AHR/CYP1A1 interplay triggers lymphatic barrier breaching in breast cancer spheroids by inducing 12(S)-HETE synthesis

摘要

A causal link between overexpression of aryl hydrocarbon receptor (AHR) and its target cytochrome P450 1A1 (CYP1A1) and metastatic outgrowth of various cancer entities has been established. Nevertheless, the mechanism how AHR/CYP1A1 support metastasis formation is still little understood. In vitro we discovered a potential mechanism facilitating tumour dissemination based on the production of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Utilising a three-dimensional lymph endothelial cell (LEC) monolayer & MDA-MB231 breast cancer cell spheroid co-culture model in combination with knock-down approach allowed elucidation of the molecular/biochemical basis of AHR/CYP1A1-induced tumour breaching through the LEC barrier. Enzyme immunoassay evidenced the potential of recombinant CYP1A1 to synthesise 12(S)-HETE in vitro and qPCR and Western blotting measured gene and protein expression in specific experimental settings. In detail, AHR induced CYP1A1 expression and 12(S)-HETE secretion in tumour spheroids, which caused LEC junction retraction thereby forming large discontinuities allowing transmigration of the tumour. This was enforced by the activating AHR ligand 6-formylindolo (3,3-b)carbazole (FICZ), or inhibited by the AHR antagonist 3,3’-diindolylmethane (DIM) as well as by siRNA against AHR and CYP1A1. AHR and NF-κB were negatively cross talking and therefore, the inhibition of AHR (but not CYP1A1) induced RELA, RELB, NFKB1, NFKB2 and the NF-κB target MMP1, which itself promotes tumour intravasation by a mechanism that is different from 12(S)-HETE. Conversely, the inhibition of NFKB2 induced AHR, CYP1A1 and 12(S)-HETE synthesis. The approved clinical drugs guanfacine and vinpocetine, which inhibit CYP1A1 and NF-κB, respectively, significantly inhibited LEC barrier breaching in vitro indicating an option to reduce metastatic dissemination.