Deregulated Genes By Abnormal Promoter Methylation In Early And Late Stages In A Linear Model Of Melanoma Progression

Abstract Metastatic melanomas are extremely aggressive tumors, accounting for 70% of skin cancer deaths, which incidence is increasing globally. Despite the advances in their treatment, metastatic melanomas are still associated with a poor prognosis, with a median survival of 6 to 12 months. The study of the molecular alterations affecting key pathways during melanoma progression may contribute to a better understanding of the biology underlying the aggressiveness of the disease and lead to the identification of biomarkers for prognosis and novel targets for therapy. In addition to mutations, epigenetic alterations are important contributors to malignant transformation and tumor progression. Aberrant DNA methylation is an epigenetic hallmark of cancer, known to play important roles in melanoma development. Although different genes were described as presenting methylation alterations in their promoters in melanomas, the dynamics of these alterations along melanocyte malignant transformation and melanoma progression is still unclear. Our laboratory developed a linear model of melanoma progression in which different pre-malignant and melanoma cell lines were established after subjecting murine melanocytes to a sustained stressful condition (adhesion impediment). In this study, we used the spontaneously immortalized melanocyte lineage (melan-a); pre-malignant melanocytes (4C), obtained after subjecting melan-a cells to 4 cycles of adhesion impediment; a non-metastatic tumorigenic cell line (4C11-), obtained after a limiting dilution of spheroids formed by 4C adhesion impediment; and a metastatic cell line (4C11+), resulting from the spontaneously loss of p53 by 4C11- cells. The aim of this work was to identify genes that have their expression altered by promoter and gene body methylation that are related to the malignant transformation of melanocytes and metastasis. Through the integrative analysis of methylome (ERRBS) with transcriptome (RNAseq) data, it was possible to identify the dynamic changes in DNA methylation in promoters and gene bodies, and their relation with gene expression in both early and late stages of melanoma progression. Among those genes are Foxd1, Lrrk2, Adcy1 and Adcy3, which had their expression validated by qPCR, and their epigenetic regulation validated by the treatment of the cells with epigenetic drugs (5-aza-2'-deoxycytidine and Trichostatin A). The gene expression and promoter methylation profiles found in the murine model of melanoma progression were correlated with clinical data from melanoma patients available in cancers cohorts, revealing potential novel prognostic markers. Supported by FAPESP, CNPQ and CAPES Citation Format: Débora Papaiz, Flávia E. Rius, Diogo Pessoa, Eduardo M. Reis, Christopher E. Mason, Gangning Liang, Miriam G. Jasiulionis. Deregulated genes by abnormal promoter methylation in early and late stages in a linear model of melanoma progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 140.