Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells

Abstract

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.

Identification of New Tumor Suppressor Genes in Triple-Negative Breast Cancer

“…Insertional inactivation of ZNF326 has also been associated with the immortalization of astroglial-like neural stem cells in SB mutagenesis studies (22). Moreover, in embryonic stem cells, ZNF326 has been shown to form complexes with NANOG and SOX2 and play an important role in stem cell self-renewal (30), suggesting that ZNF326 might also function in CSC self-renewal.…”

“…ZNF326 regulates the expression of multiple genes in the CSC pathway Interestingly, ZNF326 was also identified in a transposon screen that identified genes, which can promote the immortalization of atroglial-like cells (22). Likewise, other studies in embryonic stem cells have shown that ZNF326 forms complexes with Nanog-Sox2, which plays an important role in stem cell selfrenewal (30).…”

“…The sleeping beauty (SB) transposon system has expanded the use of insertional mutagenesis for the study of many types of cancers (11)(12)(13)(14)(15)(16)(17)(18)(19)(20); and discovered candidate cancer genes (CCG) involved in the EMT and CSC pathways (21)(22)(23). To better understand the genes and pathways involved in the pathogenesis of TNBC, we performed an siRNA validation study of 40 SB-identified breast cancer TS genes identified in an SB mutagenesis screen performed in Pten mutant mice (24).…”

Identification of RNA-Binding Protein LARP4B as a Tumor Suppressor in Glioma

“…Transposon-based insertional mutagenesis is useful as it allows for an unbiased forward genetic screen for cancer genes in mice (3,4), thereby providing a functional readout to complement cancer genome-sequencing studies. We used transposon mutagenesis to better understand the genes and pathways that are able to transform neural stem cells (NSC) into glioma-initiating cells (5), and identified La-related protein 4b (Larp4b) as a candidate tumor-suppressor gene in glioblastoma.…”

Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma

“…cells (25). This versatility and high relevance to human cancers makes SB an invaluable tool for cancer gene identification in mice.…”