Project description:Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. The initial lesion is believed to be the relatively benign precursor lesion (ICGNU), from which either highly chemosensitive seminomas or the more aggressive non-seminomas develop. Previous analyses of selected genes have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. However, 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 tumoursâ biology have not yet been established. Here genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data. This demonstrated that the seminoma cells exhibited very little aberrant DNA methylation while non-seminoma cells exhibited very high levels of DNA methylation. 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 did indeed play a role in their silencing and many of these genes were also differentially expressed in primary tumours. Through this approach the genes silenced in the various GCT cell lines were identified. Conclusions: Several pluripotency-associated genes, never before implicated in this type of cancer, were identified as a major functional group of silenced genes. Silencing of these factors that normally suppress somatic differentiation might play an important role in the progression to non-seminoma formation. Genomic DNA and RNA was extracted from cell lines representing four subtypes of GCT. RNA was subjected to Affymetric expression array analysis while DNA was bisulfite treated and analysed using Illumina Infinium 450K arrays. Statistical approaches were used to correlate methylation and expression for each gene.

Project description:Germ cell tumors (GCT) originate from germ cells at different developmental stages (GCT type I – V) They are thought to inherit their methylation profile from (early embryonic) germ cells which undergo a characteristic epigenetic “reset” during maturation. This study aims to provide insight into the developmental timing and underlying biology of the various subtypes of GCTs and their (embryonic) cells of origin by identifying specific and global methylation differences between GCT subtypes. In total, 91 type I-IV GCTs and four cell lines were profiled using the Illumina HumanMethylation450 BeadChip (450K) platform. The data were pre-processed using a validated pipeline and analyzed using an in-house generated extension of the annotation manifest. Marked methylation differences were identified between GCT subtypes both on the global and local level (i.e. differentially methylated regions, imprinting control regions, promoters, etc). GCT subtypes indeed show major similarities to specific stages of (early) developing embryonic germ cells with regard to their methylation profile. The extended annotation manifest for the Illumina 450K platform and methylation datasets are readily available on GEO (GEO accession: GSE58538, GPL18809), providing an integrated hypothesis generating data source for future research. 91 germ cell tumors of various histologies were investigated for their methylation profile. Methylation detection was performed using Illumina’s HumanMethylation450 BeadChip. 14 type I teratomas (TE) were included of which 6 were located sacral (3 male: I.TE.s.m; 3 female: I.TE.s.f), 4 were located in the testis (I.TE.t) and 3 were located in the ovary (I.TE.o). 30 pure embryonal carcinomas (EC) were included as well as 21 mixed non seminomas (mNS). 12 seminomas of the testis (SE) were included as well as 4 dysgerminomas (their couterpart in the ovary). Finally, 4 spermatocytic seminomas (SS) and three dermoid cysts were analyzed. For a detailed overview the histological classification of germ cell tumors please see the publication linked to this data or associated literature [1-3]. Four cell lines were included, al modelling type II GCTs. Cell lines derived from EC include NT2[4-8], NCCIT [5, 9] and 2102EP[4-8]. TCam-2 closely resembles SE [10-12]. 1. Oosterhuis, J.W. and L.H. Looijenga, Testicular germ-cell tumours in a broader perspective. Nat Rev Cancer, 2005. 5(3): p. 210-22. 2. Looijenga, L.H., Human testicular (non)seminomatous germ cell tumours: the clinical implications of recent pathobiological insights. J Pathol, 2009. 218(2): p. 146-62. 3. Woodward, P.J., et al., Testicular germ cell tumors, in World Health Organization Classification of Tumours Pathology and Genetics of the Urinary System and Male Genital Organs., J.N. Eble, et al., Editors. 2004, IARC Press: Lyon. p. 17-278. 4. Andrews, P.W., et al., A comparative study of eight cell lines derived from human testicular teratocarcinoma. Int J Cancer, 1980. 26(3): p. 269-80. 5. Andrews, P.W., et al., Comparative analysis of cell surface antigens expressed by cell lines derived from human germ cell tumours. Int J Cancer, 1996. 66(6): p. 806-16. 6. Wang, N., et al., Nonrandom abnormalities in chromosome 1 in human testicular cancers. Cancer Res, 1980. 40(3): p. 796-802. 7. Fogh, J. and G. Trempe, New Human Tumor Cell Lines, in Human Tumor Cells in Vitro, J. Fogh, Editor. 1975, Springer US. p. 115-159. 8. Fogh, J., Cultivation, characterization, and identification of human tumor cells with emphasis on kidney, testis, and bladder tumors. Natl Cancer Inst Monogr, 1978(49): p. 5-9. 9. Teshima, S., et al., Four new human germ cell tumor cell lines. Lab Invest, 1988. 59(3): p. 328-36. 10. Eckert, D., et al., TCam-2 but not JKT-1 cells resemble seminoma in cell culture. Cell Tissue Res, 2008. 331(2): p. 529-38. 11. de Jong, J., et al., Further characterization of the first seminoma cell line TCam-2. Genes Chromosomes Cancer, 2008. 47(3): p. 185-96. 12. Mizuno, Y., et al., [Establishment and characterization of a new human testicular germ cell tumor cell line (TCam-2)]. Nihon Hinyokika Gakkai Zasshi, 1993. 84(7): p. 1211-8.

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Genome-wide methylation analysis and gene expression profiling identifies genes silenced in non-seminoma cell lines [methylation]

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