RESUMO
Neuroblastoma is a neural crest-derived embryonal tumour of the postganglionic sympathetic nervous system and a disease with several different chromosomal gains and losses, which include MYCN-amplified neuroblastoma on chromosome 2, deletions of parts of the chromosomes 1p and 11q, gain of parts of 17q and triploidy. Recently, activating mutations of the ALK (Anaplastic Lymphoma Kinase) RTK (Receptor Tyrosine Kinase) gene have been described in neuroblastoma. A meta-analysis of neuroblastoma cases revealed that ALK mutations (49 of 709 cases) in relation to genomic subtype were most frequently observed in MYCN amplified tumours (8.9%), correlating with a poor clinical outcome. MYCN proteins target proliferation and apoptotic pathways, and have an important role in the progression of neuroblastoma. Here, we show that both wild-type and gain-of-function mutants in ALK are able to stimulate transcription at the MYCN promoter and initiate mRNA transcription of the MYCN gene in both neuronal and neuroblastoma cell lines. Further, this stimulation of MYCN gene transcription and de novo MYCN protein expression is abrogated by specific ALK inhibitors, such as crizotinib (PF-2341066), NVP-TAE684, and by small interfering RNA to ALK resulting in a decrease in proliferation rate. Finally, co-transfection of ALK gain-of-function mutations together with MYCN leads to an increase in transformation potential. Taken together, our results indicate that ALK signalling regulates initiation of transcription of the MYCN gene providing a possible explanation for the poor clinical outcome observed when MYCN is amplified together with activated ALK.
Assuntos
Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Cicloeximida/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Humanos , Camundongos , Mutação/genética , Proteína Proto-Oncogênica N-Myc , Células NIH 3T3 , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Células PC12 , Regiões Promotoras Genéticas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transcrição Gênica/efeitos dos fármacosRESUMO
Many different types of cancer originate from aberrant signaling from the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), arising through different translocation events and overexpression. Further, activating point mutations in the ALK domain have been recently reported in neuroblastoma. To characterize signaling in the context of the full-length receptor, we have examined whether ALK is able to activate Rap1 and contribute to differentiation/proliferation processes. We show that ALK activates Rap1 via the Rap1-specific guanine-nucleotide exchange factor C3G, which binds in a constitutive complex with CrkL to activated ALK. The activation of the C3G/Rap1 pathway results in neurite outgrowth of PC12 cells, which is inhibited by either overexpression of Rap1GAP or siRNA-mediated knockdown of Rap1 itself or the guanine nucleotide exchange factor C3G. Significantly, this pathway also appears to function in the regulation of proliferation of neuroblastoma cells such as SK-N-SH and SH-SY5Y, because abrogation of Rap1 activity by Rap1-specific siRNA or overexpression of Rap1GAP reduces cellular growth. These results suggest that ALK activation of Rap1 may contribute to cell proliferation and oncogenesis of neuroblastoma driven by gain-of-function mutant ALK receptors.
Assuntos
Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Neuroblastoma/patologia , Proteínas Tirosina Quinases/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quinase do Linfoma Anaplásico , Animais , Proliferação de Células , Transformação Celular Neoplásica , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Ativadoras de GTPase/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Fator 2 de Liberação do Nucleotídeo Guanina/deficiência , Fator 2 de Liberação do Nucleotídeo Guanina/genética , Humanos , Camundongos , Neuritos/metabolismo , Neuroblastoma/genética , Proteínas Nucleares/metabolismo , Células PC12 , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Ratos , Receptores Proteína Tirosina Quinases , Proteínas rap1 de Ligação ao GTP/deficiência , Proteínas rap1 de Ligação ao GTP/genéticaAssuntos
Transformação Celular Neoplásica , Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia , Tirosina Quinase 3 Semelhante a fms/fisiologia , Animais , Linhagem Celular , Proliferação de Células , Humanos , Camundongos , Camundongos Endogâmicos C3H , RNA Interferente Pequeno/genética , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidoresRESUMO
Two new prenylflavonol glycosides epimedokoreanoside I ( 7) and epimedokoreanoside II ( 8) were isolated from EPIMEDIUM KOREANUM Nakai (Berberidaceae) beside five other known prenylflavonol glycosides with hypotensive activity. The structures were elucidated by spectroscopic and chemical methods.
