Althoughincreased NF-κB activity is associated with a pro-inflammatory microenvironmentin aggressive thyroid cancer, the mechanism underlying NF-κB activation remainsunclear. Recently, we evidenced that Toll-like receptor 4 (TLR4) stimulationtrigger NF-κB signaling in thyroid cancer.

To investigate thyroidcancer-secreted endogenous TLR4 agonists able to trigger NF-κB signaling.

Experiments were performed onPCCl3 cells, PCCl3 cells conditionally expressing BRAF^V600E (PC/BRAF^V600E),and BRAF^V600E-mutant 8505c thyroid cancer cells. Gene reporterassays were used to evaluate NF-κB transcriptional activity. Transcriptomicanalysis was based on multiple public access datasets deposited on The CancerGenome Atlas and Gene Expression Omnibus.

Conditioned media (CM) fromPC/BRAF^V600E stimulate NF-κB transcriptional activity in PCCl3 cellscarrying an NF-κB reporter. This effect is TLR4-dependent as pre-incubation ofPCCl3 cells with the TLR4 signaling inhibitor TAK-242 abrogate NF-κBtranscriptional activity. CM from 8505c cells stimulate NF-κB signaling inPCCl3 cells. Pre-incubation of 8505c cells with the BRAF^V600Einhibitor PLX4032 abrogate CM-stimulated NF-κB signaling in PCCl3 cells.Transcriptomic analysis revealed a significant upregulation of endogenous TLR4agonists (Fibronectin-1, Tenascin-C) in papillary and anaplastic carcinomas. Nosignificant modulation of known endogenous TLR4 agonists was evidenced infollicular carcinomas.

Endogenous TLR4 agonists secretedto the tumor microenvironment may stimulate TLR4-dependent NF-κBtranscriptional activity, thus promoting a pro-inflammatory environmentassociated with thyroid cancer progression