The tumor microenvironment is increasingly recognized as key player in cancer progression. Investigating heterotypic interactions between cancer cells and their microenvironment is important for understanding how specific cell types support cancer. Forming the vasculature, endothelial cells (ECs) are a prominent cell type in the microenvironment of both normal and neoplastic breast gland. Here, we sought out to analyze epithelial–endothelial cross talk in the breast using isogenic non-tumorigenic vs. tumorigenic breast epithelial cell lines and primary ECs. The cellular model used here consists of D492, a breast epithelial cell line with stem cell properties, and two isogenic D492-derived EMT cell lines, D492M and D492HER2. D492M was generated by endothelial-induced EMT and is non-tumorigenic while D492HER2 is tumorigenic, expressing the ErbB2/HER2 oncogene. To investigate cellular cross talk, we used both conditioned medium (CM) and 2D/3D co-culture systems. Secretome analysis of D492 cell lines was performed using mass spectrometry and candidate knockdown (KD), and overexpression (OE) was done using siRNA and CRISPRi/CRISPRa technology. D492HER2 directly enhances endothelial network formation and activates a molecular axis in ECs promoting D492HER2 migration and invasion, suggesting an endothelial feedback response. Secretome analysis identified extracellular matrix protein 1 (ECM1) as potential angiogenic inducer in D492HER2. Confirming its involvement, KD of ECM1 reduced the ability of D492HER2-CM to increase endothelial network formation and induce the endothelial feedback, while recombinant ECM1 (rECM1) increased both. Interestingly, NOTCH1 and NOTCH3 expression was upregulated in ECs upon treatment with D492HER2-CM or rECM1 but not by CM from D492HER2 with ECM1 KD. Blocking endothelial NOTCH signaling inhibited the increase in network formation and the ability of ECs to promote D492HER2 migration and invasion. In summary, our data demonstrate that cancer-secreted ECM1 induces a NOTCH-mediated endothelial feedback promoting cancer progression by enhancing migration and invasion. Targeting this interaction may provide a novel possibility to improve cancer treatment.
Subscribe to Journal
Get full journal access for 1 year
only $41.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Hassiotou F, Geddes D. Anatomy of the human mammary gland: current status of knowledge. Clinical Anat. 2013;26:29–48.
Sigurdsson V, Fridriksdottir AJ, Kjartansson J, Jonasson JG, Steinarsdottir M, Petersen OW, et al. Human breast microvascular endothelial cells retain phenotypic traits in long-term finite life span culture. In Vitro Cell Dev Biol Anim. 2006;42:332–40.
Ingthorsson S, Sigurdsson V, Fridriksdottir A Jr., Jonasson JG, Kjartansson J, Magnusson MK, et al. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture. BMC Res Notes. 2010;3:184.
Gudjonsson T, Ronnov-Jessen L, Villadsen R, Bissell MJ, Petersen OW. To create the correct microenvironment: three-dimensional heterotypic collagen assays for human breast epithelial morphogenesis and neoplasia. Methods. 2003;30:247–55.
Bergthorsson JT, Magnusson MK, Gudjonsson T. Endothelial-rich microenvironment supports growth and branching morphogenesis of prostate epithelial cells. Prostate. 2013;73:884–96.
Bissell MJ, Hall HG, Parry G. How does the extracellular matrix direct gene expression? J Theor Biol. 1982;99:31–68.
Ghajar CM, Peinado H, Mori H, Matei IR, Evason KJ, Brazier H, et al. The perivascular niche regulates breast tumour dormancy. Nat Cell Biol. 2013;15:807–17.
Erler JT, Weaver VM. Three-dimensional context regulation of metastasis. Clin Exp Metastasis. 2009;26:35–49.
Hoye AM, Erler JT. Structural ECM components in the premetastatic and metastatic niche. Am J Physiol Cell Physiol. 2016;310:C955–67.
Hanahan D, Coussens LM. Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell. 2012;21:309–22.
Oskarsson T, Batlle E, Massague J. Metastatic stem cells: sources, niches, and vital pathways. Cell Stem Cell. 2014;14:306–21.
