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Identification of 19 novel loci reveals gene regulatory mechanisms determining susceptibility to testicular germ cell tumour

ABSTRACT: Testicular germ cell tumour (TGCT) is the most common cancer in young men1,2, and is characterised by strong inherited genetic risk factors3. Here we have undertaken large-scale genome wide association study (GWAS) for TGCT, encompassing ~7,500 cases and ~23,000 controls, through the Oncoarray consortium. We identified 19 novel loci, approximately doubling the number of known TGCT risk loci to 44 (P<5x10-8)4-14 and conduct deep, high-throughput functional annotation of all risk loci. We establish a network of physical interactions for all risk SNPs to candidate casual genes in 3D space, using high-throughput chromosome conformation capture techniques (HiC) in TGCT cells. Firstly, functional evidence reveals widespread disruption of developmental transcriptional regulators, consistent with failed primordial germ cell differentiation as an initiating step in TGCT oncogenesis15. We secondly observe multiple risk loci associated with defective microtubule assembly, compatible with the high level of aneuploidy observed in TGCTs, suggesting gross chromosomal instability. Finally KIT-MAPK signalling features as a recurrently dysregulated pathway. In summary our findings substantially increase the number of known TGCT risk alleles, and provides a functional basis for disease susceptibility.

PROVIDER: EGAS00001001836 | EGA |

REPOSITORIES: EGA

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Project description:Malignant (type II) testicular germ cell tumors (TGCT) are considered tumors of embryonic germ cell ancestry that diverge morphologically as seminoma (SE), and nonseminomas (NS), the latter including embryonal carcinoma (EC), teratoma (TE), yolk sac tumor (YS) and choriocarcinoma. We have analyzed the genomes and epigenomes of pure histological forms of TGCT (n=130) and 128 matched adjacent normal testicular tissues for TGCT core and subtype-specific DNA methylome profiles and somatic copy number aberrations (SCNA). Original data were compared with published data sets focused on differentiated and pluripotent states. First, we identified pan-TGCT recurrent erasure of maternal and paternal germline imprints and DPPA3 (STELLA), consistent with an embryonic germ cell ancestry. Beyond these conserved properties, we identified TGCT subtype-dependent epigenomic congruency with pluripotent versus somatic reference lineages, thereby establishing competency for lineage-conforming methylation reprogramming in the multi-potent common ancestor of TGCT. Notable subtype programming distinctions are as follows: the SE methylome reflects a ground-state of germ cell erasure; EC is found to harbor pervasive embryonic stem cell (ESC)-like CpH (non-CpG) methylation, absent in other TGCT and non-germ cell tumors and differentiated tissues. EC was further distinguished by ESC-like hypomethylation of NANOG, contrasting with NANOG methylation in TE, YS, and somatic tissues. Thus, EC methylomes present a novel mixed ESC/embryonic germ cell epigenomic state. TE global methylation was most convergent with somatic tissue; however, and unique among TGCT subtypes, TE manifested hypermethylation of the H19 paternal germline DMR. The YS methylome most closely resembled extra-embryonic trophoblast. The total set of findings provides evidence for a multi-potent TGCT stem cell whose progeny may harbor pluripotent, primordial germ/gonocyte, and somatic lineage-defining methylation marks. Despite such competency, no TGCT subtype restored erased parental germline imprints. TE is the exception, with H19 hypermethylation. Benign testis adjacent to TGCT exhibited a bimodal epigenotype distribution determined by spermatogenesis and significantly associated with high versus low Johnsen score. Overall, TGCT methylomes are trapped in a core embryonic germ cell-like state of DPPA3/STELLA and imprint erasure, while differential somatic and pluripotent reprogramming profiles are otherwise established.

Project description:Malignant germ-cell-tumours (GCTs) are characterised by microRNA (miRNA/miR-) dysregulation, with universal over-expression of miR-371~373 and miR-302/367 clusters regardless of patient age, tumour site, or subtype (seminoma/yolk-sac-tumour/embryonal carcinoma). These miRNAs are released into the bloodstream, presumed within extracellular-vesicles (EVs) and represent promising biomarkers. Here, we comprehensively examined the role of EVs, and their miRNA cargo, on (fibroblast/endothelial/macrophage) cells representative of the testicular GCT (TGCT) tumour microenvironment (TME). Small RNA next-generation-sequencing was performed on 34 samples, comprising representative malignant GCT cell lines/EVs and controls (testis fibroblast [Hs1.Tes] cell-line/EVs and testis/ovary samples). TME cells received TGCT co-culture, TGCT-derived EVs, and a miRNA overexpression system (miR-371a-OE) to assess functional relevance. TGCT cells secreted EVs into culture media. MiR-371~373 and miR-302/367 cluster miRNAs were overexpressed in all TGCT cells/subtypes compared with control cells and were highly abundant in TGCT-derived EVs, with miR-371a-3p/miR-371a-5p the most abundant. TGCT co-culture resulted in increased levels of miRNAs from the miR-371~373 and miR-302/367 clusters in TME (fibroblast) cells. Next, fluorescent labelling demonstrated TGCT-derived EVs were internalised by all TME (fibroblast/endothelial/macrophage) cells. TME (fibroblast/endothelial) cell treatment with EVs derived from different TGCT subtypes resulted in increased miR-371~373 and miR-302/367 miRNA levels, and other generic (eg, miR-205-5p/miR-148-3p) and subtype-specific (seminoma, eg, miR-203a-3p; yolk-sac-tumour, eg, miR-375-3p) miRNAs. MiR-371a-OE in TME cells resulted in increased collagen contraction (fibroblasts) and angiogenesis (endothelial cells), via direct mRNA downregulation and alteration of relevant pathways. TGCT cells communicate with nontumour stromal TME cells through release of EVs enriched in oncogenic miRNAs, potentially contributing to tumour progression.

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Identification of 19 novel loci reveals gene regulatory mechanisms determining susceptibility to testicular germ cell tumour

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