Published OnlineFirst March 8, 2019; DOI: 10.1158/1078-0432.CCR-18-1294 Translational Cancer Mechanisms and Therapy Clinical Cancer Research A Novel ACKR2-Dependent Role of Fibroblast- Derived CXCL14 in Epithelial-to-Mesenchymal Transition and Metastasis of Breast Cancer Elin Sjoberg€ 1, Max Meyrath2, Laura Milde3, Mercedes Herrera1, John Lovrot€ 1, Daniel Hagerstrand€ 1, Oliver Frings1, Margarita Bartish1, Charlotte Rolny1, Erik Sonnhammer4, Andy Chevigne2, Martin Augsten1, and Arne Ostman€ 1 Abstract Purpose: Fibroblasts expressing the orphan chemokine graft model. Furthermore, tumor cells primed by CXCL14 CXCL14 have been previously shown to associate with poor fibroblasts displayed enhanced lung colonization after tail- breast cancer prognosis and promote cancer growth. This study vein injection. By loss-of function experiments, the atypical explores the mechanism underlying the poor survival associa- G-protein–coupled receptor ACKR2 was identified to mediate tions of stromal CXCL14. CXCL14-stimulated responses. Downregulation of ACKR2, or Experimental Design: Tumor cell epithelial-to-mesenchymal CXCL14-induced NOS1, attenuated the pro-EMT and migra- transition (EMT), invasion, and metastasis were studied in in tory capacity. CXCL14/ACKR2 expression correlated with EMT vitro and in vivo models together with fibroblasts overexpres- and survival in gene expression datasets. sing CXCL14. An approach for CXCL14 receptor identification Conclusions: Collectively, the findings imply an autocrine included loss-of-function studies followed by molecular and fibroblast CXCL14/ACKR2 pathway as a clinically relevant functional endpoints. The clinical relevance was further stimulator of EMT, tumor cell invasion, and metastasis. The explored in publicly available gene expression datasets. study also identifies ACKR2 as a novel mediator for CXCL14 Results: CXCL14 fibroblasts stimulated breast cancer EMT, function and thereby defines a pathway with drug target migration, and invasion in breast cancer cells and in a xeno- potential. Introduction expression and shorter survival in a population-based breast cancer cohort. Notably, epithelial expression of CXCL14 did not Death of patients with breast cancer is almost exclusively due have an impact on breast cancer outcome (1). to metastatic disease. Metastasis develops through a multistep During the early steps of metastasis development, tumor cells process, involving tissue invasion, intravasation, survival in the lose cell-to-cell contacts and epithelial characteristics and instead bloodstream and lymph system, extravasation, and tissue colo- gain mesenchymal traits that allow them to invade the surround- nization. This study develops recent correlative studies that have ing tissue and metastasize; a process termed epithelial-to-mesen- indicated clinical relevance, in breast cancer, of stroma-derived chymal transition (EMT; ref. 2). expression of the chemokine CXCL14 by demonstrating signifi- EMT is controlled by distinct transcriptional programs activated cant and independent associations between high stromal CXCL14 by specific transcription factors, including Snail, Slug, Twist, Zeb, and Gsc. Activation of these factors ultimately leads to the loss of epithelial markers including E-cadherin and cytokeratins, and the 1 Department of Oncology-Pathology, Cancer Center Karolinska (CCK), Karo- upregulation of mesenchymal markers, such as vimentin, alpha- linska Institutet, Stockholm, Sweden. 2Department of Infection and Immunity, smooth muscle actin (aSMA), and matrix-degrading enzymes (3). Immuno-Pharmacology and Interactomics, Luxembourg Institute of Health (LIH), Esch-sur-Alzette, Luxembourg. 3Division for Vascular Oncology and Although the classical paradigm attributing EMT a crucial role in Metastasis, German Cancer Research Center (DKFZ), Heidelberg, Germany. the process of metastasis has been recently challenged by studies 4Stockholm Bioinformatics Center, Department of Biochemistry and Biophysics, in genetic mouse models, other recent studies including in vivo Stockholm University, Science for Life Laboratory, Stockholm, Sweden. imaging approaches demonstrated that cancer cells displaying an Note: Supplementary data for this article are available at Clinical Cancer EMT phenotype give rise to metastases (4–6). Research Online (http://clincancerres.aacrjournals.org/). Induction of EMT can occur in a paracrine manner by secreted M. Augsten and A. Ostman€ contributed equally to this article. factors from cells of the tumor stroma, as for example, the cancer- associated fibroblasts (CAF; refs. 7, 8). CAFs constitute a hetero- Current address for M. Augsten: amcure GmbH, Eggenstein-Leopoldshafen, geneous cell population that contributes