Regulação transcricional do gene Dnmt1 causada pelo bloqueio de ancoragem de melanócitos e o papel de Dnmt1 na progressão do melanoma
Data
2015-09-30
Autores
Silva, Camila Tainah da 
Orientadores
Jasiulionis, Miriam Galvonas
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A metilação aberrante do DNA é um dos mecanismos relacionados à transformação maligna de diversos tipos de tumor. Uma das principais enzimas envolvidas na maquinaria de metilação do DNA é a Dnmt1, que tem como papel principal a manutenção do perfil de metilação de célula-mãe para célula-filha durante o processo de mitose. Em diversos tipos de tumor, a Dnmt1 pode ter expressão alterada por mecanismos ainda não completamente compreendidos. O objetivo deste trabalho foi determinar se o aumento de Dnmt1 durante a transformação maligna de melanócitos, ocasionada por ciclos sequenciais de bloqueio de ancoragem, é decorrente da sua regulação transcricional pelos fatores de transcrição E2F1 e c-Jun. Os dados obtidos mostram que o fator de transcrição E2F1 encontra-se ligado ao promotor da Dnmt1 em melanócitos pré-malignos 1C e 4C e nas linhagens de melanoma 4C11- e 4C11+, mas não na linhagem parental de melanócitos melan-a. Em contrapartida, nossas análises demonstram que o fator de transcrição c-Jun está ligado ao promotor de Dnmt1 apenas nas linhagens tumorais 4C11- e 4C11+. Em paralelo, avaliamos o papel de Dnmt1 na progressão do melanoma. A diminuição na expressão de Dnmt1 (shRNA) fez com que as linhagens 4C, 4C11- e 4C11+ resistissem menos ao anoikis. Na linhagem de melanoma metastático 4C11+, o silenciamento de Dnmt1 resultou ainda na diminuição da capacidade clonogênica e tumoral in vivo, enquanto que na linhagem 4C há o aumento da capacidade clonogênica, indicando que Dnmt1 pode ter diferentes papeis dependendo da fase da progressão do melanoma. Identificamos 33 alvos no genoma onde Dnmt1 se associa diferencialmente nessas duas linhagens, podendo ser a chave para explicar o fenômeno observado. Este trabalho contribuiu para o entendimento da regulação e do papel de Dnmt1 na progressão do melanoma, que pode ser fundamental para esclarecer a importância da epigenética nesta doença.
Aberrant DNA methylation is a mechanism related to malignant transformation in many cancer types. One of the key enzymes involved in DNA methylation machinery is Dnmt1, which plays a main role in the maintenance of methylation pattern from parent to daughter cells during mitosis. In many types of cancer, Dnmt1 has its expression increased by not fully understood mechanisms. The aim of this study was to determine whether the increase of Dnmt1 during malignant transformation of melanocytes, caused by sequential deadhesion cycles, occurs due to its transcriptional regulation by E2F1 and c-Jun transcription factors. The data obtained shows that E2F1 transcription factor is linked to Dnmt1 promoter in pre-malignant melanocytes 1C and 4C and in malignant melanomas 4C11- and 4C11+ cell lines but not in parental melanocytes melan-a. In contrast, the transcription factor c-Jun is linked to Dnmt1 promoter only in 4C11- and 4C11+ tumor cell lines. In parallel, we evaluated the role of Dnmt1 in melanoma progression. The decrease of Dnmt1 expression (by shRNA) made 4C, 4C11- and 4C11+ cell lines less resistant to anoikis. In metastatic melanoma cells 4C11+, Dnmt1 decrease also reduced clonogenic and tumorigenic capacity in vivo, while in 4C clonogenic capacity was increased, indicating that Dnmt1 may have different roles depending on the stage of melanoma progression. We identified 33 targets in the genome where Dnmt1 is differentially associated in these two cell lines and that may be the key to explain the observed phenomenon. This study contributes to the understanding of the regulation and role of Dnmt1 in melanoma progression, which can be crucial to clarify the importance of epigenetic in this disease.
Aberrant DNA methylation is a mechanism related to malignant transformation in many cancer types. One of the key enzymes involved in DNA methylation machinery is Dnmt1, which plays a main role in the maintenance of methylation pattern from parent to daughter cells during mitosis. In many types of cancer, Dnmt1 has its expression increased by not fully understood mechanisms. The aim of this study was to determine whether the increase of Dnmt1 during malignant transformation of melanocytes, caused by sequential deadhesion cycles, occurs due to its transcriptional regulation by E2F1 and c-Jun transcription factors. The data obtained shows that E2F1 transcription factor is linked to Dnmt1 promoter in pre-malignant melanocytes 1C and 4C and in malignant melanomas 4C11- and 4C11+ cell lines but not in parental melanocytes melan-a. In contrast, the transcription factor c-Jun is linked to Dnmt1 promoter only in 4C11- and 4C11+ tumor cell lines. In parallel, we evaluated the role of Dnmt1 in melanoma progression. The decrease of Dnmt1 expression (by shRNA) made 4C, 4C11- and 4C11+ cell lines less resistant to anoikis. In metastatic melanoma cells 4C11+, Dnmt1 decrease also reduced clonogenic and tumorigenic capacity in vivo, while in 4C clonogenic capacity was increased, indicating that Dnmt1 may have different roles depending on the stage of melanoma progression. We identified 33 targets in the genome where Dnmt1 is differentially associated in these two cell lines and that may be the key to explain the observed phenomenon. This study contributes to the understanding of the regulation and role of Dnmt1 in melanoma progression, which can be crucial to clarify the importance of epigenetic in this disease.
Descrição
Citação
SILVA, Camila Tainah da. Regulação transcricional do gene Dnmt1 causada pelo bloqueio de ancoragem de melanócitos e o papel de Dnmt1 na progressão do melanoma. 2015. 99 f. Tese (Doutorado em Microbiologia e Imunologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.