The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas

Abstract

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population-embryonal carcinoma (EC)-is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra-embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho- and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.

Keywords: CD24; differentiation; embryonal carcinoma; epigenetics; germ cell tumors; microenvironment.

Fig. 1

(A, B) Flow cytometry‐ and qRT‐PCR‐based analysis of CD24 protein (REA832 antibody) (A) and CD24 mRNA (B) levels in indicated GCT cell lines and normal control cells (n = 3). As controls (gray), cells without addition of antibodies were measured. Error bars are indicated by means of standard deviations (SD). (C) Immunohistochemical staining of CD24 (SWA11 antibody) in GCT tissues. Scale bar = 50 µm; exception lower right corner incl. inlay = 200 µm. (D) Immunofluorescent staining of CD24 (SN3 antibody) in EC cell lines 2102EP, NCCIT, and NT2/D1. As a negative control, JAR cells were included. DAPI was used to stain nuclear DNA. Scale bar = 100 µm. (E) Expression microarray data (left) and qRT‐PCR analysis (right) of CD24 and GDF3 (as EC marker gene) in GCT tissues (GCNIS, seminoma, EC) and normal testis tissue as control. Error bars are indicated by means of standard deviations (SD).

Fig. 2

(A) Flow cytometric analysis of CD44, CD133, and CD184 (CXCR4) in GCT cell lines and MPAF fibroblasts. As controls (gray), cells without addition of antibodies were measured. (B) Sodium bisulfite sequencing analysis of the DNA methylation status of the CD24 CpG island in GCT tissues and cell lines. Each sample has been analyzed in quintuplicates. (C) qRT‐PCR analysis of CD24 expression in JAR, BeWo, JEG3, and TCam‐2 cells 72–96 h after 20 or 50 nm decitabine treatment (n = 3). Error bars are indicated by means of standard deviations (SD). (D) qRT‐PCR analysis of CD24 expression in GCT and fibroblast cells treated for 16 h with related IC₅₀ concentrations of the HDACi quisinostat, HDMi JIB‐04, HTMi GSK343, E3‐ULi PRT4165, and BRDi MZ‐1 and LP99. Error bars are indicated by means of standard deviations (SD). (E) SOX2‐ and SOX17‐ChIP‐seq in 2102EP (n = 3) and TCam‐2 (n = 3), respectively. Four putative SOX binding sites (region 1–4) were found within or in close proximity to the CD24 coding sequence. The CpG island region analyzed by sodium bisulfite sequencing is labeled in light blue. (F) SOX2‐ and SOX17‐ChIP‐qPCR analysis in 2102EP or TCam‐2 cells, respectively. As control, an IgG antibody was used. For qPCR, two primers were used: (1) amplifying CD24 region 4 (on target) and (2) amplifying a region with no SOX binding motif and not related to the CD24 coding region (off target). Error bars are indicated by means of standard deviations (SD). (G) qRT‐PCR analysis of CD24, GDF3, CD44, and CD133 expression in EC cell lines (NCCIT and NT2/D1) treated with 20 µm retinoic acid for 8 days. As controls and for comparison, other GCT and control cells were included (n = 3). Error bars are indicated by means of standard deviations (SD). (H) Expression microarray data of CD24 and SOX2 expression in GCT cell lines, SOX2‐deficient TCam‐2 grown in vivo for 6 weeks (T SOX2^‐/‐ 6w) and during in vivo reprogramming of TCam‐2 into an EC‐like cell fate over 6 weeks (T 1w, T 2w, T 4w, and T 6w). (I) qRT‐PCR analyses of CD24 expression in the GCT cell populations after co‐cultivation of GCT cell lines with immune cells (THP‐1‐M2 (M2 macrophages), JURKAT (T lymphocytes), endothelial cells (HUVEC), or fibroblasts (HVHF2)) for 72 h and flow cytometry‐based cell sorting (n = 3). Error bars are indicated by means of standard deviations (SD). Two‐tailed t‐tests were performed to test for significance; *P‐value < 0.05, **P‐value < 0.005, and ***P‐value < 0.0005.

