NF-κB contributes to MMP1 expression in breast cancer spheroids causing paracrine PAR1 activation and disintegrations in the lymph endothelial barrier in vitro
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Chi Huu Nguyen1,2, Daniel Senfter2, Jose Basilio3, Silvio Holzner2, Serena Stadler2, Sigurd Krieger2, Nicole Huttary2, Daniela Milovanovic2, Katharina Viola2, Ingrid Simonitsch-Klupp2, Walter Jäger1, Rainer de Martin3, Georg Krupitza2
1Department of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria
2Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria
3Department of Vascular Biology and Thrombosis Research, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Vienna, Austria
Georg Krupitza, e-mail: firstname.lastname@example.org
Keywords: RELA, NFKB1, MMP1, PAR1, lymph endothelial cell migration
Received: June 16, 2015 Accepted: October 05, 2015 Published: October 15, 2015
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 Ca2+ 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.
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