Identification of Foxr2 As an Oncogene in Medulloblastoma

Published OnlineFirst March 5, 2014; DOI: 10.1158/0008-5472.CAN-13-1523 Cancer Tumor and Stem Cell Biology Research Identification of FoxR2 as an Oncogene in Medulloblastoma Hideto Koso1,2, Asano Tsuhako1, Eli Lyons1, Jerrold M. Ward2, Alistair G. Rust3, David J. Adams3, Nancy A. Jenkins2,4, Neal G. Copeland2,4, and Sumiko Watanabe1 Abstract Medulloblastoma is the most common pediatric brain tumor, and in 25% of cases, it is driven by aberrant activation of the Sonic Hedgehog (SHH) pathway in granule neuron precursor (GNP) cells. In this study, we identified novel medulloblastoma driver genes through a transposon mutagenesis screen in the developing brain of wild-type and Trp53 mutant mice. Twenty-six candidates were identified along with established driver genes such as Gli1 and Crebbp. The transcription factor FoxR2, the most frequent gene identified in the screen, is overexpressed in a small subset of human medulloblastoma of the SHH subtype. Tgif2 and Alx4, 2 new putative oncogenes identified in the screen, are strongly expressed in the SHH subtype of human medulloblastoma. Mutations in these two genes were mutually exclusive with mutations in Gli1 and tended to cooccur, consistent with involvement in the SHH pathway. Notably, Foxr2, Tgif2, and Alx4 activated Gli-binding sites in cooperation with Gli1, strengthening evidence that they function in SHH signaling. In support of an oncogenic function, Foxr2 overexpression transformed NIH3T3 cells and promoted proliferation of GNPs, the latter of which was also observed for Tgif2 and Alx4. These findings offer forward genetic and functional evidence associating Foxr2, Tgif2, and Alx4 with SHH subtype medulloblastoma. Cancer Res; 74(8); 1–11. Ó2014 AACR. Introduction of the dorsal brainstem (2). Although mutations in the SHH and Medulloblastoma is the most common malignant brain WNT pathway seem mutually exclusive, these alterations affect tumor of childhood and tends to metastasize throughout the only 30% to 40% of medulloblastomas (3), suggesting that other central nervous system. Despite overall improvements in sur- pathways are also operative in medulloblastoma formation. To vival with multimodal treatment, a substantial proportion of better understand the pathogenesis of medulloblastomas, a patients are still incurable. Moreover, survivors often suffer comprehensive understanding of genes and pathways that considerable treatment-related morbidities, such as neurocog- regulate tumor formation in the cerebellum is essential. nitive deficits related to radiation therapy. To develop better Genome-wide insertional mutagenesis is an unbiased and fi therapies, insights into the molecular mechanisms leading to high-throughput method to pro le the landscape of driver genes the development of medulloblastomas are essential. Recent in a mouse model system (4). The Sleeping Beauty (SB) transposon molecular studies have shown that medulloblastomas are system has expanded the applicability of insertional mutagenesis composed of at least four distinct subtypes: Sonic Hedgehog for the study of various types of solid tumors (5). To identify genes (SHH), WNT, Group 3, and Group 4 (1). Tumors characterized and pathways that can transform embryonic neural stem and by aberrant activation of the SHH pathway (SHH subtype) progenitor cells to brain tumor-initiating cells, we applied SB originate from cerebellar granule neuron precursor (GNP), transposon mutagenesis to the developing mouse brain. A whereas tumors with activating mutations in the WNT effector previous SB mutagenesis study screened medulloblastoma can- CTNNB1 (WNT subtype) arise outside the cerebellum from cells didate genes by analyzing recurrent insertions in medulloblastomas that developed on Ptch1 heterozygous or Trp53 (þ/À and À/À) mutant genetic backgrounds (6) and identified genes that Authors' Affiliations: 1Division of Molecular and Developmental Biology, promote metastatic dissemination of medulloblastomas (7). In The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; contrast, we used wild-type or Trp53 mutant (R172H/þ)genetic 2 Division of Genetics and Genomics, Institute of Molecular and Cell Biol- backgrounds that do not develop medulloblastomas spontane- ogy, Agency for Science, Technology and Research, Singapore, Singa- pore; 3Experimental Cancer Genetics, Wellcome Trust Sanger Institute, ously. Transposition of the mutagenic SB transposons induced Hinxton, Cambridge, United Kingdom; and 4Cancer Research Program, medulloblastomas in the cerebellum even on the wild-type The Methodist Hospital Research Institute, Houston, Texas background. Here, we describe the identification and functional Note: Supplementary