title: Genome-wide methylation sequencing of paired primary and metastatic cell lines identifies common DNA methylation changes and a role for EBF3 as a candidate epigenetic driver of melanoma metastasis.
abstract: Epigenetic alterations are increasingly implicated in metastasis, whereas very few genetic mutations have been identified as authentic drivers of cancer metastasis. Yet, to date, few studies have identified metastasis-related epigenetic drivers, in part because a framework for identifying driver epigenetic changes in metastasis has not been established. Using reduced representation bisulfite sequencing (RRBS), we mapped genome-wide DNA methylation patterns in three cutaneous primary and metastatic melanoma cell line pairs to identify metastasis-related epigenetic drivers. Globally, metastatic melanoma cell lines were hypomethylated compared to the matched primary melanoma cell lines. Using whole genome RRBS we identified 75 shared (10 hyper- and 65 hypomethylated) differentially methylated fragments (DMFs), which were associated with 68 genes showing significant methylation differences. One gene, Early B Cell Factor 3 (EBF3), exhibited promoter hypermethylation in metastatic cell lines, and was validated with bisulfite sequencing and in two publicly available independent melanoma cohorts (n = 40 and 458 melanomas, respectively). We found that hypermethylation of the EBF3 promoter was associated with increased EBF3 mRNA levels in metastatic melanomas and subsequent inhibition of DNA methylation reduced EBF3 expression. RNAi-mediated knockdown of EBF3 mRNA levels decreased proliferation, migration and invasion in primary and metastatic melanoma cell lines. Overall, we have identified numerous epigenetic changes characterising metastatic melanoma cell lines, including EBF3-induced aggressive phenotypic behaviour with elevated EBF3 expression in metastatic melanoma, suggesting that EBF3 promoter hypermethylation may be a candidate epigenetic driver of metastasis.
authors.affiliation: Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand.|Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, New Zealand.
authors.forename: Aniruddha
authors.initials: A
authors.lastname: Chatterjee
authors.affiliation: Department of Biochemistry, University of Otago, Dunedin, New Zealand.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Stockwell
authors.affiliation: Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand.
authors.forename: Antonio
authors.initials: A
authors.lastname: Ahn
authors.affiliation: Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand.|Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, New Zealand.
authors.forename: Euan J
authors.initials: EJ
authors.lastname: Rodger
authors.affiliation: Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand.
authors.forename: Anna L
authors.initials: AL
authors.lastname: Leichter
authors.affiliation: Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand.|Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, New Zealand.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Eccles
mesh_terms: D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008545:Melanoma / Q000235:genetics*; D009362:Neoplasm Metastasis; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D012878:Skin Neoplasms / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D015854:Up-Regulation; D000096142:Cutaneous Malignant Melanoma
chemical_list: C000605678:EBF3 protein, human; D014157:Transcription Factors
keywords: DNA methylation; bisulfite sequencing; cancer epigenome; melanoma; metastasis