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Norisato Mitsutake, Makoto Miyagishi, Shin Mitsutake, Nagako Akeno, Cleo Mesa, Jeffrey A. Knauf, Lei Zhang, Kazunari Taira, James A. Fagin, BRAF Mediates RET/PTC-Induced Mitogen-Activated Protein Kinase Activation in Thyroid Cells: Functional Support for Requirement of the RET/PTC-RAS-BRAF Pathway in Papillary Thyroid Carcinogenesis, Endocrinology, Volume 147, Issue 2, 1 February 2006, Pages 1014–1019, https://doi.org/10.1210/en.2005-0280
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In human papillary thyroid cancers (PTCs), mutations of RET/PTC, NTRK, RAS, or BRAF are found in about two thirds of cases with practically no overlap, providing genetic evidence that constitutive signaling along RET-RAS-BRAF-MAPK is key to their development. The requirement for BRAF in RET/PTC-mediated MAPK activation and gene expression has not been tested functionally. There are three RAF isoforms: ARAF, BRAF, and CRAF. Compared with the others, ARAF is a much weaker stimulator of MAPK. To determine the key RAF isoform mediating RET/PTC-induced ERK phosphorylation, we stably transfected doxycycline-inducible RET/PTC3-expressing thyroid PCCL3 cells with small interfering RNA vectors to induce selective knockdown of BRAF or CRAF. Conditional RET/PTC3 expression induced comparable ERK phosphorylation in CRAF knockdown and control cells but negligible ERK phosphorylation in BRAF knockdown cells. Selective knockdown of BRAF prevented RET/PTC-dependent down-regulation of the sodium iodide symporter, a gene that confers key biological effects of RET/PTC in PTCs. Moreover, microarray analysis revealed numerous RET/PTC-regulated genes showing requirement of BRAF for appropriate expression. These data indicate that BRAF is required for RET/PTC-induced MAPK activation in thyroid cells and support the notion that BRAF inactivation may be an attractive target for PTCs.