Pollard JW. Macrophages define the invasive microenvironment in breast cancer. J Leukoc Biol. 2008;84:623–30.
Komohara Y, Jinushi M, Takeya M. Clinical significance of macrophage heterogeneity in human malignant tumors. Cancer Sci. 2014;105:1–8.
Komohara Y, Takeya M. CAFs and TAMs: maestros of the tumour microenvironment. J Pathol. 2017;241:313–5.
Shan T, Chen S, Chen X, Lin WR, Li W, Ma J, et al. Cancer-associated fibroblasts enhance pancreatic cancer cell invasion by remodeling the metabolic conversion mechanism. Oncol Rep. 2017;37:1971–9.
Zhang Z, Li X, Sun W, Yue S, Yang J, Li J, et al. Loss of exosomal miR-320a from cancer-associated fibroblasts contributes to HCC proliferation and metastasis. Cancer Lett. 2017;397:33–42.
Ghajar CM. Metastasis prevention by targeting the dormant niche. Nat Rev Cancer. 2015;15:238–47.
Lee E, Fertig EJ, Jin K, Sukumar S, Pandey NB, Popel AS. Breast cancer cells condition lymphatic endothelial cells within pre-metastatic niches to promote metastasis. Nat Commun. 2014;5:4715.
Lee E, Pandey NB, Popel AS. Crosstalk between cancer cells and blood endothelial and lymphatic endothelial cells in tumour and organ microenvironment. Expert Rev Mol Med. 2015;17:e3.
Lee E, Pandey NB, Popel AS. Lymphatic endothelial cells support tumor growth in breast cancer. Sci Rep. 2014;4:5853.
Franses JW, Baker AB, Chitalia VC, Edelman ER. Stromal endothelial cells directly influence cancer progression. Sci Transl Med. 2011;3:66ra5.
Franses JW, Drosu NC, Gibson WJ, Chitalia VC, Edelman ER. Dysfunctional endothelial cells directly stimulate cancer inflammation and metastasis. Int J Cancer. 2013;133:1334–44.
Ucuzian AA, Gassman AA, East AT, Greisler HP. Molecular mediators of angiogenesis. J Burn Care Res. 2010;31:158–75.
Laughner E, Taghavi P, Chiles K, Mahon PC, Semenza GL. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol. 2001;21:3995–4004.
Murtas D, Piras F, Minerba L, Maxia C, Ferreli C, Demurtas P, et al. Activated Notch1 expression is associated with angiogenesis in cutaneous melanoma. Clin Exp Med. 2015;15:351–60.
Guo D, Li C, Teng Q, Sun Z, Li Y, Zhang C. Notch1 overexpression promotes cell growth and tumor angiogenesis in myeloma. Neoplasma. 2013;60:33–40.
Sigurdsson V, Hilmarsdottir B, Sigmundsdottir H, Fridriksdottir AJ, Ringner M, Villadsen R, et al. Endothelial induced EMT in breast epithelial cells with stem cell properties. PLoS ONE. 2011;6:e23833.
Gudjonsson T, Villadsen R, Nielsen HL, Ronnov-Jessen L, Bissell MJ, Petersen OW. Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties. Genes Dev. 2002;16:693–706.
Villadsen R, Fridriksdottir AJ, Ronnov-Jessen L, Gudjonsson T, Rank F, LaBarge MA, et al. Evidence for a stem cell hierarchy in the adult human breast. J Cell Biol. 2007;177:87–101.
Briem E, Ingthorsson S, Traustadottir GA, Hilmarsdottir B, Gudjonsson T. Application of the D492 cell lines to explore breast morphogenesis, EMT and cancer progression in 3D culture. J Mammary Gland Biol Neoplasia. 2019;24:139–47.
Ingthorsson S, Andersen K, Hilmarsdottir B, Maelandsmo GM, Magnusson MK, Gudjonsson T. HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR. Oncogene. 2015;35:4244–55.
