Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells Hideto Kosoa,b, Haruna Takedaa,c, Christopher Chin Kuan Yewa, Jerrold M. Warda, Naoki Nariaid, Kazuko Uenod, Masao Nagasakid, Sumiko Watanabeb, Alistair G. Ruste, David J. Adamse, Neal G. Copelanda,f, and Nancy A. Jenkinsa,f,1 aDivision of Genetics and Genomics, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore 138673; bDivision of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; cDepartment of Microbiology, University of Tokyo, Tokyo 113-0033, Japan; dDepartment of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, Sendai 980-8579, Japan; eExperimental Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom; and fCancer Research Program, Methodist Hospital Research Institute, Houston, TX 77030 Contributed by Nancy A. Jenkins, September 14, 2012 (sent for review July 6, 2012) Neural stem cells (NSCs) are considered to be the cell of origin of with these GBM subtypes, providing additional support for their glioblastoma multiforme (GBM). However, the genetic alterations existence. Numerous other, often rare, mutations have also been that transform NSCs into glioma-initiating cells remain elusive. identified in GBM. Although these datasets are valuable for un- Using a unique transposon mutagenesis strategy that mutagenizes derstanding the molecular pathogenesis of GBM, it is still difficult NSCs in culture, followed by additional rounds of mutagenesis to to distinguish between mutations that contributed to tumor initi- generate tumors in vivo, we have identified genes and signaling ation and those acquired later during tumor progression. pathways that can transform NSCs into glioma-initiating cells. The cell of origin (COO) of GBM is still controversial. Neural Mobilization of Sleeping Beauty transposons in NSCs induced the stem cells (NSCs) are good candidates because the adult brain has immortalization of astroglial-like cells, which were then able to gen- very few proliferating cells capable of accumulating the numerous erate tumors with characteristics of the mesenchymal subtype of mutations required for gliomagenesis. NSCs are also more sus- GBM on transplantation, consistent with a potential astroglial origin ceptible to malignant transformation than differentiated cells in for mesenchymal GBM. Sequence analysis of transposon insertion the adult brain (6, 7). However, the genetic pathways that can sites from tumors and immortalized cells identified more than 200 transform NSCs into glioma-initiating cells still remain elusive. frequently mutated genes, including human GBM-associated genes, Transposon-based mutagenesis provides an unbiased, high- such as Met and Nf1, and made it possible to discriminate between throughput method for identifying genes important for GBM (8). genes that function during astroglial immortalization vs. later Here, we describe a unique two-step insertional mutagenesis stages of tumor development. We also functionally validated five strategy that makes it possible to identify genes and signaling GBM candidate genes using a previously undescribed high-through- pathways that are able to transform a NSC into a cancer-initiating put method. Finally, we show that even clonally related tumors cell for the mesenchymal subtype of GBM. In this two-step ap- derived from the same immortalized line have acquired distinct proach, NSCs are first mutagenized in vitro and the mutagenized combinations of genetic alterations during tumor development, cells are then transplanted into immunocompromised mice for suggesting that tumor formation in this model system involves subsequent tumor development following additional rounds of competition among genetically variant cells, which is similar to the transposon-based mutagenesis. This makes it possible to dis- Darwinian evolutionary processes now thought to generate many criminate between the genetic changes that occur early in tumor human cancers. This mutagenesis strategy is faster and simpler than initiation and those required for tumor progression. In addition conventional transposon screens and can potentially be applied to to identifying several previously undescribed GBM candidate any tissue stem/progenitor cells that can be grown and differenti- cancer genes, our studies suggest that transposon-induced ated in vitro. tumors mimic the evolutionary processes now thought to gen- erate many human cancers, in which tumors have a branched lioblastoma multiforme (GBM) is the most common form of cellular and genetic architecture reminiscent of Darwin’s iconic Gmalignant brain cancer in adults. Patients with GBM have evolutionary tree. a uniformly poor prognosis, with a