Aberrant activation of the Hedgehog (Hh) pathway can drive tumorigenesis1. To investigate the mechanism by which glioma-associated oncogene family zinc finger-1 (GLI1), a crucial effector of Hh signaling2, regulates Hh pathway activation, we searched for GLI1-interacting proteins. We report that the chromatin remodeling protein SNF5 (encoded by SMARCB1, hereafter called SNF5), which is inactivated in human malignant rhabdoid tumors (MRTs), interacts with GLI1. We show that Snf5 localizes to Gli1-regulated promoters and that loss of Snf5 leads to activation of the Hh-Gli pathway. Conversely, re-expression of SNF5 in MRT cells represses GLI1. Consistent with this, we show the presence of a Hh-Gli–activated gene expression profile in primary MRTs and show that GLI1 drives the growth of SNF5-deficient MRT cells in vitro and in vivo. Therefore, our studies reveal that SNF5 is a key mediator of Hh signaling and that aberrant activation of GLI1 is a previously undescribed targetable mechanism contributing to the growth of MRT cells.
Medulloblastomas, the most common malignant pediatric brain tumors, are comprised of four molecularly distinct subtypes. However, treatment has yet to exploit these molecular vulnerabilities. Three recent studies sequenced a total of 310 primary tumors and identified that two of the four medulloblastoma subtypes are concomitantly associated with subtype-specific mutations as previously characterized. In contrast, the overwhelming majority of mutations occurred only once in the entire cohort and just 12 genes were recurrently mutated with statistical significance. Perturbations in epigenetic regulation are emerging as a unifying theme in cancer and similarly recurring mutations in epigenetic mechanisms were distributed across all subtypes in medulloblastoma. Designing targeted therapies to such a molecularly diverse disease in the post-genomic era presents new challenges. This will require novel methods to link these nonrecurrent mutations into pathways, and preclinical models that faithfully recapitulate patient driver events. Presently, medulloblastoma reinforces epigenetic mechanisms as a tantalizing therapeutic target in cancers.
cerebellum; chromatin; epigenetics; epigenomics; medulloblastoma; Swi/Snf; systems biology
Emerging evidence demonstrates that subunits of the SWI/SNF chromatin remodeling complex are specifically mutated at high frequency in a variety of human cancer types. SNF5 (SMARCB1/INI1/BAF47), a core subunit of the SWI/SNF complex, is inactivated in the vast majority of rhabdoid tumors (RT), an aggressive type of pediatric cancer. SNF5-deficient cancers are diploid and genomically stable, suggesting that epigenetically based changes in transcription are key drivers of tumor formation caused by SNF5 loss. However, there is limited understanding of the target genes that drive cancer formation following SNF5 loss. Here we performed comparative expression analyses upon three independent SNF5-deficient cancer data sets from both human and mouse and identify downregulation of the BIN1 tumor suppressor as a conserved event in primary SNF5-deficient cancers. We show that SNF5 recruits the SWI/SNF complex to the BIN1 promoter, and that the marked reduction of BIN1 expression in RT correlates with decreased SWI/SNF occupancy. Functionally, we demonstrate that re-expression of BIN1 specifically compromises the proliferation of SNF5-deficient RT cell lines. Identification of BIN1 as a SNF5 target gene reveals a novel tumor suppressive regulatory mechanism whose disruption can drive cancer formation.
BAF47; BIN1; INI1; rhabdoid tumor; SMARCB1; SNF5
Recent evidence argues that the oncogenesis and growth of CNS tumors occurs through dysregulated molecular and cellular mechanisms of neural development. New insights have emerged that have had a significant impact on both research and treatment of these cancers.
embryonal tumors; medulloblastoma; astrocytoma; stem cells; cerebellum; cerebellar granule cells
Medulloblastomas are heterogeneous tumors that collectively represent the most common malignant brain tumor in children. To understand the molecular characteristics underlying their heterogeneity and to identify whether such characteristics represent risk factors for patients with this disease, we performed an integrated genomic analysis of a large series of primary tumors.
Patients and Methods
We profiled the mRNA transcriptome of 194 medulloblastomas and performed high-density single nucleotide polymorphism array and miRNA analysis on 115 and 98 of these, respectively. Non-negative matrix factorization–based clustering of mRNA expression data was used to identify molecular subgroups of medulloblastoma; DNA copy number, miRNA profiles, and clinical outcomes were analyzed for each. We additionally validated our findings in three previously published independent medulloblastoma data sets.