Han Z, Ni J, Smits P, Underhill CB, Xie B, Chen Y, et al. Extracellular matrix protein 1 (ECM1) has angiogenic properties and is expressed by breast tumor cells. FASEB J. 2001;15:988–94.
Sercu S, Zhang L, Merregaert J. The extracellular matrix protein 1: its molecular interaction and implication in tumor progression. Cancer Invest. 2008;26:375–84.
Wang L, Yu J, Ni J, Xu X-M, Wang J, Ning H, et al. Extracellular matrix protein 1 (ECM1) is over-expressed in malignant epithelial tumors. Cancer Lett. 2003;200:57–67.
Chen H, Jia W, Li J. ECM1 promotes migration and invasion of hepatocellular carcinoma by inducing epithelial-mesenchymal transition. World J Surg Oncol. 2016;14:195.
Gan L, Meng J, Xu M, Liu M, Qi Y, Tan C, et al. Extracellular matrix protein 1 promotes cell metastasis and glucose metabolism by inducing integrin beta4/FAK/SOX2/HIF-1alpha signaling pathway in gastric cancer. Oncogene. 2017;37:744–55.
Lee KM, Nam K, Oh S, Lim J, Kim RK, Shim D, et al. ECM1 regulates tumor metastasis and CSC-like property through stabilization of beta-catenin. Oncogene. 2015;34:6055–65.
Wang Z, Zhou Q, Li A, Huang W, Cai Z, Chen W. Extracellular matrix protein 1 (ECM1) is associated with carcinogenesis potential of human bladder cancer. Onco Targets Ther. 2019;12:1423–32.
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–22.
Guillen J. FELASA guidelines and recommendations. J Am Assoc Lab Anim Sci. 2012;51:311–21.
Rosenblat JD, Brietzke E, Mansur RB, Maruschak NA, Lee Y, McIntyre RS. Inflammation as a neurobiological substrate of cognitive impairment in bipolar disorder: evidence, pathophysiology and treatment implications. J Affect Disord. 2015;188:149–59.
Kastrup J. Can YKL-40 be a new inflammatory biomarker in cardiovascular disease? Immunobiology. 2012;217:483–91.
Vignon E. Is glycoprotein YKL40 a new marker for joint disorders? Joint Bone Spine. 2001;68:454–6.
Zhou Y, Peng H, Sun H, Peng X, Tang C, Gan Y, et al. Chitinase 3-like 1 suppresses injury and promotes fibroproliferative responses in Mammalian lung fibrosis. Sci Transl Med. 2014;6:240ra76.
Francescone RA, Scully S, Faibish M, Taylor SL, Oh D, Moral L, et al. Role of YKL-40 in the angiogenesis, radioresistance, and progression of glioblastoma. J Biol Chem. 2011;286:15332–43.
Shao R. YKL-40 acts as an angiogenic factor to promote tumor angiogenesis. Front Physiol. 2013;4:122.
Mathieu E, Meheus L, Raymackers J, Merregaert J. Characterization of the osteogenic stromal cell line MN7: identification of secreted MN7 proteins using two-dimensional i’olyacrylamide gel electrophoresis, western blotting, and microsequencing. J Bone Miner Res. 1994;9:903–13.
Oyama N, Merregaert J. The extracellular matrix protein 1 (ECM1) in molecular-based skin biology. In: Farage M., Miller K., Maibach H. (eds) Textbook of Aging Skin. Springer, Berlin, Heidelberg. 2017. p. 91–110.
Lee KM, Nam K, Oh S, Lim J, Kim YP, Lee JW, et al. Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling. Breast Cancer Res. 2014;16:479.
Sercu S, Zhang M, Oyama N, Hansen U, Ghalbzouri AE, Jun G, et al. Interaction of extracellular matrix protein 1 with extracellular matrix components: ECM1 is a basement membrane protein of the skin. J Invest Dermatol. 2008;128:1397–408.
Harjes U, Bridges E, Gharpure KM, Roxanis I, Sheldon H, Miranda F, et al. Antiangiogenic and tumour inhibitory effects of downregulating tumour endothelial FABP4. Oncogene. 2017;36:912–21.
