title: NF-κB contributes to MMP1 expression in breast cancer spheroids causing paracrine PAR1 activation and disintegrations in the lymph endothelial barrier in vitro.
abstract: RELA, RELB, CREL, NFKB1 and NFKB2, and the upstream regulators NEMO and NIK were knocked-down in lymph endothelial cells (LECs) and in MDA-MB231 breast cancer spheroids to study the contribution of NF-κB in vascular barrier breaching. Suppression of RELA, NFKB1 and NEMO inhibited "circular chemo-repellent induced defects" (CCIDs), which form when cancer cells cross the lymphatic vasculature, by ~20-30%. Suppression of RELB, NFKB2 and NIK inhibited CCIDs by only ~10-15%. In MDA-MB231 cells RELA and NFKB1 constituted MMP1 expression, which caused the activation of PAR1 in adjacent LECs. The knock-down of MMP1 in MDA-MB231 spheroids and pharmacological inhibition of PAR1 in LECs inhibited CCID formation by ~30%. Intracellular Ca(2+) release in LECs, which was induced by recombinant MMP1, was suppressed by the PAR1 inhibitor SCH79797, thereby confirming a functional intercellular axis: RELA/NFKB1 - MMP1 (MDA-MB231) - PAR1 (LEC). Recombinant MMP1 induced PAR1-dependent phosphorylation of MLC2 and FAK in LECs, which is indicative for their activity and for directional cell migration such as observed during CCID formation. The combined knock-down of the NF-κB pathways in LECs and MDA-MB231 spheroids inhibited CCIDs significantly stronger than knock-down in either cell type alone. Also the knock-down of ICAM-1 in LECs (a NF-κB endpoint with relevance for CCID formation) and knock-down of MMP1 in MDA-MB231 augmented CCID inhibition. This evidences that in both cell types NF-κB significantly and independently contributes to tumour-mediated breaching of the lymphatic barrier. Hence, inflamed tumour tissue and/or vasculature pose an additional threat to cancer progression.
authors.affiliation: Department of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria.|Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Chi Huu
authors.initials: CH
authors.lastname: Nguyen
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Senfter
authors.affiliation: Department of Vascular Biology and Thrombosis Research, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Vienna, Austria.
authors.forename: Jose
authors.initials: J
authors.lastname: Basilio
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Silvio
authors.initials: S
authors.lastname: Holzner
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Serena
authors.initials: S
authors.lastname: Stadler
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Sigurd
authors.initials: S
authors.lastname: Krieger
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Nicole
authors.initials: N
authors.lastname: Huttary
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Daniela
authors.initials: D
authors.lastname: Milovanovic
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Viola
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Simonitsch-Klupp
authors.affiliation: Department of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria.
authors.forename: Walter
authors.initials: W
authors.lastname: Jäger
authors.affiliation: Department of Vascular Biology and Thrombosis Research, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Vienna, Austria.
authors.forename: Rainer
authors.initials: R
authors.lastname: de Martin
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Georg
authors.initials: G
authors.lastname: Krupitza
mesh_terms: D029681:Arabidopsis Proteins; D051792:Basic Helix-Loop-Helix Proteins; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D042783:Endothelial Cells / Q000378:metabolism*; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D020781:Matrix Metalloproteinase 1 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D019899:Paracrine Communication; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism*; D018874:Spheroids, Cellular; D014162:Transfection
chemical_list: D029681:Arabidopsis Proteins; D051792:Basic Helix-Loop-Helix Proteins; D020781:Matrix Metalloproteinase 1; D016328:NF-kappa B; D044463:Receptor, PAR-1; C524961:PAR1 protein, Arabidopsis; C522360:MMP1 protein, human
keywords: MMP1; NFKB1; PAR1; RELA; lymph endothelial cell migration