Identified are six molecular subgroups of medulloblastoma, each with a unique combination of numerical and structural chromosomal aberrations that globally influence mRNA and miRNA expression. We reveal the relative contribution of each subgroup to clinical outcome as a whole and show that a previously unidentified molecular subgroup, characterized genetically by c-MYC copy number gains and transcriptionally by enrichment of photoreceptor pathways and increased miR-183∼96∼182 expression, is associated with significantly lower rates of event-free and overall survivals.
Our results detail the complex genomic heterogeneity of medulloblastomas and identify a previously unrecognized molecular subgroup with poor clinical outcome for which more effective therapeutic strategies should be developed.
Despite significant progress in the molecular understanding of medulloblastoma, stratification of risk in patients remains a challenge. Focus has shifted from clinical parameters to molecular markers, such as expression of specific genes and selected genomic abnormalities, to improve accuracy of treatment outcome prediction. Here, we show how integration of high-level clinical and genomic features or risk factors, including disease subtype, can yield more comprehensive, accurate, and biologically interpretable prediction models for relapse versus no-relapse classification. We also introduce a novel Bayesian nomogram indicating the amount of evidence that each feature contributes on a patient-by-patient basis.
Patients and Methods
A Bayesian cumulative log-odds model of outcome was developed from a training cohort of 96 children treated for medulloblastoma, starting with the evidence provided by clinical features of metastasis and histology (model A) and incrementally adding the evidence from gene-expression–derived features representing disease subtype–independent (model B) and disease subtype–dependent (model C) pathways, and finally high-level copy-number genomic abnormalities (model D). The models were validated on an independent test cohort (n = 78).
On an independent multi-institutional test data set, models A to D attain an area under receiver operating characteristic (au-ROC) curve of 0.73 (95% CI, 0.60 to 0.84), 0.75 (95% CI, 0.64 to 0.86), 0.80 (95% CI, 0.70 to 0.90), and 0.78 (95% CI, 0.68 to 0.88), respectively, for predicting relapse versus no relapse.
The proposed models C and D outperform the current clinical classification schema (au-ROC, 0.68), our previously published eight-gene outcome signature (au-ROC, 0.71), and several new schemas recently proposed in the literature for medulloblastoma risk stratification.
Recent studies have established miR-34a as a key effector of the p53 signaling pathway and have implicated its role in multiple cancer types. Here, we establish that miR-34a induces apoptosis, G2 arrest, and senescence in medulloblastoma and renders these cells more sensitive to chemotherapeutic agents. These effects are mediated in part by the direct post-transcriptional repression of the oncogenic MAGE-A gene family. We demonstrate that miR-34a directly targets the 3′ untranslated regions of MAGE-A genes and decreases MAGE-A protein levels. This decrease in MAGE-A results in a concomitant increase in p53 and its associated transcriptional targets, p21/WAF1/CIP1 and, importantly, miR-34a. This establishes a positive feedback mechanism where miR-34a is not only induced by p53 but increases p53 mRNA and protein levels through the modulation of MAGE-A genes. Additionally, the forced expression of miR-34a or the knockdown of MAGE-A genes by small interfering RNA similarly sensitizes medulloblastoma cells to several classes of chemotherapeutic agents, including mitomycin C and cisplatin. Finally, the analysis of mRNA and micro-RNA transcriptional profiles of a series of primary medulloblastomas identifies a subset of tumors with low miR-34a expression and correspondingly high MAGE-A expression, suggesting the coordinate regulation of these genes. Our work establishes a role for miR-34a in modulating responsiveness to chemotherapy in medulloblastoma and presents a novel positive feedback mechanism involving miR-34a and p53, via direct targeting of MAGE-A.
chemosensitivity; MAGE-A; medulloblastoma; miR-34a; p53; positive feedback mechanism
Medulloblastoma, a small blue cell malignancy of the cerebellum, is a major cause of morbidity and mortality in pediatric oncology. Current mechanisms for clinical prognostication and stratification include clinical factors (age, presence of metastases, and extent of resection) as well as histological subgrouping (classic, desmoplastic, and large cell/anaplastic histology). Transcriptional profiling studies of medulloblastoma cohorts from several research groups around the globe have suggested the existence of multiple distinct molecular subgroups that differ in their demographics, transcriptomes, somatic genetic events, and clinical outcomes. Variations in the number, composition, and nature of the subgroups between studies brought about a consensus conference in Boston in the fall of 2010. Discussants at the conference came to a consensus that the evidence supported the existence of four main subgroups of medulloblastoma (Wnt, Shh, Group 3, and Group 4). Participants outlined the demographic, transcriptional, genetic, and clinical differences between the four subgroups. While it is anticipated that the molecular classification of medulloblastoma will continue to evolve and diversify in the future as larger cohorts are studied at greater depth, herein we outline the current consensus nomenclature, and the differences between the medulloblastoma subgroups.