Bruning U, Morales-Rodriguez F, Kalucka J, Goveia J, Taverna F, Queiroz KCS, et al. Impairment of angiogenesis by fatty acid synthase inhibition involves mTOR malonylation. Cell Metab. 2018;28:866.e15–80.e15.
Kalucka J, Bierhansl L, Conchinha NV, Missiaen R, Elia I, Bruning U, et al. Quiescent endothelial cells upregulate fatty acid beta-oxidation for vasculoprotection via redox homeostasis. Cell Metab. 2018;28:881.e13–94.e13.
Wu Q, Chen D, Luo Q, Yang Q, Zhao C, Zhang D, et al. Extracellular matrix protein 1 recruits moesin to facilitate invadopodia formation and breast cancer metastasis. Cancer Lett. 2018;437:44–55.
Gomez-Contreras P, Ramiro-Diaz JM, Sierra A, Stipp C, Domann FE, Weigel RJ, et al. Extracellular matrix 1 (ECM1) regulates the actin cytoskeletal architecture of aggressive breast cancer cells in part via S100A4 and Rho-family GTPases. Clin Exp Metastasis. 2017;34:37–49.
Martin-Pardillos A, Valls Chiva A, Bande Vargas G, Hurtado Blanco P, Pineiro Cid R, Guijarro PJ, et al. The role of clonal communication and heterogeneity in breast cancer. BMC Cancer. 2019;19:666.
Ferraro DA, Patella F, Zanivan S, Donato C, Aceto N, Giannotta M, et al. Endothelial cell-derived nidogen-1 inhibits migration of SK-BR-3 breast cancer cells. BMC Cancer. 2019;19:312.
Smits P, Bhalerao J, Merregaert J. Molecular cloning and characterization of the mouse Ecm1 gene and its 5’ regulatory sequences. Gene. 1999;226:253–61.
Ye H, Yu X, Xia J, Tang X, Tang L, Chen F. MiR-486-3p targeting ECM1 represses cell proliferation and metastasis in cervical cancer. Biomed Pharmacother. 2016;80:109–14.
Furuhashi M, Saitoh S, Shimamoto K, Miura T. Fatty acid-binding protein 4 (FABP4): pathophysiological insights and potent clinical biomarker of metabolic and cardiovascular diseases. Clin Med Insights Cardiol. 2014;8(Suppl 3):23–33.
Traustadottir GA, Jensen CH, Thomassen M, Beck HC, Mortensen SB, Laborda J, et al. Evidence of non-canonical NOTCH signaling: delta-like 1 homolog (DLK1) directly interacts with the NOTCH1 receptor in mammals. Cell Signal. 2016;28:246–54.
Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science. 1999;284:770–6.
We thank the FingerPrints Proteomics Facility at the University of Dundee for performing the mass spectrometry and the Center for Systems Biology at the University of Iceland for the technical support during mass spectrometry data analysis. Further, we want to thank the HI-STEM laboratory and the sequencing core facility at the German Cancer Research Center, DKFZ Heidelberg, Germany, for technical support and performing the RNA microarray. Finally, we thank Prof. Dr Haraldur Haraldsson, University of Iceland for providing primary human umbilical vascular ECs. Human umbilical vascular ECs were obtained from umbilical chords at the childbirth department, University Hospital Iceland (ethical application nr. 35/2013). This work was supported by Grants from Landspitali University Hospital Science Fund, University of Iceland Research Fund (Grant of Excellence: #152144051 Doctoral Grant: #185042051), and Icelandic Science and Technology Policy.
Conflict of interest
The authors declare that they have no conflict of interest. The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Steinhaeuser, S.S., Morera, E., Budkova, Z. et al. ECM1 secreted by HER2-overexpressing breast cancer cells promotes formation of a vascular niche accelerating cancer cell migration and invasion. Lab Invest 100, 928–944 (2020). https://doi.org/10.1038/s41374-020-0415-6