Mitochondrial Localization and Regulation of BRAFV600E in Thyroid Cancer: A Clinically Used RAF Inhibitor Is Unable to Block the Mitochondrial Activities of BRAFV600E

摘要

Context: The oncogenic BRAF~(V600E) mutation results in an active structural conformation characterized by greatly elevated ERK activity. However, additional cellular effects caused by subcellular action of BRAF~(V600E) remain to be identified.Objective: To explore these effects, differences in the subcellular localization of wild-type and mutant BRAF in thyroid cancer were investigated.Results: A significant proportion of endogenous and exogenous BRAF~(V600E), but not wild-type BRAF, was detected in the mitochondrial fraction, similar to other BRAF mutants including BRAF~(V600D), BRAF~(V600K), BRAF~(V600R), and BRAF~(G469A), which showed elevated kinase activity and mitochondrial localization. Induced expression of BRAF~(V600E) suppressed the apoptotic responses against staurosporine and TNFα/cycloheximide. Interestingly, the mitochondrial localization and antiapoptotic activities of BRAF~(V600E) were unaffected by sorafenib and U0126 suppression of MAPK kinase (MEK) and ERK activities. Similarly, although the RAF inhibitor sorafenib effectively inhibited MEK/ERK activation, it did not block the mitochondrial localization of BRAF~(V600E). In addition, inducible expression of BRAF~(V600E) increased the glucose uptake rate and decreased O_(2) consumption, suggesting that BRAF~(V600E) reduces mitochondrial oxidative phosphorylation, a signature feature of cancer cells. Again, these metabolic alterations resulted by BRAF~(V600E) expression were not affected by the treatment of thyroid cells by sorafenib. Therefore, RAF and MEK inhibitors are unable to block the antiapoptotic activity of BRAF~(V600E) or correct the high glucose uptake rate and glycolytic activity and suppressed mitochondrial oxidative phosphorylation induced by BRAF~(V600E).Conclusions: The mitochondrial localization observed in oncogenic BRAF mutants might be related to their altered responses to apoptotic stimuli and characteristic metabolic phenotypes found in thyroid cancer. The inability of MEK and RAF inhibitors, U0126 and sorafenib, respectively, to block the mitochondrial localization of BRAF~(V600E) has additional therapeutic implications for BRAF~(V600E)-positive thyroid cancers.