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.

Embryonal carcinomas (ECs) and seminomas are testicular germ cell tumors. ECs display expression of SOX2, while seminomas display expression of SOX17. In somatic differentiation, SOX17 drives endodermal cell fate. However, seminomas lack expression of endoderm markers, but show features of pluripotency. Here, we use chromatin immunoprecipitation sequencing to report and compare the binding pattern of SOX17 in seminoma‐like TCam‐2 cells to SOX17 in somatic cells and SOX2 in EC‐like 2102EP cells. In seminoma‐like cells, SOX17 was detected at canonical (SOX2/OCT4), compressed (SOX17/OCT4) and noncomposite SOX motifs. SOX17 regulates TFAP2C, PRDM1 and PRDM14, thereby maintaining latent pluripotency and suppressing somatic differentiation. In contrast, in somatic cells canonical motifs are rarely bound by SOX17. In sum, only 12% of SOX17‐binding sites overlap in seminoma‐like and somatic cells. This illustrates that binding site choice is highly dynamic and cell type specific. Deletion of SOX17 in seminoma‐like cells resulted in loss of pluripotency, marked by a reduction of OCT4 protein level and loss of alkaline phosphatase activity. Furthermore, we found that in EC‐like cells SOX2 regulates pluripotency‐associated genes, most likely by partnering with OCT4. In conclusion, SOX17 (in seminomas) functionally replaces SOX2 (in ECs) to maintain expression of the pluripotency cluster.

Embryonal carcinomas (ECs) and seminomas are testicular germ cell tumours. ECs display expression of SOX2, while seminomas display expression of SOX17. In somatic differentiation, SOX17 drives endodermal cell fate. However, seminomas lack expression of endoderm markers, but show features of pluripotency. Here, we use ChIP-sequencing to report and compare the binding pattern of SOX17 in seminoma-like TCam-2 cells to SOX2 in EC-like 2102EP cells and SOX17 in somatic cells. In seminoma-like cells, SOX17 was detected at canonical (SOX2/OCT4), compressed (SOX17/OCT4) and other SOX family member motifs. SOX17 directly regulates TFAP2C, PRDM1 and PRDM14, thereby maintaining latent pluripotency and supressing somatic differentiation. In contrast, in somatic cells canonical motifs are not bound by SOX17. In sum only 10% of SOX17 binding sites overlap in seminoma-like and somatic cells. This illustrates that binding site choice is highly dynamic and cell type specific. Deletion of SOX17 in seminoma-like cells resulted in loss of pluripotency, marked by a reduction of OCT4 protein level and loss of alkaline phosphatase activity. Further, we found that in EClike cells SOX2 regulates pluripotency-associated genes, predominantly by partnering with OCT4. In conclusion, SOX17 (in seminomas) functionally replaces SOX2 (in ECs) to maintain expression of the pluripotency cluster.

LARP1 is an oncogenic RNA-binding protein required for ribosome biogenesis and cancer cell survival. From published in vitro studies, there is disparity over which of two different LARP1 protein isoforms (termed the long LI-LARP1 and short SI-LARP1) is the canonical. Here, after conducting a series of biochemical and cellular assays, we conclude that LI-LARP1 (NM_033551.3 > NP_056130.2) is the dominantly expressed form. We observe that SI-LARP1 (NM_015315.5> NP_056130.2) is epigenetically repressed and that this repression is evolutionarily conserved in all but a small subclade of mammalian species. As with other LARP family members, there are multiple potential LARP1 mRNA isoforms that appear to be censored within the nucleus. The capacity of the cell to modulate splicing and expression of these apparently 'redundant' mRNAs hints at contextually specific mechanisms of LARP1 expression.