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.

Aniruddha Chatterjee,Michael R Eccles,Anna L Leichter,Peter A Stockwell,Antonio Ahn,Euan J Rodger

doi:10.18632/oncotarget.14042

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.

Highlights

Cutaneous melanoma is a highly aggressive malignancy that originates from melanocytes and accounts for 75% of skin cancer related deaths [1]
To identify epigenetic changes occurring in metastatic melanoma, compared to the primary tumor of origin, we profiled whole genome methylation in three primary cutaneous melanoma and three matched metastatic melanoma cell lines
The primary aim of our study was to identify candidate “driver” DNA methylation changes, using pairs of matched primary and metastatic melanoma cell lines, focusing on metastasis-related changes, while at the same time minimizing the detection of random epigenetic differences resulting from inter-patient or intra-tumoral cellular heterogeneity

Summary

Introduction

Cutaneous melanoma is a highly aggressive malignancy that originates from melanocytes and accounts for 75% of skin cancer related deaths [1]. Globally cancer related deaths have decreased over the last two decades, the death rate from melanoma continues to increase [2]. Better understanding of melanoma metastasis is important to develop treatments to inhibit metastasis. Remarkable progress has been made over the last two decades in understanding the genetic basis of melanoma tumorigenesis. Very few mutations have been identified that play a key role in contributing to metastasis [6]. Melanoma is strongly predisposed to metastasis, but relatively little is known about the role that epigenetic changes play during the metastatic processes, during which the tumor cells acquire new properties [7]

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