RESUMO
The presence of mutant BRAF V600E correlates with the risk of recurrence in papillary thyroid cancer (PTC) patients. However, not all PTC patients with BRAF V600E are associated with poor prognosis. Thus, understanding the mechanisms by which certain PTC patients with nuclear BRAF V600E become aggressive and develop resistance to a selective BRAF inhibitor, PLX-4032, is urgently needed. The effect of nuclear localization of BRAFV600E using in vitro studies, xenograft mouse-model and human tissues was evaluated. PTC cells harboring a nuclear localization signal (NLS) of BRAFV600E were established and examined in nude mice implanted with TPC1-NLS-BRAFV600E cells followed by PLX-4032 treatment. Immunohistochemical (IHC) analysis was performed on 100 PTC specimens previously confirmed that they have BRAFV600E mutations. Our results demonstrate that 21 of 100 (21%) PTC tissues stained with specific BRAFV600E antibody had nuclear staining with more aggressive features compared to their cytosolic counterparts. In vitro studies show that BRAFV600E is transported between the nucleus and the cytosol through CRM1 and importin (α/ß) system. Sequestration of BRAFV600E in the cytosol sensitized resistant cells to PLX-4032, whereas nuclear BRAFV600E was associated with aggressive phenotypes and developed drug resistance. Proteomic analysis revealed Arp2/3 complex members, actin-related protein 2 (ACTR2 aliases ARP2) and actin-related protein 3 (ACTR3 aliases ARP3), as the most enriched nuclear BRAFV600E partners. ACTR3 was highly correlated to lymph node stage and extrathyroidal extension and was validated with different functional assays. Our findings provide new insights into the clinical utility of the nuclear BRAFV600E as a prognostic marker for PTC aggressiveness and determine the efficacy of selective BRAFV600E inhibitor treatment which opens new avenues for future treatment options.
