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.
a- Division of Genetics and Genomics, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore 138673
b- Division of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
c- Department of Microbiology, University of Tokyo, Tokyo 113-0033, Japan
d- Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, Sendai 980-8579, Japan
e- Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom
f- Cancer Research Program, Methodist Hospital Research Institute, Houston, TX 77030
Published ahead of print in PNAS on 8th October 2012.
Neural stem cells (NSCs) are considered to be the cell of origin of glioblastoma multiforme (GBM). However, the genetic alterations that transform NSCs into glioma-initiating cells remain elusive. Using a unique transposon mutagenesis strategy that mutagenizes NSCs in culture, followed by additional rounds of mutagenesis to generate tumors in vivo, we have identified genes and signaling pathways that can transform NSCs into glioma-initiating cells. Mobilization of Sleeping Beauty transposons in NSCs induced the immortalization of astroglial-like cells, which were then able to generate tumors with characteristics of the mesenchymal subtype of GBM on transplantation, consistent with a potential astroglial origin for mesenchymal GBM. Sequence analysis of transposon insertion sites from tumors and immortalized cells identified more than 200 frequently mutated genes, including human GBM-associated genes, such as Met and Nf1, and made it possible to discriminate between genes that function during astroglial immortalization vs. later stages of tumor development. We also functionally validated five GBM candidate genes using a previously undescribed high-throughput method. Finally, we show that even clonally related tumors derived from the same immortalized line have acquired distinct combinations of genetic alterations during tumor development, suggesting that tumor formation in this model system involves competition among genetically variant cells, which is similar to the Darwinian evolutionary processes now thought to generate many human cancers. This mutagenesis strategy is faster and simpler than conventional transposon screens and can potentially be applied to any tissue stem/progenitor cells that can be grown and differentiated in vitro.
Figure Legend: Model for transposon-induced tumor development.
Immortalization CIS genes (e.g., Gli3, Nf1) promote immortalization of NSCs. Immortalized astroglial-like cells have a large repertoire of insertions represented by different colors. Transposons are continuously jumping after transplantation, and transplanted cells become cancer-initiating cells (green) by randomly acquiring new insertions in tumor CIS genes (e.g., Met, Pdgfrb, Gab1) and clonally expand to form tumors. Several immortalization CIS genes are lost during tumor formation by continuous mobilization of transposons.