Medulloblastoma; Consensus; Subgroups; SHH; WNT; Group 3; Group 4
Neuralized (Neurl) is a highly conserved E3 ubiquitin ligase, which in Drosophila acts upon Notch ligands to regulate Notch pathway signaling. Human Neuralized1 (NEURL1) was investigated as a potential tumor suppressor in medulloblastoma (MB). The gene is located at 10q25.1, a region demonstrating frequent loss of heterozygosity in tumors. In addition, prior publications have shown that the Notch pathway is functional in a proportion of MB tumors and that Neurl1 is only expressed in differentiated cells in the developing cerebellum. In this study, NEURL1 expression was downregulated in MB compared with normal cerebellar tissue, with the lowest levels of expression in hedgehog-activated tumors. Control of gene expression by histone modification was implicated mechanistically; loss of 10q, sequence mutation, and promoter hypermethylation did not play major roles. NEURL1-transfected MB cell lines demonstrated decreased population growth, colony-forming ability, tumor sphere formation, and xenograft growth compared with controls, and a significant increase in apoptosis was seen on cell cycle and cell death analysis. Notch pathway inhibition occurred on the exogenous expression of NEURL1, as shown by decreased expression of the Notch ligand, Jagged1, and the target genes, HES1 and HEY1. From these studies, we conclude that NEURL1 is a candidate tumor suppressor in MB, at least in part through its effects on the Notch pathway.
deazaneplanocin; Jagged1; Neuralized1; Notch; tumor suppressor
The diagnosis of medulloblastoma likely encompasses several distinct entities, with recent evidence for the existence of at least four unique molecular subgroups that exhibit distinct genetic, transcriptional, demographic, and clinical features. Assignment of molecular subgroup through routine profiling of high-quality RNA on expression microarrays is likely impractical in the clinical setting. The planning and execution of medulloblastoma clinical trials that stratify by subgroup, or which are targeted to a specific subgroup requires technologies that can be economically, rapidly, reliably, and reproducibly applied to formalin-fixed paraffin embedded (FFPE) specimens. In the current study, we have developed an assay that accurately measures the expression level of 22 medulloblastoma subgroup-specific signature genes (CodeSet) using nanoString nCounter Technology. Comparison of the nanoString assay with Affymetrix expression array data on a training series of 101 medulloblastomas of known subgroup demonstrated a high concordance (Pearson correlation r = 0.86). The assay was validated on a second set of 130 non-overlapping medulloblastomas of known subgroup, correctly assigning 98% (127/130) of tumors to the appropriate subgroup. Reproducibility was demonstrated by repeating the assay in three independent laboratories in Canada, the United States, and Switzerland. Finally, the nanoString assay could confidently predict subgroup in 88% of recent FFPE cases, of which 100% had accurate subgroup assignment. We present an assay based on nanoString technology that is capable of rapidly, reliably, and reproducibly assigning clinical FFPE medulloblastoma samples to their molecular subgroup, and which is highly suited for future medulloblastoma clinical trials.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-011-0899-7) contains supplementary material, which is available to authorized users.
Medulloblastoma; Molecular classification; Clinical trials; NanoString
Epigenetic alterations have been increasingly implicated in oncogenesis. Analysis of Drosophila mutants suggests that Polycomb and SWI/SNF complexes can serve antagonistic developmental roles. However, the relevance of this relationship to human disease is unclear. Here we have investigated functional relationships between these epigenetic regulators in oncogenic transformation. Mechanistically, we show that loss of the SNF5 tumor suppressor leads to elevated expression of the Polycomb gene EZH2 and that Polycomb targets are broadly H3K27-trimethylated and repressed in SNF5-deficient fibroblasts and cancers. Further, we show antagonism between SNF5 and EZH2 in the regulation of stem cell-associated programs and that Snf5 loss activates those programs. Finally, using conditional mouse models, we show that inactivation of Ezh2 blocks tumor formation driven by Snf5 loss.
SNF5; EZH2; SWI/SNF; oncogenesis; Polycomb
Tumour cells release an abundance of microvesicles containing a selected set of proteins and RNAs. Here, we show that tumour microvesicles also carry DNA, which reflects the genetic status of the tumour, including amplification of the oncogene c-Myc. We also find amplified c-Myc in serum microvesicles from tumour-bearing mice. Further, we find remarkably high levels of retrotransposon RNA transcripts, especially for some human endogenous retroviruses, such as LINE-1 and Alu retrotransposon elements, in tumour microvesicles and these transposable elements could be transferred to normal cells. These findings expand the nucleic acid content of tumour microvesicles to include: elevated levels of specific coding and non-coding RNA and DNA, mutated and amplified oncogene sequences and transposable elements. Thus, tumour microvesicles contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer.
