Identification of differentially expressed and developmentally regulated genes in medulloblastoma using suppression subtraction hybridization NaokiYokota 1,2,3 ,ToddGMainprize 1,2 ,MichaelDTaylor 1,2 ,TomohikoKohata 1,2 , MichaelLoreto 1,2 ,ShigeoUeda 1,2 ,WieslawDura 4 ,WiesiaGrajkowska 4 ,JohnSKuo 1,2 andJamesTRutka 1,2, * 1 The Arthur and Sonia Labatt Brain Tumor Research Centre, The University of Toronto, Toronto, Ontario, Canada; 2 The Division of Neurosurgery, The University of Toronto, Toronto, Ontario, Canada; 3 The Department of Neurosurgery, Hamamatsu University School of Medicine, Hamamatsu, Japan; 4 Department of Pathology, University of Warsaw, Warsaw, Poland To increase our understanding of the molecular pathogen- esisofmedulloblastoma(MB),weutilizedthetechniqueof suppression subtractive hybridization (SSH) to identify genes that are dysregulated in MB when compared to cerebellum. SSH-enriched cDNA libraries from both human and Ptch þ / heterozygous murine MBs were generated by subtracting common cDNAs from corre- sponding non-neoplastic cerebellum. For the human classic MB library, total human cerebellar RNA was used as control tissue; for the Ptch þ / heterozygous MB, non-neoplastic cerebellum from an unaffected Ptch þ / littermate was used as the control. Through differential screening of these libraries, over 100 upregulated tumor cDNA fragments were isolated, sequenced and identified with the NCBI BLAST program. From these, we selected genes involved in cellular proliferation, antiapoptosis, and cerebellardifferentiationforfurtheranalysis.Upregulated genes identified in the human MB library included Unc33- like protein (ULIP), SOX4, Neuronatin (NNAT), the mammalian homologue of Drosophila BarH-like 1(BARHL1), the nuclear matix protein NRP/B (ENC1), and the homeobox OTX2 gene. Genes found to be upregulated in the murine MB library included cyclin D2 (Ccnd2), thymopoietin (Tmpo), Musashi-1 (Msh1), protein phosphatase 2A inhibitor-2 (I-2pp2a), and Unc5h4(D). Using semiquantitative reverse transcrip- tion–polymerase chain reaction (RT–PCR), the mRNA expression levels for these genes were markedly higher in humanMBsthanincerebellum.Westernblotanalysiswas used to further confirm the overexpression of a subset of these genes at the protein level. Notch pathway over- activity was demonstrated in the TE671 MB cell line expressing high levels of MSH1 through HES1-Lucifer- ase transfections. This study has revealed a panel of developmentally regulated genes that may be involved in the pathogenesis of MB. Oncogene (2004) 23, 3444–3453.doi:10.1038/sj.onc.1207475 Publishedonline5April2004 Keywords: medulloblastoma;developmentallyregulated gene; differentially expressed gene; subtractive suppres- sionhybridization;cerebellum;externalgranulecell Introduction Despite the paucity of knowledge regarding the mole- cular oncogenesis of most human brain tumors, our understanding of the molecular pathways causing medulloblastoma (MB), the most common malignant braintumorinchildren,hasadvancedconsiderably.We now know that MB can occur in the setting of two major inherited genetic syndromes. In these two hereditary diseases, Turcot and Gorlin syndrome, the Wnt and Hedgehog (Hh) signaling pathways respec- tively,aredysregulatedandmayleadtoMBaswellas otherdevelopmentalmalformationsandcancers(Taylor et al., 2000; Wechsler-Reya and Scott, 2001; Yokota etal.,2002).MBisalsofoundinapproximately20%of offspringfromheterozygoustransgenicmicecarryinga targeted deletion of the Ptch gene that results in constitutive Hh signaling (Goodrich et al., 1997; Wetmore et al., 2001). Both Wnt and Hh signaling pathways play crucial roles in the proliferation and differentiation of cerebellar granule cells, from which MBsarethoughttoarise(Yokota et al.,1996;Buhren et al.,2000). Primitive neuroectodermal tumors (PNETs), such as MB, are characterized by dysregulation of cellular proliferation, apoptosis, or differentiation signals that normally occur at critical developmental stages in neurogenesis (Wechsler-Reya and Scott, 2001). By histology, MB is composed mainly of undifferentiated small, round blue cells. However, many MBs exhibit markersofneuronaldifferentiationincludingexpression of neuronal transcriptional factors from the PAX (Kozmik etal.,1995;Vincent etal.,1996), ZIC (Yokota et al.,1996;Michiels et al.,1999;Pomeroy et al.,2002), and NEUROD gene families (Rostomily et al., 1997). Theseneuronaltranscriptionalfactorsaredevelopmen- tally regulated and govern the expression of several genesthatinfluencecellfateinthecerebellargranulecell Received7September2003;revised5January2004;accepted5January 2004;Publishedonline5April2004 *Correspondence: JT Rutka, The Division of Neurosurgery, Suite 1502, The Hospital for Sick Children, 555 University Avenue, Toronto,Ontario,CanadaM5G1X8; E-mail:james.rutka@sickkids.ca Oncogene (2004) 23, 3444–3453 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc