In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 44 ( 2012-10-30)
Abstract:
We next examined the combinations of CIS genes in tumors derived from a single immortalized line. Notably, each tumor showed a distinct combination of insertional mutations in RTK pathway genes, even though all tumors were derived from a single line, indicating that transplanted cells become tumorigenic by acquiring new insertions in genes that promote tumor development, followed by clonal expansion and the generation of tumors with distinct combinations of mutations in tumor CIS genes ( Fig. P1 ). Functional analysis of the tumor CIS genes identified in our screen, such as Met , Pdgfrb , and Gab1 , showed that these genes play functionally overlapping roles in tumor development. Together, these findings suggest that tumor formation in this model system mimics the evolutionary processes now thought to generate many human cancers, in which the pathway to tumor development has a branched architecture reminiscent of Darwin’s iconic evolutionary tree ( 5 ). Our dataset of GBM-causing genes provides a rich resource for cross-species comparative analyses of forthcoming human sequencing data from GBMs. These analyses will undoubtedly aid in the identification of potential therapeutic targets for this deadly disease. We also identified genes specific to tumors. Analysis of these genes associated them with the receptor tyrosine kinase (RTK) signaling pathway, which is the most frequently mutated pathway identified in human GBM. The protooncogene Met and the tumor suppressor gene Nf1 are most frequently mutated in the mouse tumors. Frequent NF1 mutations and increased MET expression are characteristic of mesenchymal GBM ( 4 ), indicating that the mouse tumors genetically resemble mesenchymal GBM. We next sequenced the transposon insertion sites from 25 immortalized lines and 67 tumors and identified common insertion sites (CISs), which are regions in the genome that harbor a higher number of transposon insertions than predicted by random chance, and therefore are most likely to harbor genes responsible for cancer progression. We identified 140 and 148 CIS genes in immortalized cells and tumors, respectively ( Fig. P1 ). Thirty-four genes were common to both immortalized cells and tumors, and the rest were specific to either, suggesting different mechanisms for immortalization and tumor formation. Analysis of CIS genes from immortalized lines identified signaling pathways that involved cytoskeletal organization and transcriptional regulation, which play key roles in regulating self-renewal vs. differentiation of NSCs. In contrast, 34 CIS genes, which were common, enriched genes associated with mitosis and cell division. The mutagenic Sleeping Beauty ( SB ) transposons, which were designed to elicit loss-of-function mutations as well as gain-of-function mutations, were mobilized specifically in the NSC compartment by using genetically engineered mice ( 3 ). NSCs were expanded in vitro, induced to differentiate, and serially passaged to select for the immortalized cells that exhibit an unlimited proliferative potential in culture. The frequency of immortalization was higher in cells undergoing active SB transposition than in cells that were not. Gene expression profiling of the immortalized cells showed that these cells were significantly enriched for genes differentially expressed in astroglial cells, which represent an immature stage of the astrocyte lineage. DNA analysis showed that continuously mobilized transposons generated a large repertoire of immortalized cells with unique combinations of insertions ( Fig. P1 ). More than half of the immortalized lines with active SB transposition induced tumors after s.c. transplantation, whereas only one of six immortalized lines that lacked active SB transposition was tumorigenic. Analysis of DNA microarrays showed that tumors were significantly enriched for genes specific for the mesenchymal subtype of GBM, consistent with an astroglial origin for mesenchymal GBM ( 4 ). Glioblastoma multiforme (GBM) is the most common form of malignant brain cancer in adults, whose mean survival is 1 y. Evidence indicates the presence of self-renewing, stem-like cells within human gliomas ( 1 ). Neural stem cells (NSCs) are considered the cell of origin of GBM, the normal cell that acquires the first glioma-promoting mutation(s), because the adult brain has very few proliferating cells capable of accumulating mutations required for gliomagenesis. To identify these genetic alterations, we used mobile DNA segments called transposons that can affect gene function by inserting in or near genes, thus providing an unbiased, high-throughput method for identifying genes important for cancer ( 2 ). Here, we used this approach to identify genes and signaling pathways that are able to transform NSCs into cancer-initiating cells for GBM.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1215899109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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