The miR-17/92 cluster is recurrently amplified in medulloblastoma
We profiled 201 primary human MBs using Affymetrix SNP arrays to delineate recurrent copy number aberrations (CNAs) that may contribute to MB pathogenesis (8
). We identified two MBs with recurrent, focal, high-level amplification on chromosome 13q31.3, sharing a minimal common region that spans ~1.82 Mb (). The only gene mapping to this amplified locus is miR-17/92
(NCBI Build 36.1). Amplification of this miR cluster has not previously been reported in MB. Further examination of the tumours harboring miR-17/92
amplification revealed amplification of MYCN
(). We subsequently carried out interphase fluorescence in situ hybridization (FISH) on a MB tissue microarray (TMA) to determine the incidence of miR-17/92 amplification in a non-overlapping series of 80 MBs. Low-level amplification of miR-17/92 was identified in ~6% (5/80) of cases (). Taken together, these results suggest that miR-17/92 functions as an oncogene in a subset of MBs.
Recurrent amplification of the miR-17/92 locus in primary human medulloblastomas
miR-17/92 is over-expressed in human and murine MBs
We performed a genome-wide survey of 427 mature miRNAs in a series of 90 primary human MBs and 10 normal human cerebella (CB; 5 fetal, 5 adult). Unsupervised hierarchical clustering of samples and differentially expressed miRNAs in the dataset could easily discriminate MBs from normal CB samples (). Notably, components of miR-17/92 including miR-18a, miR-19b, and miR-20a, as well as the paralogous miR-106a (miR-106a/363 cluster) clustered together, were expressed at low levels in the normal CB, and showed considerably higher levels of expression in most MBs ().
Over-expression of miR-17/92 in human and murine medulloblastomas
Statistical comparison of miRNA profiles for MBs versus normal CB samples revealed consistent over-expression of miR-17/92 and its related paralogs (miR-106a/363 and miR-106b/25) in MB (; Supplementary Table 1
). Re-analysis of the data after removing the 22 probe sets which detect components of miR-17/92 or paralogous clusters, shows that there are relatively few remaining over-expressed miRs in MB compared to normal CB ( right panel).
As miR-17/92 was over-expressed in a large percentage of human MBs compared to normal CB, we examined its expression in murine MB. Medulloblastomas from NeuroD2-SmoA1 and Ptc+/- mice showed marked over-expression of the miR-17/92 cluster compared to cerebellum from age-matched tumour-free littermates ().
miR-17/92 up-regulation is associated with activated Sonic hedgehog signaling in human MB
Aberrant activation of the Shh pathway through mutation of pathway members has been documented in 25-30% of medulloblastomas (5
). To sub-classify the 90 MBs employed in miRNA expression profiling above, we performed mRNA expression analysis for 17,881 mRNAs on the same cohort of MBs. Unsupervised hierarchical clustering using 1300 differentially expressed mRNAs segregated four unique molecular subgroups: WNT (blue), SHH (red), Group C (yellow), and Group D (green) (, Supplementary Figure 1A, B
). These four subgroups were supported by their expression pattern (Supplementary Table 2
) and specific genomic features, including monosomy 6 (WNT), chromosome 9q loss (SHH), and isochromosome 17q (Group C and Group D) (). MiR-17/92 was most highly expressed in the SHH subgroup, followed by Group C, and the WNT subgroup (, Supplementary Figure 2
, Supplementary Table 3
miR-17/92 is over-expressed in SHH-dependent medulloblastomas and tumours with elevated MYC family expression
Confirming previous reports (10
), we observed high MYCN expression in the SHH tumours, whereas MYC levels were most elevated in WNT and Group C tumours (, Supplementary Figure 3
). MYC and MYCN have both been reported to transcriptionally regulate miR-17/92 (11
). We compared miR-17/92 expression between tumours with higher MYCN/MYC expression to tumours with lower expression to determine whether miR-17/92 regulation might also be myc-dependent in MB. As shown in , components of miR-17/92 (miR-17, miR-20a, miR-92a) and related paralogs (miR-106a, miR-20b, miR-25, miR-93) represented the majority of up-regulated miRNAs in MBs with higher MYCN/MYC (n=51) expression as compared to lower expressing MYCN/MYC (n=39) tumours (Supplementary Table 4
We carried out TaqMan miRNA assays to validate the correlation between MYCN/MYC and miR-17/92 expression observed on the array platforms. Samples were divided into 3 groups of 10 tumours each: higher MYCN, higher MYC, and lower MYCN/MYC and then qRT-PCR was performed for MYCN, MYC, miR-17, and miR-18. As predicted from the mRNA array data, the high MYCN (p=3.33E-08) and high MYC (p=3.33E-08) tumours did not overlap (, top and middle panels). Importantly, miR-17 (p=4.92E-05) and miR-18 (p=3.75E-05) were significantly up-regulated in both the higher MYCN and higher MYC expressing groups as compared to the lower MYCN/MYC expressing group (, lower panel). These results provide strong evidence that up-regulation of the miR-17/92 polycistron may be MYCN/MYC-dependent in MBs.
