Cripto: Expression, epigenetic regulation and potential diagnostic use in testicular germ cell tumors

Abstract

Type II germ cell tumors arise after puberty from a germ cell that was incorrectly programmed during fetal life. Failure of testicular germ cells to properly differentiate can lead to the formation of germ cell neoplasia in situ of the testis; this precursor cell invariably gives rise to germ cell cancer after puberty. The Nodal co-receptor Cripto is expressed transiently during normal germ cell development and is ectopically expressed in non-seminomas that arise from germ cell neoplasia in situ, suggesting that its aberrant expression may underlie germ cell dysregulation and hence germ cell cancer. Here we investigated methylation of the Cripto promoter in mouse germ cells and human germ cell cancer and correlated this with the level of CRIPTO protein expression. We found hypomethylation of the CRIPTO promoter in undifferentiated fetal germ cells, embryonal carcinoma and seminomas, but hypermethylation in differentiated fetal germ cells and the differentiated types of non-seminomas. CRIPTO protein was strongly expressed in germ cell neoplasia in situ along with embryonal carcinoma, yolk sac tumor and seminomas. Further, cleaved CRIPTO was detected in media from seminoma and embryonal carcinoma cell lines, suggesting that cleaved CRIPTO may provide diagnostic indication of germ cell cancer. Accordingly, CRIPTO was detectable in serum from 6/15 patients with embryonal carcinoma, 5/15 patients with seminoma, 4/5 patients with germ cell neoplasia in situ cells only and in 1/15 control patients. These findings suggest that CRIPTO expression may be a useful serological marker for diagnostic and/or prognostic purposes during germ cell cancer management.

Keywords: Cripto; Diagnostic; Methylation; Testicular germ cell cancer.

Figure 1

Bisulfite analysis of CpG methylation in the promoter region of Cripto in 13.5 and 17.5 dpc germ cells. (A) The mouse Cripto promoter has two CpG islands in the proximal promoter region: Region 1 at position ‐1383 – 642 contains 15 CpG sites. Region 2 at position −50 – +50 contains 18 CpG sites. (B)Cripto is expressed maximally in male gonads at 12.5 dpc and has declined by 14.5 dpc. Expression is similar in male and female gonads at 11.5 dpc before becoming downregulated by 12.5 dpc in the female. qRT‐PCR of pooled gonad‐only male and female samples at 11.5–17.5 dpc. Expression normalized to Tbp; error bars represent ±S.E.M; n = 3. (C) Methylation (expressed as % methylation) for each CpG site are shown for purified male and female germ cells at 13.5 and 17.5 dpc. For each sample ≥10 clones were sequenced.

Figure 2

Bisulfite analysis of GpG methylation in the promoter region of CRIPTO in GCC pathologies. (A) The human CRIPTO promoter has two CpG islands in the proximal promoter region: Region 1 at position −800 to −662 contains 13 CpG sites. Region 2 at position −225 to −84 contains 7 CpG sites. (B) qRT‐PCR analysis of CRIPTO expression (Log2 scale) in normal testis (control) and NS and SE samples; n = 5, 4, 9, 10, 7, 10. Methylation for each CpG site is shown for CH (C), TE (D), EC (E), YST (F) SE (G) and JKT‐1 control cell line (H). For each sample, graphs represent colony sequencing of ≥10 clones of one sample, each bar representing one CpG site (expressed at % methylation). Direct sequencing of 2–4 different tumor samples is presented as circles at each CpG site. Open circles represent unmethylated site, closed circles represent a methylated site and half‐filled circles represent heterogeneity of methylation at this site within a given sample.

Figure 3

Immunohistochemistry for CRIPTO expression (brown staining) in EC (A), YST (B), SE (C), TE (D) and GCNIS (E) pathologies, counterstained with haematoxylin. Images are representative of ≥8 biopsies analyzed for each subtype at a magnification of both 100 and 200× (left and right images, respectively).

Figure 4

(A) Immunohistochemistry for CRIPTO expression in JKT‐1, TCam‐2, TERA‐1 and NT2 cell lines (B) CRIPTO concentration (ng/mL) assessed by ELISA in cell culture media collected from cell lines NT2 (range: 2.85–30.35), TERA‐1 (range: 1.58–18.28), TCam2 (range: 0.78–4.19) and JKT‐1 (range: 0.0–0.0) at varying confluences (ranging 10–100%). Error bars represent ±S.E.M; n = 13,6,6,6.

Figure 5

CRIPTO concentration (ng/mL) assessed by ELISA in patient serum of control (no testicular malignancy), SE, EC, YST, GCNIS‐only, CH and TE pathologies. Error bars represent ±S.E.M; n = 15,15,15,3,5,1,5.

Figure 6

Schematic of CRIPTO/NODAL/GDF3 signaling (A) and the combinations of receptor/ligand expression in normal germ cells, embryonic stem cells and the subtypes of GCC (B). Symbols represent positive expression (+), negative expression (−) and heterogeneous expression (−/+).