Perurena et al. Journal of Hematology & Oncology (2017) 10:23 DOI 10.1186/s13045-017-0399-x RESEARCH Open Access EPCR promotes breast cancer progression by altering SPOCK1/testican 1-mediated 3D growth Naiara Perurena1, Carolina Zandueta1, Susana Martínez-Canarias1, Haritz Moreno1, Silvestre Vicent1,4,5, Ana S. Almeida8, Elisabet Guruceaga2, Roger R. Gomis7, Marta Santisteban6, Mikala Egeblad8, José Hermida3 and Fernando Lecanda1,4,5* Abstract Background: Activated protein C/endothelial protein C receptor (APC/EPCR) axis is physiologically involved in anticoagulant and cytoprotective activities in endothelial cells. Emerging evidence indicates that EPCR also plays a role in breast stemness and human tumorigenesis. Yet, its contribution to breast cancer progression and metastasis has not been elucidated. Methods: Transcriptomic status of EPCR was examined in a cohort of 286 breast cancer patients. Cell growth kinetics was evaluated in control and EPCR and SPARC/osteonectin, Cwcv, and kazal-like domains proteoglycan (SPOCK1/testican 1) silenced breast cancer cells in 2D, 3D, and in co-culture conditions. Orthotopic tumor growth and lung and osseous metastases were evaluated in several human and murine xenograft breast cancer models. Tumor-stroma interactions were further studied in vivo by immunohistochemistry and flow cytometry. An EPCR- induced gene signature was identified by microarray analysis. Results: Analysis of a cohort of breast cancer patients revealed an association of high EPCR levels with adverse clinical outcome. Interestingly, EPCR knockdown did not affect cell growth kinetics in 2D but significantly reduced cell growth in 3D cultures. Using several human and murine xenograft breast cancer models, we showed that EPCR silencing reduced primary tumor growth and secondary outgrowths at metastatic sites, including the skeleton and the lungs. Interestingly, these effects were independent of APC ligand stimulation in vitro and in vivo. Transcriptomic analysis of EPCR-silenced tumors unveiled an effect mediated by matricellular secreted proteoglycan SPOCK1/testican 1. Interestingly, SPOCK1 silencing suppressed in vitro 3D growth. Moreover, SPOCK1 ablation severely decreased orthotopic tumor growth and reduced bone metastatic osteolytic tumors. High SPOCK1 levels were also associated with poor clinical outcome in a subset breast cancer patients. Our results suggest that EPCR through SPOCK1 confers a cell growth advantage in 3D promoting breast tumorigenesis and metastasis. Conclusions: EPCR represents a clinically relevant factor associated with poor outcome and a novel vulnerability to develop combination therapies for breast cancer patients. Keywords: Matricellular, Metastasis, Microenvironment, Sphere cultures, Extracellular matrix * Correspondence: [email protected] 1Adhesion and Metastasis Laboratory, Program Solid Tumors and Biomarkers, Center for Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain 4IdiSNA, Navarra Institute for Health Research, Pamplona, Spain Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Perurena et al. Journal of Hematology & Oncology (2017) 10:23 Page 2 of 12 Background APC-EPCR blocking antibodies [18]. In contrast, overex- Endothelial protein C receptor (EPCR) is an endothelial pression of EPCR in MDA-MB-231 cells resulted in re- type 1 transmembrane receptor that enhances the activa- duced final tumor volumes in a xenograft model despite tion of protein C (PC) by the thrombin (IIa)-thrombomo- favoring tumor growth at initial stages [19]. The effect of dulin (TM) complex [1]. EPCR-dissociated activated EPCR at different stages of tumor progression remains protein C (APC) negatively regulates the coagulation poorly defined. process, while EPCR-bound APC induces cytoprotective In this study, we addressed the functional role of signaling through the proteolytic cleavage of protease- EPCR in primary and metastatic tumor growth in breast activated receptor 1 (PAR1), leading to anti-inflammatory cancer using several human and murine xenograft and anti-apoptotic responses [2]. models. We found that EPCR silencing impaired ortho- Recently, research in EPCR has gained considerable topic tumor growth and metastatic activity to the skel- momentum by the identification of new EPCR ligands eton and lungs. Moreover, high EPCR expression levels [2]. An EPCR domain distinct from the APC binding site associated with a poor clinical outcome in a cohort of was shown