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
Since the establishment of a protocol for molecular analysis of hereditary medullary thyroid carcinoma (MTC) in southern Brazil, in 1997, 17 independent families with RET germline mutation have been identified. Because neither molecular diagnosis nor the pentagastrin test were available before the establishment of this protocol, we had the opportunity to observe a large number of patients in whom the disease has evolved naturally without medical intervention, namely prophylactic thyroidectomy. We observed a wide spectrum in terms of clinical presentation and natural course of the disease even among genetically related individuals. Sixty-nine individuals from 12 different families presented a codon 634 mutation, the most prevailing missense mutation in our series. The specific mutations identified were C634Y (n = 49), C634R (n = 13), and C634W (n = 7). Individuals with the C634R mutation presented significantly more distant metastases at diagnosis than subjects with the C634Y or C634W mutations (54.5% vs. 19.4% vs. 14.3%, respectively, P = 0.03). Further analysis of the estimated cumulative frequency of lymph node and/or distant metastases by Kaplan-Meier curves showed that the appearance of lymph nodes and metastases occurred later in patients with C634Y than in those with C634R (P = 0.001). Our results suggest that specific nucleotide and amino acid exchanges at codon 634 might have a direct impact on tumor aggressiveness in MEN 2A syndrome.