Fig. 3

(A, B) qRT‐PCR (A) (n = 3) and flow cytometry (B) (NCCIT‐CD24 ^+/+ n = 1; NCCIT‐ΔCD24 n = 5) analysis of CD24 expression and CD24 protein levels (REA832 antibody) in NCCIT‐ and 2102EP‐ΔCD24 clones and parental cells. Ab = antibody. Error bars are indicated by means of standard deviations (SD). (C, D) Flow cytometry‐based measurement of cell cycle phase distribution (C) and proliferation rates (D) in NCCIT‐ and 2102EP‐ΔCD24 clones and parental cells 72 h after plating (NCCIT‐CD24 ^+/+, n = 2; NCCIT‐ΔCD24, n = 5). Error bars are indicated by means of standard deviations (SD). (E, F) Measurement of adhesion ability (E) and migratory capacity by transwell assays (F) and in NCCIT‐ and 2102EP‐ΔCD24 clones and parental cells 24 h after plating (NCCIT‐CD24 ^+/+, n = 3; NCCIT‐ΔCD24, n = 5). Error bars are indicated by means of standard deviations (SD). Two‐tailed t‐tests were performed to test for significance; *P‐value < 0.05, **P‐value < 0.005, and ***P‐value < 0.0005.

Fig. 4

(A) PCA of RNA‐seq data of NCCIT‐ΔCD24 clones and parental cells (NCCIT‐CD24 ^+/+, n = 2; NCCIT‐ΔCD24, n = 5). (B) Illustration of differentially expressed genes in NCCIT‐ΔCD24 cells compared with the parental cells. (C) DAVID‐based prediction of biological processes and molecular functions in which the genes deregulated in NCCIT‐ΔCD24 cells compared with the parental cells are involved in. (D) STRING‐based protein interaction prediction of the molecules upregulated or downregulated in NCCIT‐ΔCD24 cells compared with the parental cells. (E) qRT‐PCR validation (n = 3) of selected deregulations in gene expression found by the RNA‐seq analysis in NCCIT‐DCD24 and parental cells. Error bars are indicated by means of standard deviations (SD). Two‐tailed t‐tests were performed to test for significance; *P‐value < 0.05, **P‐value < 0.005, and ***P‐value < 0.0005.

Fig. 5

(A) PCA of 850 k DNA methylation array data in NCCIT‐ and 2102EP‐ΔCD24 clones and parental cells (NCCIT‐CD24 ^+/+, n = 1; NCCIT‐ΔCD24, n = 3). (B) Violin plots illustrating global DNA methylation levels in NCCIT‐ and 2102EP‐ΔCD24 clones and parental cells. Global DNA methylation levels of the individual CD24‐deficient clones are given on the right side of each bar (n. s. = not significant). (C) Illustration of differentially methylated genes in NCCIT‐/2102EP‐ΔCD24 cells compared with the parental cells. A two‐group comparison (t‐test) was performed to sort for significance. (D) Affected by DNA methylation gene and CpG island regions in NCCIT‐/2102EP‐ΔCD24 cells compared with the parental cells. Illustrations were taken from the ‘Illumina Infinium HumanMethylation450 BeadChip’ datasheet. (E, F) Volcano plot (E) and waterfall diagram (F) of commonly deregulated genes in NCCIT‐/2102EP‐ΔCD24 cells showing inverse correlation between DNA methylation (5mC) and gene expression (GEX). Error bars are indicated by means of standard deviations (SDs).

Fig. 6

(A) qRT‐PCR analysis (n = 3) of indicated marker genes of all three germ layers ((extra‐embryonic (exe))‐endo‐, meso‐, ectoderm) after 10 days of RA‐mediated differentiation of NCCIT‐ and 2102EP‐ΔCD24 and parental cells (NCCIT‐CD24 ^+/+, n = 1; NCCIT‐ΔCD24, n = 5). Error bars are indicated by means of standard deviations (SD). Additionally, exemplary pictures of cell morphologies +/− RA are given. (B) Flow cytometry‐based measurement (n = 3) of apoptosis rates after application of SWA11 and/or cisplatin in EC cell lines and corresponding cisplatin‐resistant subclones (‐R). Error bars are indicated by means of standard deviations (SD). Two‐tailed t‐tests were performed to test for significance; *P‐value < 0.05, **P‐value < 0.005, and ***P‐value < 0.0005.

Fig. 7

(A–C) Models summarizing key findings of this study. (A) CD24 expression characteristics and dynamics in different GCT entities. (B) Molecular and (epi)genetic mechanisms regulating or influencing CD24 expression. (C) Overview of the molecular function of CD24 in ECs.