in response to NIH-ctr or NIH-CXCL14derivatives without (shCtr) or with stable knockdown of ACKR2 (shACKR2:A and shACKR2:B). B,Western blot analysis and quantification of E-cadherin levels inMCF7 cells subsequent to coculture with control (shCtr) or ACKR2-targeting (shACKR2:A) NIH-ctr or NIH-CXCL14 fibroblasts for 48 hours. C,MCF7 cells wereallowed to migrate for 24 hours toward NIH-ctr or NIH-CXCL14 derivatives without (shCtr) or with stable knockdown of NOS1 (shNOS1:A and shNOS1:B). D,Western blot analysis of E-cadherin and Snail levels in MCF7 cells following coculture with NIH-ctr or NIH-CXCL14 fibroblasts with or without stable suppressionof NOS1 expression. E,Quantification ofWestern blots as shown in B for E-cadherin (E-cad; left) and Snail (right) expression from three independentexperiments. Representative blots are shown, and quantifications are based on three independent experiments. P values were derived from unpaired two-sidedStudent t tests. ��� , P < 0.001; �� , P < 0.01 and � , P < 0.05. Error bars, SD or SEM.
Fibroblast CXCL14/ACKR2/NOS1 Signaling in Breast Cancer
www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 OF13
downregulation of CXCR4 did not affect CXCL14-inducedMAPKsignaling in MCF7 breast cancer cells (Supplementary Fig. S10C)suggesting that the functions of CXCL14 might be more complexthan for other chemokines and might require formation ofchemokine or receptor heterocomplexes, which may explain thedifficulty to precisely define its signaling components (54).
These atypical chemokine receptors have earlier beendefined asscavenging receptors that bind chemokines with high affinity butare unable to induce signaling and cellmigration (11). Absence ofthe well-conserved DRYLAIVHA motif (DKYLEIVHA in ACKR2)has been assumed to explain the inability of atypical chemokinereceptors to induce downstream receptor signaling subsequent toligand binding.
In this study, we observe an impact of ACKR2 on themolecularsignaling, including MAPK activation (Fig. 4A–C; SupplementaryFigs. S10B and S11A–S11E) and cellular functions, includingenhanced fibroblast proliferation (Fig. 4F; SupplementaryFig. S11F) in response to CXCL14. Independent recent evidenceindeed does support signaling functions for some of the atypicalchemokine receptors. These signaling propertieswere proposed tobe possibly cell type dependent as in one study, experimentsdemonstrated coupling of ACKR3 to Gai proteins and inductionof CXCL12-dependent conformational changes, but no activationof calcium signaling (55). Another study confirmed the bindingof ACKR3 to PTX-sensitive Gai proteins and revealed activation ofcalcium mobilization, ERK signaling, and AKT signaling andenhanced migration and proliferation, subsequent to CXCL12binding in rodent astrocytes and human glioma cell lines (56).Earlier overexpression studies have also demonstrated ACKR2-induced calcium mobilization by murine ACKR2 (57). A recentstudy also proposed G-protein–independent, b-arrestin–depen-dent, activationof the cofilin pathway [Rac1-p21–activated kinase1 (PAK1)-LIM kinase 1 (LIMK1) cascade] following ACKR2stimulation suggesting that ACKR2 is not a totally silentreceptor (58). Taken together, these findings challenge thedefinition of ACKRs as exclusive nonsignaling, chemokine scav-enger receptors.
Earlier studies have identified stromal, but not epithelial,CXCL14 as a bad prognosis marker in breast cancer (1). Thecorrelative data of this study support the notion of functionalclinical relevant interaction between CXCL14 and ACKR2. Asshown in Fig. 5, the association between CXCL14 and an "EMTprofile" is enhanced when ACKR2 status is integrated in patientclassification (Fig. 5A). Similarly, the survival association ofCXCL14 in the breast cancer TCGA gene expression dataset isonly detected in analyses that also consider ACKR2 status (Fig. 5B;Supplementary Fig. S14). Importantly, the combined CXCL14/ACKR2metric is also a significant marker in multivariate analysesincluding molecular breast cancer subtypes (SupplementaryTable S4). It is recognized that the TCGA-based analyses fail
to assign the prognostically relevant ACKR2 expression to thestromal or epithelial compartment. However, the mechanisticstudies of the current report suggest that autocrine CXCL14/ACKR2 signaling in the stroma contributes to the survival andEMT associations. Compartment-specific analyses of ACKR2 areprompted by the findings of the current study.
