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ECM1 secreted by HER2-overexpressing breast cancer cells promotes formation of a vascular niche accelerating cancer cell migration and invasion

Abstract

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.

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Fig. 1: CM from D492HER2 enhances endothelial network and induces endothelial feedback increasing D492HER2 migration/invasion.
Fig. 2: Identification of ECM1 as pro-angiogenic candidate in D492HER2.
Fig. 3: Recombinant ECM1 enhances endothelial network and induces endothelial feedback increasing D492HER2 migration/invasion.
Fig. 4: ECM1 knockdown inhibits pro-angiogenic effect of D492HER2-CM and endothelial feedback on D492HER2 migration/invasion.
Fig. 5: Inhibition of endothelial NOTCH signaling prevents increased tube formation and feedback induction.
Fig. 6: Schematic summary of ECM1 inducing an endothelial feedback promoting cancer cell migration/invasion.

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Acknowledgements

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.

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Correspondence to Thorarinn Gudjonsson.

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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

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