The bromodomain inhibitor JQ1 as novel therapeutic option for type II testicular germ cell tumours [and] The role of SOX2 and SOX17 in regulating germ cell tumour pluripotency

The bromodomain inhibitor JQ1 as novel therapeutic option for type II testicular germ cell tumours Type II testicular germ cell tumours (TGCTs) represent the most common malignancy in young men (19-35 years). They are classified as seminoma or embryonal carcinoma (EC; the stem cell population of non-seminomas). TGCTs are highly sensitive to radio- and chemotherapy, however 1-5% of TGCTs may develop resistance mechanisms to standard therapy regimens. Epigenetic drugs open a new avenue to cancer therapy and may present a promising alternative to treat recurrent TGCTs. JQ1 is an inhibitor of the BET family of bromodomain reader proteins. In TGCT cell lines, JQ1 treatment leads to upregulation of stress markers (i.e. CDKN1C, DDIT4, TSC22D1, TXNIP), induction of the differentiation marker HAND1, and downregulation of pluripotency-associated genes (i.e. LIN28, DPPA4, UTF1) [1]. This results in growth arrest and apoptosis in cisplatin-sensitive and cisplatin- resistant EC cells (at doses = 100 nM) and seminoma cells (at doses = 250 nM) [1, 2]. In line, EC xenografts in nude mice show reduced tumour burden when treated with JQ1 (50 mg / kg) compared to solvent controls. Additionally, JQ1-treated tumours showed reduced blood vessel count (lower CD31+), possibly due to JQ1- mediated downregulation of VEGFB. Altogether, this reflects the therapeutic potential of bromodomain inhibition for TGCTs. However, similar to TGCT cells, somatic control cells (here: Sertoli cells) responded with cell cycle arrest and apoptosis to JQ1 treatment. Thus, a more detailed analysis of possible side effects of JQ1 administration is recommended, before commissioning the drug for clinical use. Interestingly, JQ1 treatment had similar effects on TGCT cells as the HDAC inhibitor romidepsin (i.e. induction of stress markers GADD45A, GADD45B, RHOB, ID2) [1-3]. I now showed that JQ1 and romidepsin may elicit additive or synergistic effects on cytotoxicity levels of TGCT cells in vitro and in vivo. Since a combination of both drugs may, however, also increase potential side effects, the exact efficacy vs toxicity relationship of this treatment strategy needs further evaluation.The role of SOX2 and SOX17 in regulating germ cell tumour pluripotency TGCTs can be characterized as seminoma or EC. While seminomas display limited differentiation capacities, ECs display features of pluri- to totipotency. Previous data suggests that pluripotency in EC cells is maintained by cooperative binding of SOX2- OCT4 to the canonical (SOX2/OCT4) motifs at pluripotency genes. Indeed, SOX2 binding in EC cells is enriched at canonical motifs and SOX2 target genes showed significant overlap with embryonic stem cell signatures. In contrast, seminomas lack expression of SOX2, but display high levels of OCT4 and SOX17. In embryonic stem cells cooperative binding of SOX17-OCT4 to the compressed (SOX17/OCT4) motif on DNA induces endodermal differentiation. However, seminomas maintain an undifferentiated state, indicated by expression of pluripotency genes and lack of expression of typical differentiation markers. We therefore asked, whether the SOX17-OCT4 complex in seminoma cells binds to canonical (SOX2/OCT4) binding sites to regulate and maintain seminoma pluripotency. High-throughput chromatin immunoprecipiation (ChIP)-sequencing analysis revealed that the majority of genes bound by SOX17 in seminoma cells has functions in neuronal differentiation and that 26% of SOX17 peaks contain the compressed (SOX17/OCT4) binding motif. These findings are in disagreement with the latent pluripotent state of seminoma cells. However, a small subset of SOX17-bound genes has roles in pluripotency maintenance (e.g. NANOG, POU5F1 (OCT4), PRDM1 and TFAP2C) and 10% of SOX17 peaks include the described canonical (SOX2/OCT4) binding motif. This suggests that, next to somatic genes, SOX17 regulates pluripotency genes in seminoma cells by binding to the canonical motif. In line, CRISPR/Cas9-mediated deletion of SOX17 in TCam-2 resulted in a strong reduction of OCT4 and TFAP2C protein levels, as well as alkaline phosphatase activity. qRT-PCR analysis showed that loss of SOX17 induces differentiation into trophoblast-like lineages. I conclude that SOX17 shares a similar role in seminoma cells as in primordial germ cells (PGC), which is to maintain a latent pluripotent state and to suppress cellular differentiation (i.e. via downstream activation of the PGC specifiers PRDM1 and TFAP2C and by direct activation of pluripotency genes such as NANOG and POU5F1)

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

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 repro- grammed 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 tran- sition 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 repro- gramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogram- ming, 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 sig- naling. 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