In summary, this study thus identifies a novel potentiallydruggable CXCL14/ACKR2 pathway involved in breast cancerEMT and metastasis. Important tasks for future studies includedevelopment of inhibitory agents for initial testing in experimen-tal breast cancer models and further exploration of relevance ofthis pathway in other tumor types. Furthermore, these results alsoencourage continued studies exploring biological activity ofACKRs and to decipher the exact interplay between CXCL14 withclassical and atypical chemokine receptors.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: E. Sj€oberg, M. Augsten, A. €OstmanDevelopment of methodology: E. Sj€oberg, L. Milde, D. H€agerstrand,A. Chevign�eAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E. Sj€oberg, M. Meyrath, L. Milde, M. Herrera,J. L€ovrot, D. H€agerstrand, M. Bartish, C. Rolny, A. Chevign�e, M. AugstenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):E. Sj€oberg,M.Meyrath, L.Milde,M.Herrera, J. L€ovrot,
O. Frings, M. Bartish, E. Sonnhammer, A. Chevign�e, M. Augsten, A. €OstmanWriting, review, and/or revision of the manuscript: E. Sj€oberg, M. Meyrath,J. L€ovrot, D. H€agerstrand, C. Rolny, E. Sonnhammer, A. Chevign�e, M. Augsten,
A. €OstmanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. €Ostman
Study supervision: A. €Ostman
AcknowledgmentsMembers of A. €Ostman's groupare acknowledged for support throughout the
studies. Studies were supported by grants from the Swedish Cancer Society,BRECT, the Linn�e STARGET grant from Swedish Research Council and theKI/AZ-collaborative initiative, the Luxembourg Institute of Health (LIH) MESR(grants 20160116 and 20170113), and the Luxembourg National ResearchFund PhD fellows (grants AFR-3004509 and INTER/FWO "Nanokine" - grant15/10358798). Technical support was provided by the histo-pathology unit ofCancer Centrum Karolinska. Animal experiments benefited from the expertiseof the MTC animal facility.
The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received April 26, 2018; revisedDecember 30, 2018; acceptedMarch 4, 2019;published first March 8, 2019.
References1. Sjoberg E, Augsten M, Bergh J, Jirstrom K, Ostman A. Expression of the
chemokine CXCL14 in the tumour stroma is an independent marker ofsurvival in breast cancer. Br J Cancer 2016;114:1117–24.
2. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition.J Clin Invest 2009;119:1420–8.
3. De Craene B, Berx G. Regulatory networks defining EMT during cancerinitiation and progression. Nat Rev Cancer 2013;13:97–110.
4. Zheng X, Carstens JL, Kim J, Scheible M, Kaye J, Sugimoto H, et al.Epithelial-to-mesenchymal transition is dispensable for metastasis butinduces chemoresistance in pancreatic cancer. Nature 2015;527:525–30.
5. Del Pozo Martin Y, Park D, Ramachandran A, Ombrato L, Calvo F,Chakravarty P, et al. Mesenchymal cancer cell-stroma crosstalk promotesniche activation, epithelial reversion, andmetastatic colonization. Cell Rep2015;13:2456–69.
Sj€oberg et al.
Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer ResearchOF14
6. Beerling E, SeinstraD, deWit E, Kester L, vander VeldenD,MaynardC, et al.Plasticity between epithelial and mesenchymal states unlinks EMT frommetastasis-enhancing stem cell capacity. Cell Rep 2016;14:2281–8.
7. Ostman A, Augsten M. Cancer-associated fibroblasts and tumor growth–bystanders turning into key players. Curr Opin Genet Dev 2009;19:67–73.
8. Kalluri R. The biology and function of fibroblasts in cancer. Nat Rev Cancer2016;16:582–98.
9. Mishra P, Banerjee D, Ben-Baruch A. Chemokines at the crossroads oftumor-fibroblast interactions that promote malignancy. J Leukoc Biol2011;89:31–9.