Peripheral nerve injury is followed by a wave of Schwann cell proliferation in the distal nerve stumps. To resolve the role of Schwann cell proliferation during functional recovery of the injured nerves, we used a mouse model in which injury-induced Schwann cell mitotic response is ablated via targeted disruption of cyclin D1. In the absence of distal Schwann cell proliferation, axonal regeneration and myelination occur normally in the mutant mice and functional recovery of injured nerves is achieved. This is enabled by pre-existing Schwann cells in the distal stump that persist but do not divide. On the other hand, in the wild type littermates, newly generated Schwann cells of injured nerves are culled by apoptosis. As a result, distal Schwann cell numbers in wild type and cyclin D1 null mice converge to equivalence in regenerated nerves. Therefore, distal Schwann cell proliferation is not required for functional recovery of injured nerves.
There is a growing appreciation of the role that epigenetic alterations can play in oncogenesis. However, given the large number of genetic anomalies present in most cancers, it has been difficult to evaluate the extent to which epigenetic changes contribute to cancer. SNF5 (INI1/SMARCB1/BAF47) is a tumor suppressor that regulates the epigenome as a core member of the SWI/SNF chromatin remodeling complex. While the SWI/SNF complex displays potent tumor suppressor activity, it is unknown whether this activity is exerted genetically via maintenance of genome integrity or epigenetically via transcriptional regulation. Here we show that Snf5-deficient primary cells do not show altered sensitivity to DNA damaging agents, defects in γ-H2AX induction, or an abrogated DNA damage checkpoint. Further, the aggressive malignancies that arise following SNF5 loss are diploid and genomically stable. Remarkably, we demonstrate that most human SNF5-deficient cancers lack genomic amplifications/deletions and, aside from SNF5 loss, are indistinguishable from normal cells on single-nucleotide polymorphism arrays. Finally, we show that epigenetically based changes in transcription that occur following SNF5 loss correlate with the tumor phenotype. Collectively, our results provide novel insight into the mechanisms of oncogenesis by demonstrating that disruption of a chromatin remodeling complex can largely, if not completely, substitute for genomic instability in the genesis of aggressive cancer.
To address the hypothesis that medulloblastoma or supratentorial primitive neuroectodermal tumor (sPNET) can arise through infection by polyomaviruses, we examined genomic DNA isolated from 15 primary medulloblastoma and 5 sPNET biopsy specimens and from 2 medulloblastoma cell lines for the presence of DNA sequences from the polyomaviruses simian virus 40 (SV40), JC virus, and BK virus. These polyomaviruses have oncogenic potential in animals, and their DNA sequences have been detected in other surveys of various solid tumors, including childhood brain tumors. The tumor DNA samples were analyzed by Southern blot hybridization of polymerase chain reaction products that employed probes designed to detect specific polyomavirus sequences. Neither JC virus nor BK virus DNA sequences were detected in any of the specimens. None of the primary medulloblastoma or sPNET specimens contained SV40 sequences. However, SV40 DNA coding and noncoding sequences were detected in the D283-Med (medulloblastoma) cell line. Immunocytochemical studies of D283-Med revealed nuclear expression of SV40 large T antigen. In contrast to childhood ependymomas and choroid plexus tumors, medulloblastomas and sPNETs infrequently express evidence of polyomavirus infection.
Childhood Central Nervous System Primitive Neuro-Ectodermal brain Tumours (CNS-PNETs) are highly aggressive brain tumours for which molecular features and best therapeutic strategy remains unknown. We interrogated a large cohort of these rare tumours in order to identify molecular markers that will enhance clinical management of CNS-PNET.
Transcriptional and copy number profiles from primary hemispheric CNS-PNETs were examined using clustering, gene and pathways enrichment analyses to discover tumour sub-groups and group-specific molecular markers. Immuno-histochemical and/or gene expression analyses were used to validate and examine the clinical significance of novel sub-group markers in 123 primary CNS-PNETs.