mean survival of 1 y (1). Thus, advances on all fronts, both basic and applied, are needed to Results combat this deadly disease better. Recent studies have provided Transposon Mutagenesis of NSCs Promotes the Immortalization of evidence for self-renewing, stem-like cells within human gliomas Astroglial-Like Cells. Mice homozygous for a conditional floxed- (2). These glioma-initiating cells constitute a small minority of stop Sleeping Beauty (SB) transposase allele knocked in to the fi neoplastic cells within a tumor and are de ned operationally by ubiquitously expressed Rosa26 locus (Rosa26-LSL-SB11) (9) and their ability to seed new tumors (3). To target these rare glioma- up to 350 copies of a mutagenic SB transposon (T2/Onc2 or initiating cells, a better understanding of the molecular mechanisms T2/Onc3) all linked together at a single site in the genome that regulate their formation is essential. (termed the donor transposon concatamer) (9, 10) were crossed Considerable progress has been made in understanding the mutations responsible for GBM. The Cancer Genome Atlas net- work has cataloged the recurrent genomic abnormalities in GBM Author contributions: H.K., N.G.C., and N.A.J. designed research; H.K. and H.T. performed by genome-wide DNA copy number events and sequence-based research; N.N., K.U., M.N., S.W., A.G.R., and D.J.A. contributed new reagents/analytic mutation detection for 601 genes (4). Gene expression-based tools; H.K., C.C.K.Y., J.M.W., N.N., and A.G.R. analyzed data; and H.K., N.G.C., and molecular classification has also defined four subtypes of GBM N.A.J. wrote the paper. termed proneural, neural, classical, and mesenchymal (5). Pro- The authors declare no conflict of interest. neural GBM is enriched for the oligodendrocyte gene signature, Data deposition: The array data reported in this paper have been deposited in the Gene whereas the classical group is associated with the astrocytic Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo/ (accession no. GSE3689). signature. The neural class is enriched for genes differentially 1To whom correspondence should be addressed. E-mail: [email protected]. expressed by neurons, whereas the mesenchymal class is associated See Author Summary on page 17746 (volume 109, number 44). with the cultured astroglial signature (5). Several recurrent muta- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tions, such as PDGFRA, IDH1, EGFR,andNF1, also correlate 1073/pnas.1215899109/-/DCSupplemental. E2998–E3007 | PNAS | Published online October 8, 2012 www.pnas.org/cgi/doi/10.1073/pnas.1215899109 Downloaded by guest on September 25, 2021 to Nestin-Cre (Nes-cre) transgenic mice. Nes-cre is expressed in FBS, and were then cultured for 2 wk (Fig. 1A), which led to the PNAS PLUS NSCs, and will thus activate SB transposase, and hence SB appearance of cells that stained positive for neuronal, astrocytic, transposition, specifically in the NSC compartment (11). Brains and oligodendrocytic markers (Fig. 1 D and E). The cells were were collected from embryos of various different genotypes be- then reseeded every week, counted, and plotted against time tween embryonic day 17 (E17) and postnatal day 1 (P1) of de- (Fig. 1F). During early passages, total cell numbers continuously velopment (Fig. 1A). At this stage, the subventricular zone decreased; however, after several passages, exponentially grow- (SVZ) is a rich source of NSCs (12). By culturing SVZ cells with ing populations of cells were obtained. The cells were considered EGF, multipotent self-renewing NSCs could be propagated as immortalized once the cumulative total cell number exceeded clonal aggregates, denoted as neurospheres (13) (Fig. 1B). As 108. A conditional dominant-negative mutant form of p53 (LSL- expected, excision PCR (14) showed that transposons were being p53R172H) (15) was also included in many experiments as a sen- mobilized in these neurospheres (Fig. 1C). The NSCs were then sitizing mutation because p53 is one of the most frequently induced to differentiate by removal of EGF and the addition of mutated genes in GBM (4). MEDICAL SCIENCES Fig. 1. Transposon mutagenesis in NSCs promotes the immortalization of astroglial-like cells. (A) Mutagenesis strategy. NSCs isolated from the SVZ of mice with active SB transposition were expanded in vitro and induced to differentiate to select for immortalized cells. (B) Nestin+ and Sox2+ NSCs were expanded as neurospheres. (Magnification: 400×.) (C) Transposon excision from the transposon concatamer could be detected in neurospheres containing a nestin-cre allele by the presence of a 225-bp fragment. NSCs that were induced to differentiate