10. Thelen M. Dancing to the tune of chemokines. Nat Immunol 2001;2:129–34.
11. Bonecchi R, Savino B, Borroni EM, Mantovani A, Locati M. Chemokinedecoy receptors: structure-function and biological properties. Curr TopMicrobiol Immunol 2010;341:15–36.
12. Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2004;4:540–50.
13. Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, et al.Mesenchymal stem cells within tumour stroma promote breast cancermetastasis. Nature 2007;449:557–63.
14. Acharyya S, Oskarsson T, Vanharanta S,Malladi S, Kim J,Morris PG, et al. ACXCL1 paracrine network links cancer chemoresistance and metastasis.Cell 2012;150:165–78.
15. Zhang XH, Jin X,Malladi S, Zou Y,Wen YH, Brogi E, et al. Selection of bonemetastasis seeds bymesenchymal signals in the primary tumor stroma. Cell2013;154:1060–73.
16. Su S, Liu Q, Chen J, Chen J, Chen F, He C, et al. A positive feedback loopbetween mesenchymal-like cancer cells and macrophages is essential tobreast cancer metastasis. Cancer Cell 2014;25:605–20.
18. SleemanMA, Fraser JK, Murison JG, Kelly SL, Prestidge RL, Palmer DJ, et al.B cell- and monocyte-activating chemokine (BMAC), a novel non-ELRalpha-chemokine. Int Immunol 2000;12:677–89.
19. Kurth I, Willimann K, Schaerli P, Hunziker T, Clark-Lewis I, Moser B.Monocyte selectivity and tissue localization suggests a role for breast andkidney-expressed chemokine (BRAK) in macrophage development. J ExpMed 2001;194:855–61.
20. Shellenberger TD,WangM,GujratiM, JayakumarA, StrieterRM,BurdickMD,et al. BRAK/CXCL14 is a potent inhibitor of angiogenesis and a chemotacticfactor for immature dendritic cells. Cancer Res 2004;64:8262–70.
21. Starnes T, Rasila KK, Robertson MJ, Brahmi Z, Dahl R, Christopherson K,et al. The chemokine CXCL14 (BRAK) stimulates activated NK cell migra-tion: implications for the downregulation of CXCL14 in malignancy.Exp Hematol 2006;34:1101–5.
22. Augsten M, Hagglof C, Olsson E, Stolz C, Tsagozis P, Levchenko T, et al.CXCL14 is an autocrine growth factor for fibroblasts and acts as a multi-modal stimulator of prostate tumor growth. Proc Natl Acad Sci U S A 2009;106:3414–9.
23. Allinen M, Beroukhim R, Cai L, Brennan C, Lahti-Domenici J, Huang H,et al. Molecular characterization of the tumor microenvironment in breastcancer. Cancer Cell 2004;6:17–32.
24. GuXL,OuZL, Lin FJ, YangXL, Luo JM, ShenZZ, et al. Expression ofCXCL14and its anticancer role in breast cancer. Breast Cancer Res Treat 2012;135:725–35.
25. Augsten M, Sjoberg E, Frings O, Vorrink SU, Frijhoff J, Olsson E, et al.Cancer-associated fibroblasts expressing CXCL14 rely uponNOS1-derivednitric oxide signaling for their tumor-supporting properties. Cancer Res2014;74:2999–3010.
26. Szpakowska M, Nevins AM, Meyrath M, Rhainds D, D'Huys T, Guite-VinetF, et al.Different contributions of chemokineN-terminal features attest to adifferent ligand binding mode and a bias towards activation of ACKR3/CXCR7 compared with CXCR4 and CXCR3. Br J Pharmacol 2018;175:1419–38.
27. Szpakowska M, Meyrath M, Reynders N, Counson M, Hanson J, Steyaert J,et al. Mutational analysis of the extracellular disulphide bridges of theatypical chemokine receptor ACKR3/CXCR7 uncovers multiple bindingand activation modes for its chemokine and endogenous non-chemokineagonists. Biochem Pharmacol 2018;153:299–309.
28. Sonnhammer EL, Hollich V. Scoredist: a simple and robust proteinsequence distance estimator. BMC Bioinformatics 2005;6:108.
