MicroRNA profiling in human medulloblastoma Elisabetta Ferretti 1 , Enrico De Smaele 1 , Agnese Po 1 , Lucia Di Marcotullio 1 , Emanuele Tosi 1 , Maria Salome B. Espinola 1 , Concezio Di Rocco 2 , Riccardo Riccardi 3 , Felice Giangaspero 1,4 , Alessio Farcomeni 5 , Italo Nofroni 5 , Pietro Laneve 6 , Ubaldo Gioia 6,7 , Elisa Caffarelli 6 , Irene Bozzoni 6,7,8 , Isabella Screpanti 1,7 and Alberto Gulino 1,4,7 * 1 Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy 2 Neurosurgery Institute, Catholic University, Rome, Italy 3 Pediatrics Department, Catholic University, Rome, Italy 4 Neuromed Institute, Pozzilli (IS), Italy 5 Statistics Section, Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy 6 Institute of Molecular Biology and Pathology, National Research Council, Rome, Italy 7 Pasteur Institute, Cenci-Bolognetti Foundation, Rome, Italy 8 Department of Genetics and Molecular Biology, Sapienza University of Rome, Rome, Italy Medulloblastoma is an aggressive brain malignancy with high incidence in childhood. Current treatment approaches have lim- ited efficacy and severe side effects. Therefore, new risk-adapted therapeutic strategies based on molecular classification are required. MicroRNA expression analysis has emerged as a power- ful tool to identify candidate molecules playing an important role in a large number of malignancies. However, no data are yet avail- able on human primary medulloblastomas. A high throughput microRNA expression profiles was performed in human primary medulloblastoma specimens to investigate microRNA involvement in medulloblastoma carcinogenesis. We identified specific micro- RNA expression patterns which distinguish medulloblastoma dif- fering in histotypes (anaplastic, classic and desmoplastic), in mo- lecular features (ErbB2 or c-Myc overexpressing tumors) and in disease-risk stratification. MicroRNAs expression profile clearly differentiates medulloblastoma from either adult or fetal normal cerebellar tissues. Only a few microRNAs displayed upregulated expression, while most of them were downregulated in tumor sam- ples, suggesting a tumor growth-inhibitory function. This property has been addressed for miR-9 and miR-125a, whose rescued expression promoted medulloblastoma cell growth arrest and apo- ptosis while targeting the proproliferative truncated TrkC iso- form. In conclusion, misregulated microRNA expression profiles characterize human medulloblastomas, and may provide potential targets for novel therapeutic strategies. ' 2008 Wiley-Liss, Inc. Key words: microRNA; medulloblastoma; miRNA-125a; miRNA-9 Medulloblastoma (MB) is the most frequent brain malignancy observed in childhood and originates from aberrant development of cerebellar progenitor neurons. 1,2 However, the molecular aspects of its tumorigenic pathways are so far poorly understood. MB multimodal treatments (surgical resection, chemotherapy and/ or radiotherapy) have improved survival; nevertheless, it is still in- curable in about a third of cases and survivors commonly have severe treatment-induced long-term side effects. 3 Novel and risk- adapted therapeutic strategies are thus needed and an appropriate molecular classification is essential to improve assignment to dis- ease risk classes and to identify subsets of patients responsive to targeted therapies. MBs are genetically and epigenetically hetero- geneous and are associated with several molecular alterations that can be exploited as prognostic markers. 4 Recently, a high-through- put complementary DNA (cDNA) microarray analysis of human primary MBs suggested different subsets of tumors based on clus- tered gene-expression patterns. 5 These specific mRNA expression signatures are related with known or still unknown signal trans- duction pathways, which are potential targets for specific therapies in distinct subsets of tumors. 5 In addition to protein-encoding genes, a second class of genes producing small noncoding RNAs (i.e. microRNAs) has been dis- covered over the last few years. 6 These short RNAs (18- to 24-nu- cleotides) bind to cis-regulatory elements mainly present in the 3 0 UTR of mRNAs, resulting in translational inhibition or mRNA degradation. 6 MicroRNAs (miRNAs or miRs) have emerged as important regulatory factors involved in developmental processes, such as neural progenitor cell growth and differentiation. 7 The critical role played by miRNAs is also suggested by their altered expression observed in a large number of malignancies. 8 In addi- tion, the ability of some miRNAs to target oncogenes or oncosup- pressors indicates their role in tumorigenesis. 8 Although miRNAs play a crucial role in nervous system devel- opment, so far specific changes in their expression patterns have been described only in neural crest-derived neuroblastoma 9 and glial cell-derived gliomas, 10,11 while a few cell lines were ana- lyzed from neuronal tumors of the central nervous system (CNS). 12 Here, we report the first miRNA expression profiling of human primary MB, a CNS tumor that has an ontogenetic cell lineage dif- ferent from neuronal crest-derived cells and glial progenitors. We show that specific miRNA signatures distinguish tumors from ei- ther adult or fetal normal tissues and identify distinct classes of MB. Typical miRNA expression patterns classify MB histotypes, correlating with tumor molecular features and disease risk stratifi- cation. Most miRNAs display overall downregulated expression in MB, suggesting a tumor growth-inhibitory function. This property has been validated for miR-9 and miR-125a, whose rescued expression promotes MB cell growth arrest and apoptosis while targeting the proproliferative truncated TrkC (t-TrkC) isoform. We suggest that miRNA expression signatures identify human MBs and may indicate targets for novel therapeutic strategies. Material and methods MB and control tissues Surgical specimens of primary MBs were collected from 34 patients with Institutional Review Board approval. The clinical Additional Supporting Information may be found in the online version of this article. Grant sponsor: Associazione Italiana per la Ricerca sul Cancro, Tele- thon; Grant number: GGP07118; Grant sponsor: European Union Sixth Research Framework Programs RIGHT; Grant number: LSHB-CT-2004 005276; Grant sponsor: SIROCCO; Grant number: 037900; Grant spon- sors: Ministry of University and Research, Ministry of Health, Rome Oncogenomic Center, ESF project ‘‘NuRNASu’’, AIRC/Fondazione Ital- iana per la Ricerca sul Cancro (fellowship to U.G.). Elisabetta Ferretti and Enrico De Smaele contributed equally to this work. *Correspondence to: Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy. E-mail: alberto.gulino@uniroma1.it Received 17 June 2008; Accepted after revision 11 August 2008 DOI 10.1002/ijc.23948 Published online 30 October 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 124, 568–577 (2009) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer