title: In vitro transforming potential, intracellular signaling properties, and sensitivity to a kinase inhibitor (sorafenib) of RET proto-oncogene variants Glu511Lys, Ser649Leu, and Arg886Trp.
abstract: Multiple endocrine neoplasia type 2 and a subset of apparently sporadic medullary thyroid carcinoma (AS-MTC) are caused by germ line activating point mutations of the rearranged during transfection (RET) proto-oncogene. RET encodes a receptor with tyrosine kinase activity that targets several intracellular signaling cascades, such as RAS-RAF-ERK1/2, PIK3-AKT, and STAT transcription factors. The objective of this study was to assess the function of three germ line RET variants Arg886Trp, Ser649Leu, and Glu511Lys of undetermined pathogenic significance, which were found in three kindreds of isolated AS-MTC. For this purpose, we employed vectors expressing each of the RET variants and measured the number of NIH3T3 transformation foci and soft agar colonies, the degree of activation of known RET intracellular signaling targets (ERK1/2, STAT1, STAT3, and TCF4), and the extent of ERK1/2 inhibition on sorafenib treatment. We found that RET variants Arg886Trp and Glu511Lys have shown increased in vitro transforming potential in a glial-derived neurotrophic factor-dependent manner. In contrast, the Ser649Leu variant did not significantly increased the number of foci and agar colonies relative to wild-type RET (RET-WT). The variants Glu511Lys and Arg886Trp showed 10- and 12.5-fold ERK1/2 activation respectively, that was significantly higher than that observed for RET-WT (fivefold). Increased levels of STAT1 and TCF4 activation were only observed for RET Arg886Trp (2.5- and 3-fold versus 1.2- and 2-fold in RET-WT respectively). The three RET variants analyzed here were sensitive to treatment with sorafenib. In conclusion, our results allow to classify previously uncharacterized RET genotypes, which may be of use to define follow-up and therapeutic regimens.
authors.affiliation: Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) - Cancer Biology, Rua Dr Roberto Frias, Porto, Portugal.
authors.forename: Hugo
authors.initials: H
authors.lastname: Prazeres
authors.forename: Joana P
authors.initials: JP
authors.lastname: Couto
authors.forename: Fernando
authors.initials: F
authors.lastname: Rodrigues
authors.forename: João
authors.initials: J
authors.lastname: Vinagre
authors.forename: Joana
authors.initials: J
authors.lastname: Torres
authors.forename: Vitor
authors.initials: V
authors.lastname: Trovisco
authors.forename: Teresa C
authors.initials: TC
authors.lastname: Martins
authors.forename: Manuel
authors.initials: M
authors.lastname: Sobrinho-Simões
authors.forename: Paula
authors.initials: P
authors.lastname: Soares
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000378:metabolism; D001557:Benzenesulfonates / Q000494:pharmacology*; D015153:Blotting, Western; D018276:Carcinoma, Medullary / Q000235:genetics* / Q000378:metabolism; D016022:Case-Control Studies; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D005260:Female; D018095:Germ-Line Mutation / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008297:Male; D051379:Mice; D008875:Middle Aged; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D009536:Niacinamide / Q000031:analogs & derivatives; D010375:Pedigree; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D000090063:Proto-Oncogene Mas; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics* / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology*; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D000077157:Sorafenib; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000378:metabolism; D000073940:Transcription Factor 4; D014157:Transcription Factors / Q000378:metabolism; D055815:Young Adult
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001557:Benzenesulfonates; C000715470:MAS1 protein, human; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors; D000090063:Proto-Oncogene Mas; D011725:Pyridines; D050796:STAT3 Transcription Factor; C532096:TCF4 protein, human; D000073940:Transcription Factor 4; D014157:Transcription Factors; D009536:Niacinamide; D000077157:Sorafenib; D008156:Luciferases; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D051057:Proto-Oncogene Proteins c-akt; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3