BMP Inhibition in Seminomas Initiates Acquisition of Pluripotency via NODAL Signaling Resulting in Reprogramming to an Embryonal Carcinoma

Abstract

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.

Imprints and DPPA3 are bypassed during pluripotency- and differentiation-coupled methylation reprogramming in testicular germ cell tumors

“…The most parsimonious interpretation of these observations is that the prominent burst of mCpH-pluri in ECs, having already passed through an erased PGC/GCNIS ancestry, represents a reversion to/ reactivation of pluripotency, with de novo apparition. As further evidence for such a recrudescence of pluripotency in ECs, we found that induction of pluripotent EC phenotypes from the seminomatous TCam-2 cell line following xenograft to murine testis (Nettersheim et al 2015) spawns mCpH-pluri (Supplemental Fig. S9C), which we now interpret as a key molecular event previously unrecognized in that experiment.…”

“…TGCTs' association with developmental gonadal defects supports the prevailing model by which they originate from a multipotent stem cell arising in the germline, which fails to disable pluripotency to mature into a spermatogonium (McGlynn and Cook 2009;Sheikine et al 2012). On the other hand, recent studies identifying reversion to pluripotency in the germline (Nettersheim et al 2015;Oliveros-Etter et al 2015) question whether TGCTs entail a continuation of, versus reversion to, functional pluripotency.…”

SOX2 is essential for in vivo reprogramming of seminoma-like TCam-2 cells to an embryonal carcinoma-like fate

“…In a previous study, we demonstrated that TCam-2 cells are able to transit into an EC-like state when being xenografted into the flank of nude mice [10]. Analyses revealed that the somatic microenvironment inhibits BMP signaling, which is the initial step in the reprogramming process of TCam-2 cells, leading to induction of NODAL signaling and expression of EC-related genes as well as globally increased 5mC levels ( in vitro : 18 %; 6 weeks in vivo : 70 %) [10].…”

“…Analyses revealed that the somatic microenvironment inhibits BMP signaling, which is the initial step in the reprogramming process of TCam-2 cells, leading to induction of NODAL signaling and expression of EC-related genes as well as globally increased 5mC levels ( in vitro : 18 %; 6 weeks in vivo : 70 %) [10]. During this reprogramming, we found a very rapid (after 1 week of in vivo growth) and strong upregulation of the transcription factor SOX2 (Log 2 2.16 fold) and in parallel downregulation of SOX17 (Log 2 3.75 fold) [10]. Due to the importance of SOX2 in cellular reprogramming and maintenance of pluripotency, we were interested in the role of SOX2 in the reprogramming of TCam-2.…”

Plasticity underlies tumor progression: role of Nodal signaling

“…Non-seminomas form highly heterogeneous tumors with both differentiated and undifferentiated cells forming teratomas and choriocarcinomas and display more embryonal-like features. Using a seminoma xenograft model in which seminoma cells are reprogrammed to form carcinomas with more embryonal-like features, expression of key pluripotency and chromatin remodeling genes was examined [107]. Nodal was upregulated during this transition by demethylation of the Nodal promoter.…”