A signaling cascade including ARID1A, GADD45B and DUSP1 induces apoptosis and affects the cell cycle of germ cell cancers after romidepsin treatment

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

TCam-2 Cells Deficient for SOX2 and FOXA2 Are Blocked in Differentiation and Maintain a Seminoma-Like Cell Fate In Vivo

Testicular germ cell tumors (GCTs) are very common in young men and can be stratified into seminomas and non-seminomas. While seminomas share a similar gene expression and epigenetic profile with primordial germ cells, the stem cell population of the non-seminomas, the embryonal carcinoma (EC), resembles malignant embryonic stem cells. Thus, ECs are able to differentiate into cells of all three germ layers (teratomas) and even extra-embryonic-tissue-like cells (yolk-sac tumor, choriocarcinoma). In the last years, we demonstrated that the cellular microenvironment considerably influences the plasticity of seminomas (TCam-2 cells). Upon a microenvironment-triggered inhibition of the BMP signaling pathway in vivo (murine flank or brain), seminomatous TCam-2 cells reprogram to an EC-like cell fate. We identified SOX2 as a key factor activated upon BMP inhibition mediating the reprogramming process by regulating pluripotency, reprogramming and epigenetic factors. Indeed, CRISPR/Cas9 SOX2-deleted TCam-2 cells were able to maintain a seminoma-cell fate in vivo for about six weeks, but after six weeks in vivo still small sub-populations initiated differentiation. Closer analyses of these differentiated clusters suggested that the pioneer factor FOXA2 might be the driving force behind this induction of differentiation, since many FOXA2 interacting genes and differentiation factors like AFP, EOMES, CDX1, ALB, HAND1, DKK, DLK1, MSX1 and PITX2 were upregulated. In this study, we generated TCam-2 cells double-deficient for SOX2 and FOXA2 using the CRISPR/Cas9 technique and xenografted those cells into the flank of nude mice. Upon loss of SOX2 and FOXA2, TCam-2 maintained a seminoma cell fate for at least twelve weeks, demonstrating that both factors are key players in the reprogramming to an EC-like cell fate. Therefore, our study adds an important piece to the puzzle of GCT development and plasticity, providing interesting insights in what can be expected in a patient, when GCT cells are confronted with different microenvironments

The cancer/testis-antigen PRAME supports the pluripotency network and represses somatic and germ cell differentiation programs in seminomas

Prostate transcriptome dat

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

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 repro- grammed 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 tran- sition 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 repro- gramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogram- ming, 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 sig- naling. 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

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

Oncotarge

The cancer/testis-antigen PRAME supports the pluripotency network and represses somatic and germ cell differentiation programs in seminomas

<p>Background: Cancer/testis-antigens (CTAs) are specifically expressed in human malignancies and testis tissue, but their molecular functions are poorly understood. CTAs serve as regulators of gene expression, cell cycle and spermatogenesis, as well as targets for immune-based therapies. The CTA PRAME is expressed in various cancers, antagonises retinoic acid signalling and is regulated by DNA methylation and histone acetylation.</p><p>Methods: We analysed the molecular function of the CTA PRAME in primordial germ cells (PGC) and testicular germ cell cancers (GCC). GCCs arise from a common precursor lesion termed germ cell neoplasia in situ (GCNIS), which itself is thought to originate from a defective PGC. GCNIS cells eventually develop into unipotent seminomas or totipotent embryonal carcinomas (ECs), which are capable of differentiation into teratomas, yolk-sac tumours and choriocarcinomas.</p> <p>Results: PRAME is, like the master regulator of PGCs SOX17 expressed in human PGCs, GCNIS and seminomas but absent in ECs. shRNA-mediated knockdown of PRAME in seminomatous TCam-2 cells left SOX17 levels unchanged, but resulted in downregulation of pluripotency- and PGC-related genes (LIN28, PRDM14, ZSCAN10), whereas somatic and germ cell differentiation markers were upregulated. So, PRAME seems to act downstream of SOX17 by mediating the regulation of the germ cell differentiation and pluripotency programme. Endoderm differentiation is triggered in somatic cells by SOX17, suggesting that in PGCs, PRAME represses this programme and modulates SOX17 to function as a PGC-master regulator. Surprisingly, knockdown of PRAME in TCam-2 cells did not render the cells sensitive towards retinoic acid, despite the fact that PRAME has been described to antagonise retinoic acid signalling. Finally, we demonstrate that in non-seminomas PRAME expression is silenced by DNA methylation, which can be activated by formation of euchromatin via histone-deacetylase-inhibitors.</p><p>Conclusions: We identified the CTA PRAME as a downstream factor of SOX17 and LIN28 in regulating pluripotency and suppressing somatic/germ cell differentiation in PGC, GCNIS and seminomas.<br></p

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

<div><p>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 (<i>GDF3</i>, <i>NODAL</i>, <i>DNMT3B</i>, <i>DPPA3</i>, <i>GAL</i>, <i>AK3L1</i>) 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.</p></div