Biology of childhood germ cell tumours, focussing on the significance of microRNAs

Abstract

Genomic and protein-coding transcriptomic data have suggested that germ cell tumours (GCTs) of childhood are biologically distinct from those of adulthood. Global messenger RNA profiles segregate malignant GCTs primarily by histology, but then also by age, with numerous transcripts showing age-related differential expression. Such differences are likely to account for the heterogeneous clinico-pathological behaviour of paediatric and adult malignant GCTs. In contrast, as global microRNA signatures of human tumours reflect their developmental lineage, we hypothesized that microRNA profiles would identify common biological abnormalities in all malignant GCTs owing to their presumed shared origin from primordial germ cells. MicroRNAs are short, non-protein-coding RNAs that regulate gene expression via translational repression and/or mRNA degradation. We showed that all malignant GCTs over-express the miR-371-373 and miR-302/367 clusters, regardless of patient age, histological subtype or anatomical tumour site. Furthermore, bioinformatic approaches and subsequent Gene Ontology analysis revealed that these two over-expressed microRNAs clusters co-ordinately down-regulated genes involved in biologically significant pathways in malignant GCTs. The translational potential of this finding has been demonstrated with the detection of elevated serum levels of miR-371-373 and miR-302/367 microRNAs at the time of malignant GCT diagnosis, with levels falling after treatment. The tumour-suppressor let-7 microRNA family has also been shown to be universally down-regulated in malignant GCTs, because of abundant expression of the regulatory gene LIN28. Low let-7 levels resulted in up-regulation of oncogenes including MYCN, AURKB and LIN28 itself, the latter through a direct feedback mechanism. Targeting LIN28, or restoring let-7 levels, both led to effective inhibition of this pathway. In summary, paediatric malignant GCTs show biological differences from their adult counterparts at a genomic and protein-coding transcriptome level, whereas they both display very similar microRNA expression profiles. These similarities and differences may be exploited for diagnostic and/or therapeutic purposes.

Keywords: LIN28; biomarker; germ cell tumour; let-7; miR-302/367; miR-371-373; microRNA; serum.

Figure 1

Principal component analysis of paediatric and adult malignant germ cell tumour (GCT) protein-coding gene expression data [adapted from (Palmer et al., 2008)]. Each malignant GCT sample is represented by a coloured circle; the number next to the circle shows the age of the relevant patient in years. The distance between samples represents biological (protein-coding transcriptome) differences. Consequently, those samples clustering close together demonstrate biological similarity, whereas those that are segregated are more dissimilar.

Figure 2

Differential expression of the miR-371–373 and miR-302/367 clusters in malignant germ cell tumours (GCTs) [adapted from (Palmer et al., 2010)]. Hierarchical clustering analysis based on the eight main microRNAs from the miR-371–373 and miR-302/367 clusters (rows) segregates (A) paediatric and (B) adult malignant GCT samples from non-malignant controls (comprising benign teratomas and normal gonadal controls) (columns). In the heatmap, red represents relative microRNA over-expression and blue represents under-expression. Green columns = normal gonadal controls; brown = teratoma; blue = seminoma; yellow = yolk sac tumour; red = embryonal carcinoma.

Figure 3

MicroRNAs from the miR-371–373 and miR-302/367 clusters as novel serum biomarkers of malignant germ cell tumours (GCTs) [adapted from (Murray & Coleman, 2012)]. Levels of miR-372 from the miR-371–373 cluster (left) and miR-367 from the miR-302/367 cluster (right) in the serum at the time of diagnosis in eight malignant GCTs of different patient age, anatomical site and histological subtype. Key: EG, extragonadal; TGCT, testicular germ cell tumour. Yellow columns = yolk sac tumour samples; red = embryonal carcinoma; blue = seminoma.

Figure 4

Schematic of the dysregulated LIN28/let-7 axis in malignant germ cell tumours (GCT)s. Let-7 down-regulation in malignant GCTs is attributable to abundant LIN28 expression (Murray et al., 2013). In turn, this leads to over-expression of let-7 targets (e.g. MYCN) and increased cellular proliferation. Loss of direct negative feedback from let-7 maintains high LIN28 levels and let-7 repression. Other contributions to abundant LIN28 expression include other microRNAs that bind to the 3′UTR of LIN28 transcripts, but which are down-regulated in malignant GCTs (Palmer et al., 2010). Taken together, these findings highlight the LIN28/let-7 axis as a novel therapeutic target in malignant GCTs.