Type II germ cell cancers (GCC) can be subdivided into seminomas and non-seminomas. Seminomas are similar to carcinoma in situ (CIS) cells, the common precursor of type II GCCs, with regard to epigenetics and expression, while embryonal carcinomas (EC) are totipotent and differentiate into teratomas, yolk-sac tumors and choriocarcinomas. GCCs can present as seminomas with a non-seminoma component, raising the question if a CIS gives rise to seminomas and ECs at the same time or whether seminomas can be reprogrammed to ECs. In this study, we utilized the seminoma cell line TCam-2 that acquires an EC-like status after xenografting into the murine flank as a model for a seminoma to EC transition and screened for factors initiating and driving this process. Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated. Changes in expression level of 53 genes inversely correlated to changes in DNA methylation. Interestingly, after xenotransplantation 6 genes (GDF3, NODAL, DNMT3B, DPPA3, GAL, AK3L1) were rapidly induced, followed by demethylation of their genomic loci, suggesting that these 6 genes are poised for expression driving the reprogramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling. We propose that reprogramming of seminomas to ECs is a multi-step process. Initially, the microenvironment causes inhibition of BMP signaling, leading to induction of NODAL signaling. During a maturation phase, a fast acting NODAL loop stimulates its own activity and temporarily inhibits BMP signaling. During the stabilization phase, a slow acting NODAL loop, involving WNTs re-establishes BMP signaling and the pluripotency circuitry. In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state.

In Western countries, the incidence of testicular germ cell cancers (GCC) is steadily rising over the last decades. Mostly, men between 20 and 40 years of age are affected. In general, patients suffering from GCCs are treated by orchiectomy and radio- or chemotherapy. Due to resistance mechanisms, intolerance to the therapy or denial of chemo- / radiotherapy by the patients, GCCs are still a lethal threat, highlighting the need for alternative treatment strategies.In this study, we revealed that germ cell cancer cell lines are highly sensitive to the histone deacetylase inhibitor romidepsin in vitro and in vivo, highlighting romidepsin as a potential therapeutic option for GCC patients.Romidepsin-mediated inhibition of histone deacetylases led to disturbances of the chromatin landscape. This resulted in locus-specific histone-hyper- or hypoacetylation. We found that hypoacetylation at the ARID1A promotor caused repression of the SWI/SNF-complex member ARID1A. In consequence, this resulted in upregulation of the stress-sensors and apoptosis-regulators GADD45B, DUSP1 and CDKN1A. RNAi-driven knock down of ARID1A mimicked in parts the effects of romidepsin, while CRISPR/Cas9-mediated deletion of GADD45B attenuated the romidepsin-provoked induction of apoptosis and cell cycle alterations.We propose a signaling cascade involving ARID1A, GADD45B and DUSP1 as mediators of the romidepsin effects in GCC cells.

Protamines are arginine-rich DNA-binding proteins that replace histones in elongating spermatids. This leads to hypercondensation of chromatin and ensures physiological sperm morphology, thereby protecting DNA integrity. In mice and humans, two protamines, protamine-1 (Prm1) and protamine-2 (Prm2) are expressed in a species-specific ratio. In humans, alterations of this PRM1/PRM2 ratio is associated with subfertility. By applying CRISPR/Cas9 mediated gene-editing in oocytes, we established Prm2-deficient mice. Surprisingly, heterozygous males remained fertile with sperm displaying normal head morphology and motility. In Prm2-deficient sperm, however, DNA-hypercondensation and acrosome formation was severely impaired. Further, the sperm displayed severe membrane defects resulting in immotility. Thus, lack of Prm2 leads not only to impaired histone to protamine exchange and disturbed DNA-hypercondensation, but also to severe membrane defects resulting in immotility. Interestingly, previous attempts using a regular gene-targeting approach failed to establish Prm2-deficient mice. This was due to the fact that already chimeric animals generated with Prm2+/− ES cells were sterile. However, the Prm2-deficient mouse lines established here clearly demonstrate that mice tolerate loss of one Prm2 allele. As such they present an ideal model for further studies on protamine function and chromatin organization in murine sperm.

Type II germ cell cancers (GCC) are divided into seminomas, which are highly similar to primordial germ cells and embryonal carcinomas (EC), often described as malignant counterparts to embryonic stem cells.Previously, we demonstrated that the development of GCCs is a highly plastic process and strongly influenced by the microenvironment. While orthotopic transplantation into the testis promotes seminomatous growth of the seminoma-like cell line TCam-2, ectopic xenotransplantation into the flank initiates reprogramming into an EC-like fate.During this reprogramming, BMP signaling is inhibited, leading to induction of NODAL signaling, upregulation of pluripotency factors and downregulation of seminoma markers, like SOX17. The pluripotency factor and EC-marker SOX2 is strongly induced.Here, we adressed the molecular role of SOX2 in this reprogramming. Using CRISPR/Cas9-mediated genome-editing, we established SOX2-deficient TCam-2 cells. Xenografting of SOX2-deficient cells into the flank of nude mice resulted in maintenance of a seminoma-like fate, indicated by the histology and expression of OCT3/4, SOX17, TFAP2C, PRDM1 and PRAME. In SOX2-deficient cells, BMP signaling is inhibited, but NODAL signaling is not activated. Thus, SOX2 appears to be downstream of BMP signaling but upstream of NODAL activation. So, SOX2 is an essential factor in acquiring an EC-like cell fate from seminomas.A small population of differentiated cells was identified resembling a mixed non-seminoma. Analyses of these cells revealed downregulation of the pluripotency and seminoma markers OCT3/4, SOX17, PRDM1 and TFAP2C. In contrast, the pioneer factor FOXA2 and its target genes were upregulated, suggesting that FOXA2 might play an important role in induction of non-seminomatous differentiation.

Type II testicular germ cell cancers (TGCT) are the most frequently diagnosed tumours in young men (20–40 years) and are classified as seminoma or non‐seminoma. TGCTs are commonly treated by orchiectomy and chemo‐ or radiotherapy. However, a subset of metastatic non‐seminomas (embryonal carcinomas) displays only incomplete remission or relapse and requires novel treatment options. Recent studies have shown effective application of the small‐molecule inhibitor JQ1 in tumour therapy, which interferes with the function of ‘bromodomain and extraterminal (BET)’ proteins. JQ1‐treated TGCT cell lines display up‐regulation of genes indicative for DNA damage and cellular stress response and induce cell cycle arrest. Embryonal carcinoma (EC) cell lines, which presented as JQ1 sensitive, display down‐regulation of pluripotency factors and induction of mesodermal differentiation. In contrast, seminoma‐like TCam‐2 cells tolerated higher JQ1 concentrations and were resistant to differentiation. ECs xenografted in vivo showed a reduction in tumour size, proliferation rate and angiogenesis in response to JQ1. Finally, the combination of JQ1 and the histone deacetylase inhibitor romidepsin allowed for lower doses and less frequent application, compared with monotherapy. Thus, we propose that JQ1 in combination with romidepsin may serve as a novel therapeutic option for (mixed) TGCTs.