BRAFV600E MUTATION, TISSUE INHIBITOR OF METALLOPROTEINASE-1 UPREGULATION AND NF-KB ACTIVATION: CLOSING THE LOOP ON THE PAPILLARY THYROID CANCER TRILOGY

BRAFV600E is the most common mutation in papillary thyroid carcinoma (PTC). Tissue inhibitor of metalloproteinases (TIMP-1) and Nuclear Factor (NF)-kB have been shown to play an important role in thyroid cancer. Our aim was to evaluate whether an interplay among these three factors exerts a functional role in PTCs. 56 PTC specimens were analyzed for BRAFV600E mutation, TIMP-1 expression and NF-kB activation by real-time allele-specific amplification, realtime quantitative PCR (qRT-PCR) and electroforetic mobility shift assay (EMSA), respectively. We show that BRAFV600E mutation occurs selectively in PTC nodules and determines up-regulation of TIMP-1 and hyperactivation of NF-kB. In addition we describe that CD63, a member of the tetraspanin family, which has been indicated as the TIMP-1 receptor, was selectively up-regulated in PTC nodules harboring BRAFV600E mutation. The proof of the principle was assayed in vitro using BCPAP cell line harboring BRAFV600E mutation. When we silenced BRAF gene in BCPAP cell line using a specific small interfering RNA for the mutated form (MU-A), we found a marked decrease in TIMP-1 expression and NF-kB binding activity. Furthermore, using invasion assay we found that MU-A treated cells decreased significantly their ability to invade. We demonstrate that BRAFV600E causes up-regulation of TIMP- 1 via NF-kB activation. TIMP-1 binds its surface receptor CD63, leading to Akt activation, and thus conferring an anti-apoptotic behavior which eventually promotes cell invasion. We individuated a functional trilogy which might explain how BRAFV600E determines cancer initiation, progression and invasiveness in PTC.