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.
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.
Mitsutake
Norisato
N
Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Ohio 45267-0547, USA.
Miyagishi
Makoto
M
Mitsutake
Shin
S
Akeno
Nagako
N
Mesa
Cleo
C
Knauf
Jeffrey A
JA
Zhang
Lei
L
Taira
Kazunari
K
Fagin
James A
JA
D000818:Animals; D002291:Carcinoma, Papillary / Q000201:enzymology / Q000235:genetics*; D002478:Cells, Cultured; D002999:Clone Cells; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D020868:Gene Silencing; D007527:Isoenzymes; D015514:Oncogene Proteins, Fusion / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret / Q000378:metabolism*; D012313:RNA / Q000032:analysis; D051381:Rats; D013961:Thyroid Gland / Q000166:cytology / Q000201:enzymology; D013964:Thyroid Neoplasms / Q000201:enzymology / Q000235:genetics*
D007527:Isoenzymes; D015514:Oncogene Proteins, Fusion; D012313:RNA; D011505:Protein-Tyrosine Kinases; D051096:Proto-Oncogene Proteins c-ret; C490558:RET-PTC oncoprotein, rat; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases