Introduction: Emerging evidence suggests that hyperactivityof Toll like receptors (TLRs) signaling promotes tumor survival signals, thusfavoring tumor progression. Recently, aberrant TLR4 overexpression was evidencedin papillary thyroid carcinomas (PTC).

Objective: To study the mechanisms underlyingTLR4 overexpression in PTC harboring the BRAF^V600E mutation.

Methods: TLR4 expression was studied in thyroid tissuederived from human PTCs and transgenicmice expressing BRAF^V600Ein thyrocytes (Tg-BRAF^V600E mice). BRAF^V600E-positive PTCcell line BCPAP and PCCl3 cells expressing BRAF^V600E in response to doxycycline (PC/BRAF^V600E) were used to study BRAF^V600E-drivenTLR4 expression. The Cancer Genome Atlas (TCGA) database was used to performcombined analysis.

Results: We evidenced TLR4 overexpression inPTCs compared to normal thyroid tissues. Moreover,match-samples of primary PTCs and its lymph node metastasis showed asignificant upregulation of TLR4 levels in the metastatic tissues. In agreement, TLR4 expression wasincreased in the thyroid tissue of Tg-BRAF^V600E mice compared tolittermate controls. Furthermore, we demonstrated functional TLR4 expression inPTC cells models evidencing an increased NF-κB transcriptional activity inresponse to the exogenous TLR4-agonist lipopolysaccharide.

TCGAdata analysis revealed that BRAF^V600E-positive tumors with high TLR4expression have shorter disease-free survival. Consistently with transcriptomicdata showing correlation between TLR4 expression and ERK activation score, conditionalBRAF^V600E expression in PC/BRAF^V600E cells upregulateTLR4 protein levels. Moreover, chemical blockage of MAPK/ERK signalingabrogated BRAF^V600E-induced TLR4 expression. Deletion analysis ofTLR4 promoter revealed a critical MAPK/ERK-sensitive ETS binding-site involvedin BRAF^V600E responsiveness. Furthermore, we evidenced that the ETSbinding factor ETS1 is critical for BRAF^V600E-induced MAPK/ERKsignaling-mediated TLR4 gene expression in PTCs.

Conclusions: Increased TLR4 expression in PTCs is a functional consequence of deregulatedMAPK/ERK/ETS1 signaling as result of thyroid tumors-drivers oncogenes such as BRAF^V600E. Considering the oncogenicpotential of aberrant NF-κB signaling activation in the promotion of thyroidtumor growth, our resultssuggest a pro-oncogenic potential of TLR4 downstream signaling in thyroidtumorigenesis.