Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance

Abstract

Differences in the global methylation pattern, ie hyper- as well as hypo-methylation, are observed in cancers including germ cell tumours (GCTs). Related to their precursor cells, GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n = 103) and GCTs (n = 251) by immunohistochemical staining for 5-(m)cytidine. The global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, whereas female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) are hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumours, and choriocarcinomas) show a higher degree of methylation. Embryonal carcinomas show an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam-2 before and after demethylation using 5-azacytidine. Exposure to 5-azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell-specific marker VASA, showed increased expression. Following treatment with 5-azacytidine, TCam-2 cells were analysed using a high-throughput methylation screen for changes in the methylation sites of 14,000 genes. Among the genes revealing changes, interesting targets were identified: ie demethylation of KLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs.

TDRG1 regulates chemosensitivity of seminoma TCam-2 cells to cisplatin via PI3K/Akt/mTOR signaling pathway and mitochondria-mediated apoptotic pathway

“…1A. The concentration from the N.C. cells in this study was similar with the IC50 of CDDP for 72 h in TCam-2 cells reported by Wermann H et al 22 It was reported that PI3K signaling plays a critical role in the occurrence and development of seminoma. 15 Interestingly, we found that LY294002, a PI3K signaling inhibitor, re-sensitized TCam-2 cells with TDRG1 overexpression to CDDP, while IGF-1, a PI3K activator, reversed the positive effect of TDRG1 knockdown on the chemosensitivity of TCam-2 cells to CDDP (Fig.…”

Role of epigenetics in the etiology of germ cell cancer

“…It has been shown that global methylation status of GCC subtypes differ according to the time point of their developmental arrest; more differentiated cells showed a higher degree of methylation (Okamoto and Kawakami, 2007, Smiraglia et al, 2002, Wermann et al, 2010. It is also known that CIS/GB cells show very little DNA methylation (Netto et al, 2008, Smiraglia et al, 2002, Wermann et al, 2010.…”

“…Subsequent analysis of CpG island methylation showed that there were different methylation profiles in the treated and non-treated cells. For example, the promoter region of the CFLAR (c-FLIP) gene was hypermethylated in the treated cell line (Wermann et al, 2010). CFLAR has an important role in regulation of apoptosis via the caspase pathway and therefore could be a therapeutic target (Yang, 2008), something that needs further evaluation.…”

“…Teratomas, which are the most differentiated GCC, show the highest degree of methylation and are cisplatin resistant (Krege et al, 2008, Mayer et al, 2003. In addition, cisplatin-resistant GCC showed different overall methylation profiles than non -resistant forms (Koul et al, 2004, Netto et al, 2008, Wermann et al, 2010. Hypermethylation might therefore be a diagnostic marker as well as a predictive marker of treatment response.…”

DNA methylation in germ cell tumour aetiology: current understanding and outstanding questions

“…This conclusion is based on studies using both candidate gene approaches (Koul et al 2002, Smith-Sorensen et al 2002, Honorio et al 2003, Kempkensteffen et al 2006, Lind et al 2006, Brait et al 2012) and genome-wide approaches (Smiraglia et al 2002, Netto et al 2008, Wermann et al 2010, which have mainly focused on adult testicular GCTs (Table 2). Although some of these studies focused on a limited number of genes, microarray and genome-wide studies confirm that the methylation profile distinction between seminomas and non-seminomas is not restricted to just a few specific gene promoters (Lind et al 2007).…”