Abstract
Abstract Melanoma is the most aggressive skin cancer due to its high resistance to currently available therapy. Like other kinds of cancer, melanoma presents changes in DNA methylation and epigenetic machinery such as Dnmt1, however it is still unclear why tumor cells acquire these epigenetic alterations. The aim of this study was to analyze the mechanisms involved in Dnmt1 transcriptional regulation by the transcription factor E2F1 in melanoma progression. The cell model used in this study was previously developed in our laboratory, submitting non-tumorigenic murine melanocytes (melan-a) to successive cycles of anchorage blockade. A linear cell model for melanoma was obtained consisting in pre-malignant (1C and 4C), tumorigenic non-metastatic (4C11-) and metastatic (4C11+) cell lines. Bioinformatics data show the existence of four transcripts for Dnmt1 in mice. The transcripts 1 and 2 have the same promoter, and the transcripts 3 and 4 share a different promoter. Real time PCR showed the absence of transcript 3 in all cell lines analyzed. The transcript 4 was present in all lineages, but no significant change in its expression was observed in any one compared to melan- a melanocytes. Transcripts 1 and 2 are present and their levels are increased in 1C, 4C, 4C11- and 4C11+ compared to melan-a cells. Chromatin immunoprecipitation (ChIP) showed increased binding of E2F1 in Dnmt1 promoter in 1C, 4C, 4C11- and 4C11+ compared to melan-a cells. Interestingly, the intensity of binding was stronger in the first promoter, which regulates transcripts 1 and 2, than in the second promoter, which regulates transcripts 3 and 4. These results show for the first time that E2F1 may have an important role in the transcription regulation of Dnmt1 during melanoma progression. Additionally, we observed the increase in superoxide anion (O2.-) in 1C compared to melan-a cells. Recently published data from our group showed that depleting O2.- with MnTBAP scavenger during melan-a anchorage blockade cycles, the malignant transformation is drastically compromised. Here, we showed that 1C cells treated with MnTBAP do not have Dnmt1 mRNA and protein increase compared to melan-a cells. Furthermore, E2F1 levels seem to be regulated by O2.- in 1C cells, indicating that O2.- can be a possible mediator for E2F1 regulation and Dnmt1 transcriptional regulation in melanoma progression. Together, these data suggest an important role of oxidative stress and epigenetic mechanisms in the first steps of melanoma progression. Supported by FAPESP and CNPq. Citation Format: CAMILA T. DA SILVA, Fernanda Molognoni, Fabiana H. M. de Melo, Miriam Galvonas Jasiulionis. Transcriptional regulation of dnmt1 by E2F1 in melanoma progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 426. doi:10.1158/1538-7445.AM2014-426
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