to cancer growth and Germany. spread by secretion of a variety of protumorigenic factors, includ- € Corresponding Author: Arne Ostman, Karolinska Institutet, Cancer Center ing soluble factors. Among these CAF-secreted factors implicated Karolinska, R8:03, Karolinska University Hospital, Stockholm 17176, Sweden. in EMT and metastasis are chemokines, proteins of a size between Phone: 468-5177-0232; Fax: 468-339-031; E-mail: [email protected] 8 and 14 kDa that stimulate directed cell migration by creating a doi: 10.1158/1078-0432.CCR-18-1294 gradient along which cell types expressing the corresponding Ó2019 American Association for Cancer Research. receptor travel (7, 9). Chemokines bind to the pertussis-sensitive www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst March 8, 2019; DOI: 10.1158/1078-0432.CCR-18-1294 Sjoberg€ et al. Hs578t were cultured in DMEM (Hyclone), supplemented with Translational Relevance 10% FBS (Hyclone), 1% glutamine (Hyclone) and 1% penicillin/ Autocrine fibroblast CXCL14/ACKR2 signaling is shown to streptomycin (Hyclone). DMEM-F12 (Hyclone), supplemented induce EMT, migration, and metastasis and to correlate with with horse serum (Biochrom), 1% glutamine, and 1% penicillin/ worse survival in patients with breast cancer. The identification streptomycin was used for culturing the MCF10-DCIS cell line. of ACKR2 as a novel component in the signaling of the orphan Starvation was performed in medium without serum. All cells chemokine CXCL14 is relevant for further biomarker studies were maintained at 37 C in humidified air with 5% CO2. NIH-ctr and suggests novel targeting opportunities. and NIH-CXCL14 fibroblasts have been characterized earlier, and fibroblasts secrete physiologic levels of CXCL14 (22). Cell lines were purchased from ATCC or received from collaboration part- ners and continually tested for Mycoplasma infection during the Gai-subfamily of G-protein–coupled receptors (GPCR) that study. The identity of the cell lines used was confirmed by short engage different signaling pathways including ERK1/2, PI3K/AKT, tandem repeat (STR) profiling at Uppsala Genome Center. All and calcium signaling (10). Besides the classical chemokine experiments were performed with cells of passage 3–20. receptors, there is a subfamily of atypical chemokine receptors Western blot analyses used antibodies against p-ERK (#9101), (ACKR) that are predominantly involved in sequestration of ERK (#9102), E-cadherin (#3195), Snail (#3879), NOS1 (#4234) chemokines (11). (Cell Signaling Technology), b-actin (A1978), and a-tubulin In cancer, chemokines are involved in the recruitment of (T5201; Sigma-Aldrich). Primary antibodies detecting E-cadherin various cell types into tumors and thereby affecting inflammation, (#3195), Cytokeratin 8/18 (#4546), and PDGFbR (#3169; Cell angiogenesis, tumor growth, invasion, and metastasis (12). A Signaling Technology) together by fluorescent-linked secondary paracrine chemokine cross-talk between stromal cells and tumor antibodies, were used for immunofluorescence staining of xeno- cells, involving effects on EMT, has been demonstrated to enhance graft tumors. formation of metastases (13–16). Expression of certain chemo- Pertussis toxin was purchased from Sigma-Aldrich and recom- kines in distant tissues has also been reported to determine binant CXCL14 and CCL5 from R&D Systems and PeproTech. metastasis formation in specific organs, a process termed meta- Alexa Fluor 647–coupled chemokines were purchased from static tropism (17). Almac. The orphan chemokine CXCL14, earlier designated BRAK, MIP-2g, BMAC, or KS1 stimulates migration of various immune RNA isolation, cDNA synthesis, and qRT-PCR analysis cells, including B cells, NK cells, and monocytes, but not RNA was isolated from xenograft tumors or overnight starved T cells (18–21). CXCL14 expression has been shown to be cells using GeneElute Mammalian Total RNA Miniprep Kit upregulated in CAFs, as compared with normal fibroblasts, in (Sigma-Aldrich). cDNA synthesis was performed with the Super- human breast and prostate cancer (22, 23). Experimental studies Script III First-Strand Synthesis System for RT-PCR (Invitrogen), exploring the function of CXCL14 during tumor progression and using PolydT primers, in accordance with the manufacturer's metastasis formation have demonstrated context-dependent pro- instructions. The qRT-PCR reaction using SYBR Green Universal and antitumoral effects. The reasons for these effects remain PCR Master Mix (Applied Biosystems) was performed with the largely unknown and could possibly dependent on the cell type 7500 Real-Time PCR System (Applied Biosystems). The concen- that express the chemokine and on the profile of chemokine tration of primers