to interact with a specific T cell antigen breast cancer patients. Furthermore, we showed that receptor with potential implications in immunosurveil- EPCR effects in tumor progression were APC independ- lance of tumors [3]. EPCR was also identified as the ent and were partially mediated by a novel mechanism endothelial receptor for some subtypes of the erythro- involving SPOCK1. Thus, these findings unveil a novel cyte membrane protein 1 (PfEMP1) on the surface of mechanism mediated by EPCR in tumorigenesis and me- the parasite Plasmodium falciparum, mediating its se- tastasis of breast cancer with potential clinical impact on questration in the blood vessels during severe malaria the therapeutic management of breast cancer patients. [4]. FVII/FVIIa has been shown to bind EPCR with a similar affinity as PC/APC [5], whereas the binding of Methods FX/FXa to EPCR remains an open question [6]. Cell lines and reagents Recently, EPCR has been identified as a marker of mul- One thousand eight hundred thirty-three human breast tipotent mouse mammary stem cells (MaSCs). These cancer cell line was a kind gift from Dr. Massagué EPCR+ cells (accounting for 3–7% of basal cells) exhibited (Memorial Sloan-Kettering Cancer Center, NY, USA) [20]. a mesenchymal phenotype and enhanced colony-forming ANV5 murine breast cancer cell line was previously de- abilities [7]. EPCR was also shown to be necessary for cell scribed [21, 22]. APC (Xigris®) was purchased from Eli Lilly organization and growth of human mammary epithelial (Indianapolis, IN, USA). Anti-EPCR antibodies RCR252 cells in 3D cultures [8]. and RCR1 were kindly provided by Dr. Fukudome (Saga In cancer, aberrant expression of EPCR is detected in Medical School, Japan) while 1489 was kindly gifted by tumors of different origin including the lung [9], breast Dr. Esmon (Oklahoma Medical Research Foundation, [10], ovarian [11], colon [12], glioblastoma [13], meso- Oklahoma City, USA). F(ab´)2 fractions of the RCR252 thelioma [14], and leukemia [13]. In lung tumorigenesis, antibody were obtained as previously detailed [9]. APC/EPCR drives an anti-apoptotic program that en- shRNAs cloned into PLKO.1-puro vector and the dows cancer cells with increased survival ability, enhan- empty vector were obtained from Mission® (Sigma- cing their metastatic activity to the skeleton and adrenal Aldrich). glands [9]. Moreover, high expression levels of this single gene at the primary site in early stage lung Cell proliferation assay cancer patients predict the risk of adverse clinical Cell proliferation was assessed using CellTiter 96® progression [9, 15]. AQueous One Solution Cell Proliferation Assay (MTS), In breast cancer patients, tumor cells often dissemin- according to manufacturer’s recommendations (Pro- ate to target sites including the skeleton, lungs, brain, mega). All absorbance values were normalized with the and lymph nodes [16]. This event represents a frequent absorbance values from day 0 (5 h after seeding cells). complication associated with a 5-year survival rate ~25.9%. Recent findings have unveiled novel markers in Cell cycle analysis the primary tumor that predict the development of me- Cell cycle analysis was carried out with Click-iT® EdU tastasis to target organs such as the skeleton [17]. High Flow Cytometry Assay Kit (Invitrogen). Cells were main- EPCR levels have been associated with poor disease pro- tained in culture for 24 or 48 h before adding 10 μM gression in the polyoma middle T (PyMT) breast cancer EdU for 2 h. Next, cells were harvested, fixed in formal- model, closely similar to the luminal B type in humans dehyde (Click-iT® fixative), permeabilized in 1X Click-iT® [18]. Moreover, EPCR+ sorted MDA-MB-231 human saponin-based permeabilization and wash reagent, and breast cancer cells showed stem cell-like properties and incubated with the Click-iT® reaction cocktail for 30 min enhanced tumor-initiating activity, an effect inhibited by at room temperature in the dark. After a washing step, Perurena et al. Journal of Hematology & Oncology (2017) 10:23 Page 3 of 12 cells were incubated with 0.2 μg/μlRNaseA(Sigma- PBS were injected through the tail vein of mice. For BLI, an- Aldrich) for 1 h at room temperature, in the dark. imals were anesthetized and inoculated with 50 μlof 7AAD was added to the tubes 10 min before the acquisi- 15 mg/ml D-luciferin (Promega). Images were taken during tion of cells in a FACSCanto II cytometer (BD Biosci- 1 min