12(S)-HETE induces lymph endothelial cell retraction in�vitro by upregulation of SOX18

Abstract

Metastasising breast cancer cells communicate with adjacent lymph endothelia, intravasate and disseminate through lymphatic routes, colonise lymph nodes and finally metastasize to distant organs. Thus, understanding and blocking intravasation may attenuate the metastatic cascade at an early step. As a trigger factor, which causes the retraction of lymph endothelial cells (LECs) and opens entry ports for tumour cell intravasation, MDA-MB231 breast cancer cells secrete the pro-metastatic arachidonic acid metabolite, 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid [12(S)-HETE]. In the current study, treatment of LECs with 12(S)-HETE upregulated the expression of the transcription factors SRY-related HMG-box 18 (SOX18) and prospero homeobox protein 1 (PROX1), which determine endothelial development. Thus, whether they have a role in LEC retraction was determined using a validated intravasation assay, small interfering RNA mediated knockdown of gene expression, and mRNA and protein expression analyses. Specific inhibition of SOX18 or PROX1 significantly attenuated in vitro intravasation of MDA-MB231 spheroids through the LEC barrier and 12(S)-HETE-triggered signals were transduced by the high and low affinity receptors, 12(S)-HETE receptor and leukotriene B4 receptor 2. In addition, the current findings indicate that there is crosstalk between SOX18 and nuclear factor κ-light-chain-enhancer of activated B cells, which was demonstrated to contribute to MDA-MB231/lymph endothelial intravasation. The present data demonstrate that the endothelial-specific and lymph endothelial-specific transcription factors SOX18 and PROX1 contribute to LEC retraction.

Feed‑back loops integrating RELA, SOX18 and FAK mediate the break‑down of the lymph‑endothelial barrier that is triggered by 12(S)‑HETE

“…Previous studies have reported that matrix metalloproteinase-1 activated the protein activated receptor 1 and Ca 2+ -release in LECs, inducing MLC2 and FAK, all of which are prerequisites for their retraction and CCID formation (6,12). In contrast, 12(S)-HETE has been indicated to activate the receptors 12-HETER, BLT2 and Ca 2+ -release (10,11), as well as the transcription factors NF-κB and SOX18 in LECs (18). NF-κB communicates with FAK in macrophages (26) and, conversely, NF-κB itself is controlled by FAK in endothelial and other types of cells (13,14).…”

“…This demonstrated that the constitutive expression of SOX18 and PROX1 were positively regulated by RELA in LECs. It has recently been demonstrated that RELA controls the 12(S)-HETE-stimulated upregulation of SOX18 and PROX1 (18). Since ICAM-1 is an accepted target of the NF-κB transcription factor in ECs (20,25), and specifically of RELA (18), the expression of ICAM-1 mRNA was examined, to control whether the activity of RELA was increased upon 12(S)-HETE-treatment of LECs (18) (Fig.…”

“…Instead, their targets, ICAM-1 and PROX1, were assessed, respectively. These targets were silenced using siRNAs, as they were both reported to attenuate CCID formation, similar to RELA and SOX18 (6,20,18). The specific inhibition of PROX1 or FAK reduced CCID formation by ~25% and the inhibition of ICAM-1 by ~12%.…”

“…However, how FAK is associated in the signal transduction network triggering LEC retraction, and whether FAK is regulated by 12(S)-HETE is yet to be determined. There is a cross-talk between FAK and NF-κB (13,14), and NF-κB has been reported to regulate the endothelial lineage-determining transcription factor SRY-box transcription factor 18 (SOX18), which supports endothelial cell differentiation during embryonic development (15,16), in human umbilical vein ECs (17) and LECs (18). A previous study demonstrated that SOX18, and its transcriptional target the lymph endothelial transcription factor and marker protein prospero homeobox 1 (PROX1), contributed to CCID formation in the lymph-endothelial barrier (18).…”

“…There is a cross-talk between FAK and NF-κB (13,14), and NF-κB has been reported to regulate the endothelial lineage-determining transcription factor SRY-box transcription factor 18 (SOX18), which supports endothelial cell differentiation during embryonic development (15,16), in human umbilical vein ECs (17) and LECs (18). A previous study demonstrated that SOX18, and its transcriptional target the lymph endothelial transcription factor and marker protein prospero homeobox 1 (PROX1), contributed to CCID formation in the lymph-endothelial barrier (18). The transcription factor NF-κB and its transcriptional target, the intercellular adhesion molecule 1 (ICAM-1), were also revealed to be required for CCID formation (6,19,20).…”