Cancer cell-derived 12(S)-HETE signals via 12-HETE receptor, RHO, ROCK and MLC2 to induce lymph endothelial barrier breaching

Background: The arachidonic acid metabolite 12(S)-HETE is suspected to enhance metastatic spread by inducing cancer cell-and lymph endothelial cell (LEC) motility. However, the molecular mechanisms leading to 12(S)-HETE-triggered cell migration are still elusive. Methods: To delineate the signalling pathways involved in 12(S)-HETE-mediated migration, inhibitors against RHO and ROCK, and specific siRNAs downregulating 12(S)-HETE receptor (12-HETER) and myosin light chain 2 (MLC2) were used. The breaching of the endothelial barrier was investigated by an assay measuring tumour spheroid-triggered 'circular chemorepellent-induced defects' (CCIDs), and respective signal transduction was elucidated by western blotting. Results: We provide evidence that 12(S)-HETE phosphorylated (and activated) MLC2, which regulates actin/myosin-based contraction. MLC2 activation was found to be essential for LEC retraction and CCID formation. Furthermore, we show that 12(S)-HETE activated a 12-HETER-RHO-ROCK-MYPT signalling cascade to induce MLC2 function. Conclusions: Signalling via this pathway is described for this metabolite for the first time. This may provide potential targets for the intervention of metastatic colonisation.