Genome-wide methylation analysis identifies genes silenced in non-seminoma cell lines.

Dzul Azri Mohamed Noor,Janet Shipley,Jennie N Jeyapalan,Matthew Carr,Tom Reader,Benjamin Squibb,Paul J Scotting,Safiah Alhazmi,Jaime Hughes,Claire Wallace,Christopher Tan,Martin Cusack,Denise Sheer

doi:10.1038/npjgenmed.2015.9

Abstract

Silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the genome-wide effect of DNA methylation on gene expression has been analysed in relatively few cancers. Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. Previous analyses have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. The genomic targets that are methylated, the extent to which this results in gene silencing and the identity of the silenced genes most likely to play a role in the tumours’ biology have not yet been established. In this study, genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data from primary tumours to address this question. Genome methylation was analysed using the Illumina infinium HumanMethylome450 bead chip system and gene expression was analysed using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Regulation by methylation was confirmed by demethylation using 5-aza-2-deoxycytidine and reverse transcription–quantitative PCR. Large differences in the level of methylation of the CpG islands of individual genes between tumour cell lines correlated well with differential gene expression. Treatment of non-seminoma cells with 5-aza-2-deoxycytidine verified that methylation of all genes tested played a role in their silencing in yolk sac tumour cells and many of these genes were also differentially expressed in primary tumours. Genes silenced by methylation in the various GCT cell lines were identified. Several pluripotency-associated genes were identified as a major functional group of silenced genes.

Highlights

Promoter hypermethylation of many different tumour suppressor genes is seen in a wide range of cancers.[1,2] This has been assumed, though only occasionally demonstrated, to silence the expression of those genes
Rijlarsdaam et al.[17] analysed methylation in cell lines derived from multiple types of Germ cell tumours (GCTs) but they did not determine the Relationships between genes methylated in different GCT subtypes relationship of this methylation to gene expression, while van der Zwan et al.[18] analysed both methylation and gene expression, but only in seminoma versus embryonal carcinomas (EC) cell lines
DNA methylation was analysed in four adult GCT-derived cell lines (TCam-2, GCT44, GCT27 and NT2D1, subsequently referred to as Seminoma, yolk sac tumours (YSTs), EC and Teratoma cell lines, respectively) on a

Summary

Introduction

Promoter hypermethylation of many different tumour suppressor genes is seen in a wide range of cancers.[1,2] This has been assumed, though only occasionally demonstrated, to silence the expression of those genes. Despite high cure rates in response to platinum-based chemotherapy, they still represent a fatal disease in a minority of patients presenting with disseminated disease[8,9] and the prognosis in children is much worse than in adults.[10] GCTs are an exceptional group of tumours in many respects. They are the only class of cancer that arises from a pluripotent progenitor cell (the germ cell progenitor, PGC) and that cell exhibits profoundly different DNA methylation characteristics to all somatic cell types. Non-seminomatous tumours, such as yolk sac tumours (YSTs) and embryonal carcinomas (EC), tend to be more aggressive and resistant to therapy than seminomatous tumours,[8,9,11] especially in intracranial cases seen in children.[10]

Methods

Results

Discussion

Conclusion