NF-κB mediates the 12(S)-HETE-induced endothelial to mesenchymal transition of lymphendothelial cells during the intravasation of breast carcinoma cells

Abstract

Background:The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts.Methods:To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs.Results:We show that tumour spheroids generate ‘circular chemorepellent-induced defects' (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with ∼50% attenuation of CCID formation by treatment of cells with 10 μ Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general.Interpretation:These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell–cell contacts, migration, and the generation of CCID.

Effects of Scrophularia extracts on tumor cell proliferation, death and intravasation through lymphoendothelial cell barriers

“…pro-apoptotic properties of different Scrophularia species, and beyond that we show that S. lucida inhibits LEC-CCID formation by co-cultivated MCF-7 cancer cell spheroids (14,17) and inhibited NF-κB activity. Recently we were able to demonstrate that NF-κB activity contributed to LEC-CCID formation through inhibition of VE-cadherin expression and loss of intraspecific LEC adhesion (16).…”

“…They were shown to perform inhibitory effects on enzymes of the arachidonate cascade (COX-1, COX-2) and significant reduction of LPS-induced TNF-α production without relevant effects on the ALOX5 pathway. Treating LEC monolayers with 1 µM synthetic 12(S)-HETE, a metabolite of arachidonic acid generated by ALOX12/15, caused the phosphorylation of MLC2 (16) indicating that 12(S)-HETE induced the motility of LECs thereby provoking an early step of metastasis (15,17). This observation is also consistent with an inflammatory process, which is accompanied by the acquisition of a mobile phenotype of the affected cells reflecting 'epithelial to mesenchymal transition' [EMT; (57)].…”

“…9) which are induced by exudates [i.e., 12(S)-hydoxyeicosatetraenoic acid] of MCF-7 cancer cell spheroids. CCIDs can be considered as entry gates for tumour cells and are directly responsible for lymph node-and distant metastases (15)(16)(17). The extract of S. floribunda did not prevent CCID formation but affected the viability of LECs and because of the toxic effect of 1 mg/ml MeOH extract to LECs, the precise effect on CCID formation could not be evaluated.…”

“…We could show that the inhibition of NF-κB translocation with Bay11-7082, an irreversible inhibitor of I-κBα phosphorylation, blocked MCF-7 spheroid-induced gap formation of LECs in a dose-dependent fashion (16). To check whether the significant inhibition of CCID formation in LECs caused by S. lucida extracts may be induced through inhibition of NF-κB activity, we tested the detannified extract in an NF-κB luciferase reporter gene assay.…”

“…Cell suspension (150 µl) was transferred to each well of a 96-well plate (Greiner Bio-one, Cellstar 650185, Kremsmünster, Austria) to allow spheroid formation within the following two days. Then, MCF-7 spheroids were washed in PBS and transferred to cytotracker-stained LEC monolayers that were seeded into 24-well plates (Costar 3524, Sigma) in 2 ml EGM2 MV medium (15,16).…”