miR-17/92 is up-regulated by Shh signaling in primary CGNP cultures
To determine whether the relationship between activated Shh signaling and miR-17/92 up-regulation we observed in MBs reflected co-option of developmental programs, we cultured murine CGNPs with or without exogenous Shh (+/- cyclohexamide) for 24h, then performed array-based miRNA profiling. Of 599 mouse miRNAs assayed, 19 were significantly changed, 9 up-regulated and 10 down-regulated (, Supplementary Table 5
). The miR-17/92 polycistron was up-regulated in Shh-treated CGNPs, but not in the presence of cycloheximide indicating that a new protein intermediate needs to be synthesized to regulate miR-17/92
Sonic Hedgehog and N-myc drive the expression of miR-17/92 in cerebellar neural precursor cells resulting in mitosis
Validation by qRT-PCR in , shows the six miRNAs within the miR-17/92 cluster were consistently up-regulated by Shh, which was abrogated by cyclohexamide (data not shown). These results indicate that the association between activated Shh signaling and miR-17/92 expression is conserved between normal Shh mitogenic activity in CGNPs and oncogenic Shh signaling in MB.
miR-17/92 cluster induces proliferation of CGNPs downstream of N-myc
We have previously shown that N-myc is a downstream target of Shh whose induction is not protein synthesis dependent, and which can drive CGNP proliferation in the absence of Shh signaling (7
). We asked whether miR-17/92 was regulated by N-myc in CGNPs. We infected CGNPs with retroviruses carrying N-myc or the stabilized mutant N-mycT50A
that can prolong CGNP proliferation in vitro
). N-myc transduction resulted in increased expression of the miR-17/92 cluster, in the presence and absence of Shh (). In contrast, neither Gli1 nor Gli2 expression induced miR-17/92 in the absence of Shh; indeed, Gli1 and Gli2 suppressed Shh-mediated miR-17/92 expression. These results indicate that the Shh pathway effectors N-myc and Gli regulate different microRNA targets.
Since N-myc expression alone is sufficient to drive CGNP proliferation, we asked whether miR-17/92 contributes to the N-myc-regulated proliferation program. We infected CGNPs with retroviruses expressing five of the six miRNAs within the miR-17/92 cluster (pWzl-miR-17-19b) (15
). After 48 hours, we measured CGNP proliferation by quantifying Ki67 staining. Over-expression of the miR-17/92 cluster increased proliferation in Shh-treated cells (). miR-17/92 alone was able to maintain cell proliferation in the absence of Shh, albeit not at the same levels as Shh alone, suggesting that its expression does not recapitulate the complete Shh/N-myc proliferative response.
In summary, we have shown that high levels of miR-17/92 amplification and over-expression are a hallmark of SHH-associated MB in humans and in mice, and that its expression correlates with high levels of MYC family proto-oncogenes. We also show that in normally proliferating CGNPs miR-17/92 is a Shh target whose expression is regulated by N-myc. Our finding that Shh regulates expression of an oncogenic microRNA provide additional insights as to the mechanisms through which Shh drives cell cycle progression. Our observation that miR-17/92 expression increases Shh-mediated CGNP proliferation provides insight into its role in human MB, suggesting that high levels of miR-17/92 can provide cells with a selective growth advantage through an enhanced proliferative capacity. A role for miR17-92 in tumour cell survival may also be at play, as its targets identified in lymphoma include PTEN and the pro-apoptotic p53 target TP53INP1 (16