29. Malek A, Catapano CV, Czubayko F, Aigner A. A sensitive polymerasechain reaction-based method for detection and quantification of metas-tasis in human xenograft mouse models. Clin Exp Metastasis 2010;27:261–71.
30. Miller LD, Smeds J, George J, Vega VB, Vergara L, Ploner A, et al. Anexpression signature for p53 status in human breast cancer predictsmutation status, transcriptional effects, and patient survival. Proc NatlAcad Sci U S A 2005;102:13550–5.
31. Pawitan Y, Bjohle J, Amler L, Borg AL, Egyhazi S, Hall P, et al. Geneexpression profiling spares early breast cancer patients from adjuvanttherapy: derived and validated in two population-based cohorts.Breast Cancer Res 2005;7:R953–64.
32. Wang Y, Klijn JG, Zhang Y, Sieuwerts AM, Look MP, Yang F, et al. Gene-expression profiles to predict distant metastasis of lymph-node-negativeprimary breast cancer. Lancet 2005;365:671–9.
33. Curtis C, Shah SP, Chin SF, Turashvili G, RuedaOM,DunningMJ, et al. Thegenomic and transcriptomic architecture of 2,000 breast tumours revealsnovel subgroups. Nature 2012;486:346–52.
34. Cancer Genome Atlas Network. Comprehensive molecular portraits ofhuman breast tumours. Nature 2012;490:61–70.
35. Taube JH, Herschkowitz JI, Komurov K, Zhou AY, Gupta S, YangJ, et al. Core epithelial-to-mesenchymal transition interactomegene-expression signature is associated with claudin-low and meta-plastic breast cancer subtypes. Proc Natl Acad Sci U S A 2010;107:15449–54.
36. Parker JS, Mullins M, Cheang MC, Leung S, Voduc D, Vickery T, et al.Supervised risk predictor of breast cancer based on intrinsic subtypes. J ClinOncol 2009;27:1160–7.
37. Guarino M, Rubino B, Ballabio G. The role of epithelial-mesenchymaltransition in cancer pathology. Pathology 2007;39:305–18.
38. Tsai JH, Yang J. Epithelial-mesenchymal plasticity in carcinomametastasis.Genes Dev 2013;27:2192–206.
39. Bourcy M, Suarez-Carmona M, Lambert J, Francart ME, Schroeder H,Delierneux C, et al. Tissue factor induced by epithelial-mesenchymaltransition triggers a procoagulant state that drives metastasis of circulatingtumor cells. Cancer Res 2016;76:4270–82.
40. DeVries ME, Kelvin AA, Xu L, Ran L, Robinson J, Kelvin DJ. Defining theorigins and evolution of the chemokine/chemokine receptor system.J Immunol 2006;176:401–15.
41. Kuang H, ChenQ, Fan X, Zhang Y, Zhang L, Peng H, et al. CXCL14 inhibitstrophoblast outgrowth via a paracrine/autocrine manner during earlypregnancy in mice. J Cell Physiol 2009;221:448–57.
42. Zhao L, Ji G, Le X, Wang C, Xu L, Feng M, et al. Long noncodingRNA LINC00092 acts in cancer-associated fibroblasts to driveglycolysis and progression of ovarian cancer. Cancer Res 2017;77:1369–82.
43. Sobolik T, Su YJ, Wells S, Ayers GD, Cook RS, Richmond A. CXCR4drives the metastatic phenotype in breast cancer through induction ofCXCR2 and activation of MEK and PI3K pathways. Mol Biol Cell 2014;25:566–82.
44. Li F, Zou Z, Suo N, Zhang Z, Wan F, Zhong G, et al. CCL21/CCR7axis activating chemotaxis accompanied with epithelial-mesenchy-mal transition in human breast carcinoma. Med Oncol 2014;31:180.
45. Biswas S, Sengupta S, Roy Chowdhury S, Jana S, Mandal G, Mandal PK,et al. CXCL13-CXCR5 co-expression regulates epithelial to mesenchymaltransition of breast cancer cells during lymph node metastasis.Breast Cancer Res Treat 2014;143:265–76.