Three molecular sub-groups of CNS-PNETs distinguished by primitive neural (Group 1), oligo-neural (Group 2) and mesenchymal lineage (Group 3) gene expression signature were identified. Tumour sub-groups exhibited differential expression of cell lineage markers, LIN28 and OLIG2, and correlated with distinct demographics, survival and metastatic incidence. Group 1 tumours affected primarily younger females; male: female ratios were respectively 0.61 (median age 2.9 years; 95% CI: 2.4–5.2; p≤ 0.005), 1.25 (median age 7.9 years; 95% CI: 6–9.7) and 1.63 (median age 5.9 years; 95% CI: 4.9–7.8) for group 1, 2 and 3 patients. Overall outcome was poorest in group 1 patients which had a median survival of 0.8 years (95% CI: 0.47–1.2; p=0.019) as compared to 1.8 years (95% CI: 1.4–2.3) and 4.3 years; (95% CI: 0.82–7.8) respectively for group 2 and 3 patients. Group 3 tumours had the highest incidence of metastases at diagnosis; M0: M+ ratio were respectively 0.9 and 3.9 for group 3, versus group 1 and 2 tumours combined (p=0.037).
LIN28 and OLIG2 represent highly promising, novel diagnostic and prognostic molecular markers for CNS PNET that warrants further evaluation in prospective clinical trials.
We discovered a high-level amplicon involving the chr19q13.41 microRNA (miRNA) cluster (C19MC) in 11/45(~25%) primary CNS-PNET which results in striking over-expression of miR-517c and 520g. Constitutive expression of miR-517c or 520g promotes in vitro and in vivo oncogenicity, modulates cell survival and robustly enhances growth of untransformed human neural stem cells (hNSCs) in part by upregulating WNT pathway signaling and restricting differentiation of hNSCs. Remarkably, the C19MC amplicon, which is very rare in other brain tumors (1/263), identify an aggressive sub-group of CNS-PNET with distinct gene expression profiles, characteristic histology and dismal survival. Our data implicate miR-517c and 520g as oncogenes and promising biological markers for CNS-PNET and provide important insights into oncogenic properties of the C19MC locus.
Medulloblastoma is the most common malignant brain tumor in childhood. Molecular studies from several groups around the world demonstrated that medulloblastoma is not one disease but comprises a collection of distinct molecular subgroups. However, all these studies reported on different numbers of subgroups. The current consensus is that there are only four core subgroups, which should be termed WNT, SHH, Group 3 and Group 4. Based on this, we performed a meta-analysis of all molecular and clinical data of 550 medulloblastomas brought together from seven independent studies. All cases were analyzed by gene expression profiling and for most cases SNP or array-CGH data were available. Data are presented for all medulloblastomas together and for each subgroup separately. For validation purposes, we compared the results of this meta-analysis with another large medulloblastoma cohort (n = 402) for which subgroup information was obtained by immunohistochemistry. Results from both cohorts are highly similar and show how distinct the molecular subtypes are with respect to their transcriptome, DNA copy-number aberrations, demographics, and survival. Results from these analyses will form the basis for prospective multi-center studies and will have an impact on how the different subgroups of medulloblastoma will be treated in the future.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-012-0958-8) contains supplementary material, which is available to authorized users.
Medulloblastoma; Pediatric brain tumor; Subgroups; Meta-analysis
We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific β-tubulin isotype III, result in a spectrum of human nervous system disorders we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves, and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate normal TUBB3 is required for axon guidance and maintenance in mammals.
Medulloblastoma is the most common malignant brain tumor of childhood. Improvements in clinical outcome require a better understanding of the genetic alterations to identify clinically significant biological factors and to stratify patients accordingly. In the present study, we applied cytogenetic characterization to guide the identification of biologically significant genes from gene expression microarray profiles of medulloblastoma.
We analyzed 71 primary medulloblastomas for chromosomal copy number aberrations (CNAs) using comparative genomic hybridization (CGH). Among 64 tumors that we previously analyzed by gene expression microarrays, 27 were included in our CGH series. We analyzed clinical outcome with respect to CNAs and microarray results. We filtered microarray data using specific CNAs to detect differentially expressed candidate genes associated with survival.
The most frequent lesions detected in our series involved chromosome 17; loss of 16q, 10q, or 8p; and gain of 7q or 2p. Recurrent amplifications at 2p23-p24, 2q14, 7q34, and 12p13 were also observed. Gain of 8q is associated with worse overall survival (p = 0.0141), which is not entirely attributable to MYC amplification or overexpression. By applying CGH results to gene expression analysis of medulloblastoma, we identified three 8q-mapped genes that are associated with overall survival in the larger group of 64 patients (p < 0.05): eukaryotic translation elongation factor 1D (EEF1D), ribosomal protein L30 (RPL30), and ribosomal protein S20 (RPS20).
The complementary use of CGH and expression profiles can facilitate the identification of clinically significant candidate genes involved in medulloblastoma growth. We demonstrate that gain of 8q and expression levels of three 8q-mapped candidate genes (EEF1D, RPL30, RPS20) are associated with adverse outcome in medulloblastoma.