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

Cassy M Spiller,Ad J.M Gillis,Guillaume Burnet,Hans Stoop,Peter Koopman,Josephine Bowles,Leendert H.J Looijenga

doi:10.1016/j.molonc.2015.11.003

Cassy M Spiller, Ad J.M Gillis + Show 5 more

Open Access

https://doi.org/10.1016/j.molonc.2015.11.003

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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.

Highlights

Testicular germ cell cancers (GCCs), known as Type II germ cell tumors (Oosterhuis and Looijenga, 2005), account for w60% of all malignancies in men aged 20e40 (Adami et al, 1994; van de Geijn et al, 2009)
Because we have shown that Cripto is expressed for a short time in fetal germ cells but is ectopically expressed in GCC (Spiller et al, 2012), we wanted to investigate the mechanism by which Cripto expression is properly silenced under normal conditions using the mouse model
To assess methylation in germ cells, we isolated male and female germ cells from Oct4DPE:eGFP mouse embryos (Szabo et al, 2002) using fluorescence-activated cell sorting (FACS) at 13.5 dpc (Cripto is expressed in the male) and at 17.5 dpc

Summary

Introduction

Testicular germ cell cancers (GCCs), known as Type II germ cell tumors (Oosterhuis and Looijenga, 2005), account for w60% of all malignancies in men aged 20e40 (Adami et al, 1994; van de Geijn et al, 2009). The ‘fetal origins hypothesis’ of GCNIS predicts developmental pathways that control fetal germ cell pluripotency/differentiation contribute to their malignant potential. We recently discovered that the TGFb signaling molecule Nodal and its obligate receptor Cripto are expressed at a critical point during fetal XY germ cell development in mice and that Nodal/ Cripto signaling is active, apparently acting to maintain pluripotency and oppose differentiation (Spiller et al, 2012). We found that Nodal/Cripto signaling is ectopically activated in NS and we hypothesize that ectopic activation of Nodal signaling, or failure to silence it, contributes to GCC formation (Spiller et al, 2013)

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