data for this article are available at Cancer Research analyses of candidate genes that may regulate the initiation Online (http://cancerres.aacrjournals.org/). and/or progression of medulloblastoma. Corresponding Authors: Sumiko Watanabe, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5663; Fax: 81-3-5449-5474; E-mail: Materials and Methods [email protected]; and Neal G. Copeland, [email protected] Mouse strains doi: 10.1158/0008-5472.CAN-13-1523 Mouse strains used in this study are described previously (8). Ó2014 American Association for Cancer Research. All manipulations were performed with institutional animal www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst March 5, 2014; DOI: 10.1158/0008-5472.CAN-13-1523 Koso et al. care and use committee approval (Institute of Molecular and (Promega) and sequenced. Real-time PCR analysis was car- Cell Biology). ried out on Light Cycler 1.5 (Roche) using primers in Supplementary Table S1. Cell culture Mouse embryonic fibroblasts (MEF) were generated from Results ICR mouse embryos at embryonic day 13.5. Primary cerebellar Sleeping Beauty mutagenesis promotes medulloblastoma cultures were prepared from the cerebellum of ICR mice at formation in the cerebellum postnatal day 5 as described (9). Briefly, the cerebellum was To identify novel driver genes for medulloblastoma, we treated with 0.25% trypsin with 0.1% DNaseI (Invitrogen), and mobilized SB in the brains of wild-type and Trp53R172H mutant then dissociated by trituration. The dissociated cells were mice. Trp53R172H is an inducible lox-stop-lox allele with both cultured in DMEM/F12 medium supplemented with 10% FBS dominant-negative and gain-of-function properties (15). Brief- and penicillin/streptomycin. MEFs, cerebellar cells, and ly, Nestin-cre (Nes-Cre) transgenic mice (16) were crossed with NIH3T3 cells (American Type Culture Collection, ATCC) were Trp53R172H mutant mice (15) to generate compound hetero- infected with retrovirus and selected for puromycin resistance. zygous Nes-cre/þ; Trp53R172H/þ mice. These mice were then For cotransduction experiments, doubly infected cells were crossed to mice homozygous for a transposon concatemer selected with puromycin and G418. Foci formation assay and carrying up to 350 copies of the mutagenic transposon T2/ cell-proliferation assay in low serum media were performed as Onc2 (17) and an inducible LSL-SB transposase (SBase) tar- described (10). Cerebellar cells were plated to 6-well plates for geted to the Rosa26 locus (18) to generate triple (Nes-cre/þ;T2/ colony formation assay (more than 50 colonies were counted Onc2/þ; SBase/þ) and quadruple (Trp53R172H/þ; Nes-cre/þ; from three samples), or to 8-well chamber slide for immunos- T2/Onc2/þ; SBase/þ) mice. Trp53R172H was used as a sensi- taining (more than 100 Math1-positive cells were counted in tizing mutation because mutant Trp53 is one of the key drivers three samples). of medulloblastoma (19), whereas Nes-cre was used to activate Trp53R172H and SB transposition in neural stem and progenitor Immunohistochemical analysis cells. Triple transgenic mice showed enlargement of the head Immunohistochemistry was performed as described (8). at approximately 2 to 4 months of age and a gait disorder, Each tumor was obtained from a different mouse. Anti-Math1 whereas necropsy of these mice revealed tumors within the antibody is a generous gift from Dr. J. Johnson (University of cerebellum (Fig. 1A–C). In contrast, control animals (T2/Onc2/ Texas Southwestern Medical Center, Dallas TX; ref. 11). The þ; SBase/þ) remained tumor free (Fig. 1A, black line). Hema- detection of senescence-associated b-galactosidase activity toxylin and eosin (H&E) staining of tumors showed classical was performed as described (12). features of medulloblastoma, including small round tumors composed of sheets of undifferentiated cells with minimal Production of retrovirus stock cytoplasm (Fig. 1D and E). Tumors were negative for glioma Retroviral expression vectors (pMXs-IRES-Puro and pMXs- markers such as GFAP and Nestin (Fig. 1F and G), providing IRES-Neo) were obtained from Cell Biolabs, Inc. A plasmid further evidence they are medulloblastomas. Trp53R172H/þ; coding for full-length Foxr2 was obtained from OriGene. A Nes-cre/þ mice also succumbed to tumors but the tumors truncated Gli1 was excised from full-length Gli1 (OriGene) with were of skin cell origin (15). Trp53R172H mutant mice with active KpnI (located in exon 5) and XhoI to remove the N-terminal 125 SB transposition also developed medulloblastomas in the amino acids required for interaction with Sufu (13). A trun- cerebellum but at a higher frequency than in triple transgenic cated form

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