46. Wang N, Liu W, Zheng Y, Wang S, Yang B, Li M, et al. CXCL1 derivedfrom tumor-associated macrophages promotes breast cancer metasta-sis via activating NF-kappaB/SOX4 signaling. Cell Death Dis 2018;9:880.
47. D'Angelo RC, Liu XW, Najy AJ, Jung YS, Won J, Chai KX, et al. TIMP-1 viaTWIST1 induces EMT phenotypes in human breast epithelial cells.Mol Cancer Res 2014;12:1324–33.
48. Tardaguila M, Mira E, Garcia-Cabezas MA, Feijoo AM, Quintela-FandinoM, Azcoitia I, et al. CX3CL1 promotes breast cancer via transactivation ofthe EGF pathway. Cancer Res 2013;73:4461–73.
49. WangY,WengX,WangL,HaoM, Li Y,HouL, et al.HIC1deletionpromotesbreast cancer progression by activating tumor cell/fibroblast crosstalk.J Clin Invest 2018;128:5235–50.
Fibroblast CXCL14/ACKR2/NOS1 Signaling in Breast Cancer
www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 OF15
50. Cereijo R, Gavalda-Navarro A, Cairo M, Quesada-Lopez T, Villarroya J,Moron-Ros S, et al. CXCL14, a brown adipokine that mediates brown-fat-to-macrophage communication in thermogenic adaptation. Cell Metab2018;28:750–63.
51. LindeN,Casanova-AcebesM, SosaMS,Mortha A, RahmanA, Farias E, et al.Macrophages orchestrate breast cancer early dissemination andmetastasis.Nat Commun 2018;9:21.
52. TanegashimaK, Suzuki K, Nakayama Y, Tsuji K, Shigenaga A,Otaka A, et al.CXCL14 is a natural inhibitor of the CXCL12-CXCR4 signaling axis.FEBS Lett 2013;587:1731–5.
53. OtteM, Kliewer A, SchutzD, Reimann C, Schulz S, StummR. CXCL14 is nodirect modulator of CXCR4. FEBS Lett 2014;588:4769–75.
54. Kleist AB, Getschman AE, Ziarek JJ, Nevins AM, Gauthier PA, Chevigne A,et al. New paradigms in chemokine receptor signal transduction: Movingbeyond the two-site model. Biochem Pharmacol 2016;114:53–68.
55. Levoye A, Balabanian K, Baleux F, Bachelerie F, Lagane B. CXCR7 hetero-dimerizes with CXCR4 and regulates CXCL12-mediated G protein signal-ing. Blood 2009;113:6085–93.
56. Odemis V, Lipfert J, Kraft R, Hajek P, Abraham G, Hattermann K, et al. Thepresumed atypical chemokine receptor CXCR7 signals through G(i/o)proteins in primary rodent astrocytes and human glioma cells. Glia2012;60:372–81.
57. Nibbs RJ, Wylie SM, Pragnell IB, Graham GJ. Cloning and characterizationof a novelmurine beta chemokine receptor,D6. Comparison to three otherrelated macrophage inflammatory protein-1alpha receptors, CCR-1, CCR-3, and CCR-5. J Biol Chem 1997;272:12495–504.
58. Borroni EM, Cancellieri C, Vacchini A, Benureau Y, Lagane B, Bachelerie F,et al. beta-arrestin-dependent activation of the cofilin pathway is requiredfor the scavenging activity of the atypical chemokine receptorD6. Sci Signal2013;6:ra30.
Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer ResearchOF16
Published OnlineFirst March 8, 2019.Clin Cancer Res Elin Sjöberg, Max Meyrath, Laura Milde, et al. Metastasis of Breast CancerCXCL14 in Epithelial-to-Mesenchymal Transition and A Novel ACKR2-Dependent Role of Fibroblast-Derived
Updated version
10.1158/1078-0432.CCR-18-1294doi:
Access the most recent version of this article at:
Material
Supplementary
http://clincancerres.aacrjournals.org/content/suppl/2019/04/19/1078-0432.CCR-18-1294.DC2Access the most recent supplemental material at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's
.http://clincancerres.aacrjournals.org/content/early/2019/05/22/1078-0432.CCR-18-1294To request permission to re-use all or part of this article, use this link
