RESUMO
BACKGROUND: We have previously described a p.G533C substitution in the rearranged during transfection (RET) oncogene in a large family with medullary thyroid carcinoma. Here, we explore the functional transforming potential of RET p.G533C mutation. METHODS: Plasmids expressing RET mutants (p.G533C and p.C634Y) and RET wild type were stable transfected into a rat thyroid cell line (PCCL3). Biological and biochemical effects of RET p.G533C were investigated both in vitro and in vivo. Moreover, we report the first case of pheochromocytoma among the RET p.G533C-carriers in this Brazilian family and explore the RET mutational status in DNA isolated from pheochromocytoma. RESULTS: Ectopic expression of RET p.G533C and p.C634Y activates RET/MAPK/ERK pathway at similar levels and significantly increased cell proliferation, compared with RET wild type. We additionally show that p.G533C increased cell viability, anchorage-independent growth, and micronuclei formation while reducing apoptosis, hallmarks of the malignant phenotype. RET p.G533C down-regulates the expression of thyroid specific genes in PCCL3. Moreover, RET p.G533C-expressing cells were able to induce liver metastasis in nude mice. Finally, we described two novel RET variants (G548V and S556T) in the DNA isolated from pheochromocytoma while they were absent in the DNA isolated from blood. CONCLUSIONS: Our in vitro and in vivo analysis indicates that this mutation confers a malignant phenotype to PCCL3 cells. These findings, in association with the report of first case of pheochromocytoma in the Brazilian kindred, suggest that this noncysteine mutation may be more aggressive than was initially considered.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Transformação Celular Neoplásica/genética , Neoplasia Endócrina Múltipla Tipo 2a/genética , Mutação , Síndromes Neoplásicas Hereditárias/genética , Feocromocitoma/genética , Proteínas Proto-Oncogênicas c-ret/genética , Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/genética , Adolescente , Neoplasias das Glândulas Suprarrenais/enzimologia , Neoplasias das Glândulas Suprarrenais/patologia , Análise de Variância , Animais , Apoptose , Brasil , Carcinoma Medular/congênito , Adesão Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Camundongos Nus , Micronúcleos com Defeito Cromossômico , Pessoa de Meia-Idade , Neoplasia Endócrina Múltipla Tipo 2a/enzimologia , Neoplasia Endócrina Múltipla Tipo 2a/secundário , Síndromes Neoplásicas Hereditárias/enzimologia , Linhagem , Fenótipo , Feocromocitoma/enzimologia , Feocromocitoma/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-ret/metabolismo , Ratos , Ratos Endogâmicos F344 , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/secundário , TransfecçãoRESUMO
CONTEXT: We previously described a six-generation family with G533C RET mutation and medullary thyroid carcinoma, in the largest family reported do date. Of particular interest, phenotype variability regarding the age of onset and clinical presentation of the disease, was observed. OBJECTIVE: We evaluate whether single SNPs within RET oncogene or haplotype comprising the RET variants (defined by Haploview) could predispose to early development of MTC in this family and influence the clinical manifestation. DESIGN: Eight SNPs were selected based on their previous association with the clinical course of hereditary or sporadic MTC, in particular promoting an early onset of disease. The variants were initially tested in 77 G533C-carriers and 100 controls using either PCR-direct sequencing or PCR-RFLP. Association between a SNP or haplotype and age at diagnosis or presence of lymph node metastasis was tested in 34 G533C-carries with MTC. Different bioinformatic tools were used to evaluate the potential effects on RNA splicing. RESULTS: An association was found between IVS1-126G > T and age at diagnosis. The variant [IVS8 +82A > G; 85-86 insC] was associated with the presence of lymph node metastases at diagnosis. In silico analysis suggested that this variant may induce abnormal splicing. This in silico analysis predicted that the [IVS8 +82A > G; 85-86 insC] could alter the splicing by disrupting and/or creating exonic splicing enhancer motifs. CONCLUSIONS: We here identified two RET variants that were associated with phenotype variability in G533C-carriers, which highlights the fact that the modifier effect of a variant might depend on the type of mutation.