CONICET | Buscador de Institutos y Recursos Humanos

Background. Toll Like Receptors (TLRs) are transmembrane proteins that recognize specific patterns of pathogens, and its deregulation has been implicated in cancer. We have shown that TLR4 is functionally expressed in normal thyrocytes, although its role in human thyroid pathologies remains unidentified. Objective. Study the role of TLR4 in the development and progression of thyroid cancer. Material and methods. PC-BRAF cell line, a TET-ON system in which PCCL3 thyroid cells express the BRAF V600E oncogene when treated with doxycycline; WB; immunohistochemistry; promoter activity (luciferase) and RT-qPCR. Results. Immunohistochemistry of thyroid carcinoma tissues showed an aberrant TLR4 over-expression compared to normal tissue. Doxycycline-treated PC-BRAF cells increased TLR4 expression in a time-dependent manner, which proved to be functional by responding to treatment with lipopolysaccharide increasing the expression of the responsive genes IL-6 and iNOS. The augmented TLR4 expression involves, at least in part, the transcriptional level as demonstrated by promoter and mRNA assays. WB analysis showed an increased serine 276 phosphorylation in response to BRAF V600E expression. Moreover, co-transfection assays of a NF-κB reporter vector together with mutant p65 expression vector in which serine 276 is non-phosphorylatable, corroborated its role in BRAF V600E-induced p65 activation. Interestingly, PCCL3 normal cells cultured with BRAF V600E-transfected cells-conditioned mediums showed an increased p65 activity, demonstrating the presence of soluble factors secreted by transformed-cells able to alter normal cell behavior. Conclusions. TLR4 functional over-expression was demonstrated in BRAF V600E-transformed thyroid cells, entailing new evidence related to its participation in the development and progression of thyroid cancer disease.