The miR-17~92 cluster collaborates with the Sonic Hedgehog pathway in medulloblastoma

Abstract

Medulloblastomas (MBs) are the most common brain tumors in children. Some are thought to originate from cerebellar granule neuron progenitors (GNPs) that fail to undergo normal cell cycle exit and differentiation. Because microRNAs regulate numerous aspects of cellular physiology and development, we reasoned that alterations in miRNA expression might contribute to MB. We tested this hypothesis using 2 spontaneous mouse MB models with specific initiating mutations, Ink4c-/-; Ptch1+/- and Ink4c-/-; p53-/-. We found that 26 miRNAs showed increased expression and 24 miRNAs showed decreased expression in proliferating mouse GNPs and MBs relative to mature mouse cerebellum, regardless of genotype. Among the 26 overexpressed miRNAs, 9 were encoded by the miR-17 approximately 92 cluster family, a group of microRNAs implicated as oncogenes in several tumor types. Analysis of human MBs demonstrated that 3 miR-17 approximately 92 cluster miRNAs (miR-92, miR-19a, and miR-20) were also overexpressed in human MBs with a constitutively activated Sonic Hedgehog (SHH) signaling pathway, but not in other forms of the disease. To test whether the miR-17 approximately 92 cluster could promote MB formation, we enforced expression of these miRNAs in GNPs isolated from cerebella of postnatal (P) day P6 Ink4c-/-; Ptch1+/- mice. These, but not similarly engineered cells from Ink4c-/-; p53-/- mice, formed MBs in orthotopic transplants with complete penetrance. Interestingly, orthotopic mouse tumors ectopically expressing miR-17 approximately 92 lost expression of the wild-type Ptch1 allele. Our findings suggest a functional collaboration between the miR-17 approximately 92 cluster and the SHH signaling pathway in the development of MBs in mouse and man.

Fig. 1.

MiRNA signature during cerebellar development and in mouse MB models. Illumina deep sequencing reveals 50 miRNAs differentially expressed in whole 1-month-old and P6 cerebella, and in purified P6 GNPs from cerebella of wild-type (WT), Ink4c−/−; Ptch1+/−, Ink4c−/−; p53−/− mice, or in MBs (red) from both genotypes. Tumor and P6 GNP samples, considered together, were contrasted against adult cerebella and miRNAs were filtered to require a 1% FDR and a 4-fold change between the groups.

Fig. 2.

The miR-17∼92 cluster family is differentially expressed in proliferating GNPs and GNP-like tumor cells compared to postmitotic 1-month-old cerebella. Schematic expression profiles of the 3 miRNA clusters reveals increasing levels of the miR-17∼92 cluster family in proliferating GNPs and GNP-like tumor cells. Expression levels as sequencing read counts are indicated as color-scaled boxes. Each profile as a group was an average of multiple samples. Expression levels of miR-124a and miR-21, nonmembers of the miR-17∼92 cluster family, are shown as controls. wc, whole cerebellum; WT, wild type.

Fig. 3.

Human MBs with a SHH/PATCHED gene signature express individual miRs from the miR-17∼92 cluster. Quantitative RT-PCR analysis of selected microRNAs from the miR-17∼92 cluster using RNA extracted from human MBs, purified GNPs from P6 mice, and normal human cerebella tissue. MB tumor patient samples were previously molecularly characterized (13).

Fig. 4.

Molecular characterization of “engineered” MBs in mice after enforced expression of the miR-17∼92 cluster in GNPs from P6 Ink4c−/−; Ptch1+/− mice. Enforced expression of miR-17∼92 in P6 GNPs from Ink4c−/−; Ptch1+/− mice induced MBs in recipient mice. QRT-PCR analysis was performed in (A) and (B) using RNA extracted from purified GNPs from P6 mouse cerebella, 1-month (1M) whole mouse cerebellum, and GNP-like tumor cells from MBs engineered and spontaneously arising in Ink4c−/−; Ptch1+/− mice (MB 65939) (the numbers indicate the recipient mice). (A) miR-17∼92 expression in induced tumors was confirmed by analysis of selected individual microRNAs from the cluster (miR-19a, miR-92). (B) Induced tumors express high levels of Math1 and Gli1 mRNA. (C) GNP-like tumor cells overexpressing the miR-17∼92 cluster remain sensitive to cyclopamine: tumor cells from 2 independently derived MBs (tumor nos. 64199 and 64389) treated or not with cyclopamine were measured for proliferation by BrdU incorporation (Left panel) and for expression of Gli1 and Math1 RNA expression by QRT-PCR (Right panel).