Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups

Abstract

The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1(lacZ/+)), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1(lacZ/+) controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups.

Fig. 1.

Sleeping Beauty (SB) mutagenesis accelerated tumorigenesis and penetrance of medulloblastoma (MB) in the Ptch1 heterozygous mouse model. Three cohorts of mice were monitored for tumor development, with all three cohorts carrying the SB transposon and SB transposase. Inactivation of tumor suppressor Ptch1 was required for MB development, with β-actin Cre required for expression of SB transposase. Ptch1^lacZ/+ SB mutagenized mice succumbed significantly earlier to MB (n = 94 of 135) than animals with inactivated Ptch1^lacZ/+ alone (n = 8 of 27) (P < 0.0001, log-rank test).

Fig. 2.

Clustering of human medulloblastoma (MB) samples based on the expression of CIS genes, CIS network, and human gene signatures. Expression of the CISs identified from Sleeping Beauty (SB)-induced MB accurately clustered samples into the four known molecular subgroups of human MB. Unsupervised clustering is based on (A) 49 human protein coding genes that map to the CIS list, with principal component analysis (PCA) reducing these data to the first five principal components; (B) 49 human proteins from A, plus the local interaction network of these proteins; and (C) the human signature gene list from ref. , which had been previously optimized to separate the four molecular subtypes of human MB. RI scores for the goodness of clustering of each of these gene sets are (A) 0.469, (B) 0.685, and (C) 0.699. The colored bar below the dendrogram shows the subgroup annotation provided with the expression data (8).

Fig. 3.

Heat map and hierarchical clustering of the human MB samples using 49 human protein coding orthologs of the mouse genes from the CIS list.

Fig. 4.

Circos plot of MB common insertion sites (CISs) identified by the Gaussian kernel convolution (GKC) method. Mouse chromosomes are illustrated on the outer perimeter of the plot. GKC CISs are illustrated on the second most outer ring of the plot marking SB insertions in both the forward and reverse orientation. The number of unique insertions at each CIS is represented by orange bars, with the number of tumors containing each CIS represented by blue bars. The black lines in the center connect CISs that significantly co-occur in tumors (Fisher exact test, P < 0.01).

Fig. 5.

Local protein interaction network for the human orthologs of protein coding genes identified in the SB mutagenesis screen. Local protein interactome hubs for MAP3K1, CREBBP, and NFIA are circled as A, B, and C, respectively. Nodes colored in red represent proteins encoded by CIS-derived candidate genes that overlap with the Wu et al. (15) dataset, whereas yellow nodes represent the proteins that interact with these CIS-derived candidates. Nodes with a diamond shape represent proteins with an annotated role in DNA-dependent transcription.

Fig. 6.

Inactivation of Nfia accelerates MB formation. Kaplan–Meier survival curves comparing Math1Cre; Nfia^+/−; Ptch1^lox/+ (blue line, n = 12 of 21) experimental mice and control Math1Cre; Nfia^+/+; Ptch1^lox/+ (red line, n = 6 of 16) mice (P = 0.038, log-rank test).