Ectopic activation of WNT signaling in human embryonal carcinoma cells and its effects in short- and long-term in vitro culture

Yaser Atlasi,Rosalie Joosten,J Wolter Oosterhuis,Riccardo Fodde,Leendert H J Looijenga,Rebecca T Van Dorsten,Andrea Sacchetti

doi:10.1038/s41598-019-48396-7

Abstract

Human embryonal carcinoma (EC) cells comprise the pluripotent stem cells of malignant non-seminomatous germ cell tumors (GCTs) and represent the malignant counterpart of embryonic stem cells (ESCs). WNT/β-catenin signaling has been implicated in regulating adult and embryonic stem cells although its role in EC cells is less investigated. Here, we studied WNT signaling in a panel of representative pluripotent and nullipotent human EC cell lines. We found that EC cell lines show distinct levels of intrinsic WNT signaling and respond differently to ectopic WNT activation. Short-term activation of WNT signaling induced a differentiation-response in the pluripotent EC cells (NT2 and NCCIT) whereas the nullipotent EC cells (TERA1 and 2102Ep) were refractory and maintained high levels of OCT4 and SSEA4 expression. Long-term activation of WNT signaling in NCCIT and, to a lesser extent, TERA1 cells led to (re)gain of OCT4 expression and a switch from SSEA4 to SSEA1 surface antigens ultimately resulting in OCT4+/SSEA4−/SSEA1+ profile. Cisplatin treatment indicated that the OCT4+/SSEA4−/SSEA1+ NCCIT cells became more resistant to chemotherapy treatment. Our findings are of particular interest for the GCT and ES cell biology and shed light on the role of WNT signaling in human EC cells.

Highlights

Type II malignant germ cell tumors (GCTs), known as Germ Cell Cancers (TGCCs), are the most common malignancies in Caucasian young adults and include Seminomas (SE) and Non-Seminoma (NS) tumors[1,2]
We found that the pluripotent NT2 cells display the highest level of WNT signaling, whereas the NCCIT, TERA1 and 2102Ep embryonal carcinoma (EC) cell lines show basal WNT activity (Fig. 1a)
In accordance with the above TOP-Flash reporter results, we found that NT2 cell line encompasses the largest subpopulation of GFP+WNT+ cells (24%), whereas the other EC cell lines have hardly detectable GFP-positive populations

Summary

Introduction

Type II malignant GCTs, known as (testicular) Germ Cell Cancers (TGCCs), are the most common malignancies in Caucasian young adults and include Seminomas (SE) and Non-Seminoma (NS) tumors[1,2]. WNT signaling is involved in different stages of embryonic development and controls, among others, the self-renewal of adult and embryonic stem cells. Activation of WNT signaling results in the destabilization of this complex and increased β-catenin levels in the cytoplasm and nucleus. In mouse ES and EC cells, WNT signaling supports self-renewal and represses multi-lineage differentiation[9,10,11,12,13]. In TGCCs, global gene expression profiling together with β-catenin immunohistochemical analysis revealed enrichment of WNT signaling activity in EC and YSTs19–21. We ectopically activated WNT signaling in different hEC cell lines and evaluated its subsequent effects in short- and long-term cultures. Our findings describe the divergent effects of WNT signaling in hEC lines and report new intermediate states that can be captured in these cells

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Results

Conclusion