Summary Germ cell tumors (GCTs) are cancers of the testis, ovary or extragonadal sites that occur in infants, children and adults. Testicular GCT is the most common cancer in young men aged 15–40. Abnormalities in developmental signaling pathways such as wnt/β-catenin, TGF-β/BMP and Hedgehog have been described in many childhood tumors. To date, however, the status of BMP signaling in germ cell tumors has not been described. Here, we examine BMP-SMAD signaling in a set of clinically-annotated pediatric germ cell tumors. We find that BMP signaling activity is absent in undifferentiated tumors such as seminomas and dysgerminomas, but robustly present in most yolk sac tumors, a differentiated tumor type. Gene expression profiling of TGF-β/BMP pathway genes in germinomas and yolk sac tumors reveals a set of genes that distinguish the two tumor types. There is significant intertumoral heterogeneity between tumors of the same histologic subclass, implying that the BMP pathway can be differentially regulated in individual tumors. Finally, through miRNA expression profiling, we identify differential regulation of a set of miRNAs predicted to target the TGF-β/BMP pathway at multiple sites. Taken together, these results suggest that the BMP signaling pathway may represent a new therapeutic target for childhood germ cell tumors.

Germ cell tumors (GCT) are malignant tumors that arise from pluripotent embryonic germ cells and occur in children and young adults. GCTs are treated with cisplatin-based regimens which, while overall effective, fail to cure all patients and cause significant adverse late effects. The seminoma and nonseminoma forms of GCT exhibit distinct differentiation states, clinical behavior, and response to treatment; however, the molecular mechanisms of GCT differentiation are not fully understood. We tested whether the activity of the mTORC1 and MAPK pathways were differentially active in the two classes of GCT. Here we show that nonseminomatous germ cell tumors (NSGCT, including embryonal carcinoma, yolk sac tumor, and choriocarcinoma) from both children and adults display activation of the mTORC1 pathway, while seminomas do not. In seminomas, high levels of REDD1 may negatively regulate mTORC1 activity. In NSGCTs, on the other hand, EGF and FGF2 ligands can stimulate mTORC1 and MAPK signaling, and members of the EGF and FGF receptor families are more highly expressed. Finally, proliferation of NSGCT cells and is significantly inhibited by combined treatment with the clinically available agents erlotinib and rapamycin, which target EGFR and mTORC1 signaling, respectively. These results provide an understanding of the signaling network that drives GCT growth and a rationale for therapeutic targeting of GCTs with agents that antagonize the EGFR and mTORC1 pathways. .

<p>Supplementary Table 1. Combined immunohistochemistry scores, yolk sac tumor and seminoma Supplementary Table 2. Immunohistochemistry scores for all tumors Supplementary Table 3. Sequences of primers used for quantitative RT-PCR Supplementary Figure 1. Complete blots of NCCIT and NTERA-2 cells treated with ligands. See Figure 4A for more details. Supplementary Figure 2. Complete blots of cells treated with rapamycin. See Figure 5 for more details. Supplementary Figure 3. Complete blots of NCCIT and NTERA-2 cells treated with erlotinib. See Figure 5 for more details. Supplementary Figure 4. Effect of EGFR and mTORC1 inhibitors on the viability of NCCIT cells. Supplementary Figure 5. Model depicting signaling pathways inhibited by erlotinib and rapamycin in NSGCTs</p>

<p>Supplementary Table 1. Combined immunohistochemistry scores, yolk sac tumor and seminoma Supplementary Table 2. Immunohistochemistry scores for all tumors Supplementary Table 3. Sequences of primers used for quantitative RT-PCR Supplementary Figure 1. Complete blots of NCCIT and NTERA-2 cells treated with ligands. See Figure 4A for more details. Supplementary Figure 2. Complete blots of cells treated with rapamycin. See Figure 5 for more details. Supplementary Figure 3. Complete blots of NCCIT and NTERA-2 cells treated with erlotinib. See Figure 5 for more details. Supplementary Figure 4. Effect of EGFR and mTORC1 inhibitors on the viability of NCCIT cells. Supplementary Figure 5. Model depicting signaling pathways inhibited by erlotinib and rapamycin in NSGCTs</p>

<div>Abstract<p>Germ cell tumors (GCT) are malignant tumors that arise from pluripotent embryonic germ cells and occur in children and young adults. GCTs are treated with cisplatin-based regimens which, while overall effective, fail to cure all patients and cause significant adverse late effects. The seminoma and nonseminoma forms of GCT exhibit distinct differentiation states, clinical behavior, and response to treatment; however, the molecular mechanisms of GCT differentiation are not fully understood. We tested whether the activity of the mTORC1 and MAPK pathways were differentially active in the two classes of GCT. Here we show that nonseminomatous germ cell tumors (NSGCT, including embryonal carcinoma, yolk sac tumor, and choriocarcinoma) from both children and adults display activation of the mTORC1 pathway, while seminomas do not. In seminomas, high levels of REDD1 may negatively regulate mTORC1 activity. In NSGCTs, on the other hand, EGF and FGF2 ligands can stimulate mTORC1 and MAPK signaling, and members of the EGF and FGF receptor families are more highly expressed. Finally, proliferation of NSGCT cells <i>in vitro</i> and <i>in vivo</i> is significantly inhibited by combined treatment with the clinically available agents erlotinib and rapamycin, which target EGFR and mTORC1 signaling, respectively. These results provide an understanding of the signaling network that drives GCT growth and a rationale for therapeutic targeting of GCTs with agents that antagonize the EGFR and mTORC1 pathways. <i>Mol Cancer Ther; 17(5); 1079–89. ©2018 AACR</i>.</p></div>