RESUMO
Pigment cells and neuronal cells both are derived from the neural crest. Here, we describe the Pit-Oct-Unc (POU) domain transcription factor Brn3a, normally involved in neuronal development, to be frequently expressed in melanoma, but not in melanocytes and nevi. RNAi-mediated silencing of Brn3a strongly reduced the viability of melanoma cell lines and decreased tumour growth in vivo. In melanoma cell lines, inhibition of Brn3a caused DNA double-strand breaks as evidenced by Mre11/Rad50-containing nuclear foci. Activated DNA damage signalling caused stabilization of the tumour suppressor p53, which resulted in cell cycle arrest and apoptosis. When Brn3a was ectopically expressed in primary melanocytes and fibroblasts, anchorage-independent growth was increased. In tumourigenic melanocytes and fibroblasts, Brn3a accelerated tumour growth in vivo. Furthermore, Brn3a cooperated with proliferation pathways such as oncogenic BRAF, by reducing oncogene-induced senescence in non-malignant melanocytes. Together, these results identify Brn3a as a new factor in melanoma that is essential for melanoma cell survival and that promotes melanocytic transformation and tumourigenesis.
Assuntos
Pontos de Checagem do Ciclo Celular , Fator de Transcrição Brn-3A/metabolismo , Apoptose , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Transformação Celular Neoplásica , Senescência Celular , Quebras de DNA de Cadeia Dupla , Humanos , Melanócitos/citologia , Melanócitos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição Brn-3A/antagonistas & inibidores , Fator de Transcrição Brn-3A/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
A variety of genetic lesions, including chromosomal translocations, internal tandem duplications, and mutations, have been described in acute myeloid leukemia (AML). Expression profiling has shown that chromosomal translocations, in particular, are associated with distinctive patterns of gene expression. AML exhibiting the translocation t(8;21), which fuses the AML1 and ETO genes, has such a characteristic expression profile. One gene whose expression is highly correlated with the presence of the AML1/ETO fusion is POU4F1, which encodes the POU homeodomain transcription factor BRN3A. Here we show using specific siRNA in t(8;21) cells and overexpression studies in progenitor cells that AML1/ETO promotes expression of POU4F1/BRN3A. This effect requires DNA-binding function of AML1/ETO, and accordingly, AML1/ETO is bound to the POU4F1 locus in t(8;21) cells. Functionally, whereas overexpression of Brn3a in murine hematopoietic progenitor cells induces terminal myeloid differentiation, coexpression of AML1/ETO or AML1/ETO9a blocks this effect. Furthermore, Brn3a reduction by shRNA impairs AML1/ETO-induced immortalization of murine progenitors. In summary, we identify POU4F1/BRN3A as a novel potential upregulated AML1/ETO target gene whose dramatically high expression may cooperate with AML1/ETO in t(8;21) cells.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição Brn-3A/metabolismo , Adulto , Animais , Sequência de Bases , Western Blotting , Diferenciação Celular , Imunoprecipitação da Cromatina , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Ensaio de Desvio de Mobilidade Eletroforética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Feto , Imunofluorescência , Células-Tronco Hematopoéticas/fisiologia , Humanos , Técnicas Imunoenzimáticas , Leucemia Mieloide Aguda/patologia , Fígado/citologia , Fígado/metabolismo , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Proteína 1 Parceira de Translocação de RUNX1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Brn-3A/antagonistas & inibidores , Fator de Transcrição Brn-3A/genética , Transfecção , Translocação Genética/genéticaRESUMO
Osteoclastic bone resorption is a central mechanism in skeletal development, remodeling and pathology. RANKL is a mandatory factor controlling osteoclastogenesis; however, the underlying signaling pathways are only partially characterized. Using a screening array for the investigation of differential transcription factor activation, we identified activation of the Brn3 transcription factor family as a downstream event of RANKL signaling during terminal osteoclastogenesis. RANKL stimulation induces expression of Brn3a and b and maximal transcriptional activity of Brn3 family members concurrent with osteoclastic giant cell formation. Immunohistochemical analysis revealed both nuclear and cytoplasmic localization of Brn3a and b in mature osteoclasts. Functional inhibition of Brn3 transcription factors resulted in inhibition of pre-osteoclast fusion and reduction in bone resorbing activity of mature osteoclasts. Furthermore, we identified synaptotagmin-1, a regulator of membrane and vesicular fusion, as downstream target of Brn3 with a role in osteoclast function. We conclude that Brn-3 represents a novel molecular differentiation factor that controls osteoclast maturation and function, suggesting an important role in bone metabolism.
