Abstract
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.
Highlights
All type II testicular germ cell tumors (GCTs) derive from a common precursor lesion—the germ cell neoplasia in situ (GCNIS), which itself is thought to be the results of a defective primordial germ cell (PGC) development [1,2]
Using CRISPR/Cas9 gene editing we demonstrated that deletion of SOX2 severely impairs reprogramming [9]
TCam-2 cells generated in a previous study [9] and re-transfected cells with the CRISPR/Cas9 components including three guideRNAs targeting the coding region of FOXA2 (Figure S1A)
Summary
All type II testicular germ cell tumors (GCTs) derive from a common precursor lesion—the germ cell neoplasia in situ (GCNIS), which itself is thought to be the results of a defective primordial germ cell (PGC) development [1,2]. Seminomas and embryonal carcinomas (ECs; the stem cell population of the non-seminomas) differ considerably in Cancers 2019, 11, 728; doi:10.3390/cancers11050728 www.mdpi.com/journal/cancers. While ECs resemble malignant embryonic stem cells, seminomas are more similar to PGCs and GCNIS [1]. We and others have shown that the cell line TCam-2 serves as a reliable proxy for seminomas and GCNIS. TCam-2 cells express typical primordial germ cell and GCNIS marker genes (SOX17, PRAME, cKIT, TFAP2C, PRDM1/BLIMP1) and show a typical GCNIS/seminoma morphology (big roundish cells with a big nucleus and clear cytoplasm) [3,4,5,6].
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