Transcription factor TFAP2C regulates major programs required for murine fetal germ cell maintenance and haploinsufficiency predisposes to teratomas in male mice.

Maintenance and maturation of primordial germ cells is controlled by complex genetic and epigenetic cascades, and disturbances in this network lead to either infertility or malignant aberration. Transcription factor TFAP2C has been described to be essential for primordial germ cell maintenance and to be upregulated in several human germ cell cancers. Using global gene expression profiling, we identified genes deregulated upon loss of Tfap2c in embryonic stem cells and primordial germ cell-like cells. We show that loss of Tfap2c affects many aspects of the genetic network regulating germ cell biology, such as downregulation of maturation markers and induction of markers indicative for somatic differentiation, cell cycle, epigenetic remodeling and pluripotency. Chromatin-immunoprecipitation analyses demonstrated binding of TFAP2C to regulatory regions of deregulated genes (Sfrp1, Dmrt1, Nanos3, c-Kit, Cdk6, Cdkn1a, Fgf4, Klf4, Dnmt3b and Dnmt3l) suggesting that these genes are direct transcriptional targets of TFAP2C in primordial germ cells. Since Tfap2c deficient primordial germ cell-like cells display cancer related deregulations in epigenetic remodeling, cell cycle and pluripotency control, the Tfap2c-knockout allele was bred onto 129S2/Sv genetic background. There, mice heterozygous for Tfap2c develop with high incidence germ cell cancer resembling human pediatric germ cell tumors. Precursor lesions can be observed as early as E16.5 in developing testes displaying persisting expression of pluripotency markers. We further demonstrate that mice with a heterozygous deletion of the TFAP2C target gene Nanos3 are also prone to develop teratomas. These data highlight TFAP2C as a critical and dose-sensitive regulator of germ cell fate.

Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development.

During mammalian development the fertilized zygote and primordial germ cells lose their DNA methylation within one cell cycle leading to the concept of active DNA demethylation. Recent studies identified the TET hydroxylases as key enzymes responsible for active DNA demethylation, catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. Further oxidation and activation of the base excision repair mechanism leads to replacement of a modified cytosine by an unmodified one. In this study, we analyzed the expression/activity of TET1-3 and screened for the presence of 5 mC oxidation products in adult human testis and in germ cell cancers. By analyzing human testis sections, we show that levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are decreasing as spermatogenesis proceeds, while 5-methylcytosine levels remain constant. These data indicate that during spermatogenesis active DNA demethylation becomes downregulated leading to a conservation of the methylation marks in mature sperm. We demonstrate that all carcinoma in situ and the majority of seminomas are hypomethylated and hypohydroxymethylated compared to non-seminomas. Interestingly, 5-formylcytosine and 5-carboxylcytosine were detectable in all germ cell cancer entities analyzed, but levels did not correlate to the 5-methylcytosine or 5-hydroxymethylcytosine status. A meta-analysis of gene expression data of germ cell cancer tissues and corresponding cell lines demonstrates high expression of TET1 and the DNA glycosylase TDG, suggesting that germ cell cancers utilize the oxidation pathway for active DNA demethylation. During xenograft experiments, where seminoma-like TCam-2 cells transit to an embryonal carcinoma-like state DNMT3B and DNMT3L where strongly upregulated, which correlated to increasing 5-methylcytosine levels. Additionally, 5-hydroxymethylcytosine levels were elevated, demonstrating that de novo methylation and active demethylation accompanies this transition process. Finally, mutations of IDH1 (IDH1 (R132)) and IDH2 (IDH2 (R172)) leading to production of the TET inhibiting oncometabolite 2-hydroxyglutarate in germ cell cancer cell lines were not detected.

Establishment of a versatile seminoma model indicates cellular plasticity of germ cell tumor cells.

In western countries, 60% of all malignancies diagnosed in men between 17-45 years of age are germ cell tumors (GCT). GCT arise from the common precursor lesion carcinoma in situ, which transforms within an average of 9 years into invasive Type-II GCTs. Seminomas are considered to be the default developmental pathway of carcinoma in situ cells and the seminoma-like cell line TCam-2 has been used to study seminoma biology in vitro. However, the generation of an animal model, which would allow for the in vivo analysis of seminoma formation, remained elusive. We applied transplantation approaches using TCam-2 cell transfer into ectopic (skin, brain) and orthopic (testis) sites of immunodeficient mice. We demonstrate that a transplantation into the seminiferous tubules results in formation of a carcinoma in situ/seminoma. In contrast, TCam-2 cells adopt an embryonal carcinoma-like fate when grafted to the flank or corpus striatum and display downregulation of the seminoma marker SOX17 and upregulation of the embryonal carcinoma markers SOX2 and CD30. Grafted TCam-2 cells reduce AKT-, ERK-, EphA3-, and Tie2/TEK-signaling to levels comparable to embryonal carcinoma cells. Hence, TCam-2 cell transplantation into the testis generated a carcinoma in situ/seminoma mouse model, which enables addressing the biology of these tumors in vivo. The fact that TCam-2 cells give rise to a carcinoma in situ/seminoma or embryonal carcinoma in a transplantation site specific manner implies that conversion of carcinoma in situ/seminoma to an embryonal carcinoma does not require additional genetic aberrations but relies on signals from the tumor-microenvironment.