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

Abstract

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.

Keywords: SOX2/SOX17; TCam-2; embryonal carcinoma; reprogramming; seminoma.

Figure 1

(A) Macroscopical appearance of tumors from xenografted TCam-2, 2102EP and TCam-2-ΔSOX2 clones. (B) HE staining of tumor tissues from xenografted TCam-2-ΔSOX2 clones, parental TCam-2 and 2102EP. A seminoma and an EC tissue were stained as controls. Scale bars: 50 μm. (C) Meta-analysis of expression microarray data of indicated samples including unsupervised hierarchical clustering. Samples labelled in green were generated for this study, samples in black are part of a previous publication [10] and re-analyzed in context of this study. The yellow box highlights genes differentially expressed between TCam-2-ΔSOX2 clones and the other samples.

Figure 2

(A, B) Expression microarray (A) and qRT-PCR (B) data of indicated genes related to the in vivo reprogramming, pluripotency, seminoma-ness, signaling pathways and epigentics. For TCam-2-ΔSOX2 clones, expression data of five clones was averaged. Standard deviation is indicated above each bar. Genes showing expression values below the grey line in (A) were considered as not expressed (Log₂6.8).

Figure 3. IHC staining of indicated proteins in TCam-2-ΔSOX2 clones

Tumor tissues from xenografted 2102EP cells were stained as control. Scale bars: 50 μm.

Figure 4. ChIP followed by qPCR-analysis of SOX2-enrichment at the SOX2 binding sites within indicated genes

3% input chromatin was used for normalization and an IgG antibody was used as negative ChIP control. In qPCR, oligonucleotides were used to amplify a PCR-fragment around the SOX2 binding site (on target) and a PCR-fragment within the same gene, but without a SOX2 binding site (off target).

Figure 5

(A) Quantification of 5mC levels in tumors from xenografted TCam-2-ΔSOX2 clones, TCam-2 and 2102EP cells. In vitro cultivated TCam-2 and 2102EP served as controls. (B) Commonly expressed genes in TCam-2-ΔSOX2 (vs. TCam-2 in vitro) and non-seminomas (n = 7) (vs. seminomas (n = 4)) based on microarray data. Green labeled genes are related to FOXA2. (C) STRING analysis of FOXA2 interacting genes. (D) Co-IP experiment in TCam-2 demonstrating binding of SOX17 to OCT3/4. (E) ChIP experiment in TCam-2, demonstrating binding of SOX17 to the SOX2 / OCT3/4 binding motif within the NANOG promoter.

Figure 6

(A) Model of the differentiation abilities of TCam-2 and TCam-2-ΔSOX2 cells in vitro and in vivo. (B) Molecular mechanisms of promoting EC- or seminoma-ness and differentiation into endodermal-, mesodermal- and ectodermal-lineage (EnMeEc diff.) and trophectoderm (Troph. diff.). (C) Detailed mechanism of the role of SOX2 in reprogramming of TCam-2 to an EC-like state in vivo. Models are based on the results of this study and [8, 10, 20, 30].