Toll likereceptors (TLRs) comprise a family of transmembrane proteins related to the Interleukin-1receptor. Emerging evidence suggests that deregulated TLRs expression in tumortissue promotes tumor survival signals, thus favoring tumor progression.Recently, aberrant TLR4 overexpression was demonstrated in papillary thyroidcancer (PTC).
To study themechanisms underlying TLR4 overexpression in PTC harboring the BRAFV600Emutation.
TLR4 expression wasevaluated in thyroid tissue derived from human PTCs and transgenicmice expressing BRAFV600E in thyrocytes (Tg-BRAFV600E mice)(immunohistochemistry and RT/qPCR). PCCl3 cells expressing BRAFV600Ein response to doxycycline (PC-BRAFV600E) and BRAFV600E-positive PTC cell line BCPAP were used to study BRAFV600E-drivenTLR4 expression (western blot, RT/qPCR, and gene reporter assays).
Immunohistochemistryanalysis showed TLR4 overexpression in primary and metastatic human PTCs comparedto normal thyroid tissue. Moreover, TLR4 expression was increased in thyroidtissue from Tg-BRAFV600E mice compared to littermate controls.
Stimulation ofdoxycycline-treated PC-BRAFV600E and BCPAP cells with the TLR4agonist lipopolyssacharide induced the activation of the transcription factorNF-κB, suggesting functional TLR4 signaling.
Doxycycline-inducedBRAFV600E expression in PC-BRAFV600E cells upregulatedTLR4 protein levels. BRAFV600E increased TLR4 expression attranscriptional level by stimulating TLR4 promoter activity. Deletion analysis ofthe TLR4 promoter revealed a distal mitogen-activated protein kinase(MAPK)-sensitive ETS binding-site critical for BRAFV600E-inducedTLR4 expression. Consistently, pharmacological inhibition of BRAFV600Eand MEK/ERK signaling reduced TLR4 mRNA expression in the BRAFV600E-positivePTC cell line BCPAP.
Our findings revealedthat the oncogene BRAFV600E induces functional TLR4 overexpressionin thyroid cancer involving a MEK/ERK-dependent TLR4 gene transcriptional activation.Altogether, these data raise an intriguing question regarding the role of TLR4 signalingin the development and progression of PTC, opening new possibilities for thedesign of therapeutic approaches.
