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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

Division of Endocrinology and Metabolism (N.M., S.M., N.A., C.M., J.A.K., L.Z., J.A.F.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; Department of Chemistry and Biotechnology (M.M., K.T.), University of Tokyo School of Engineering, Hongo, Tokyo 113-8656, Japan

Address all correspondence and requests for reprints to: James A. Fagin, Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, P.O. Box 670547, Cincinnati, Ohio 45267-0547. E-mail: james.fagin{at}uc.edu.

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.

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