A Sleeping Beauty transposon-mediated screen identifies murine susceptibility genes for adenomatous polyposis coli (Apc)-dependent intestinal tumorigenesis

Department of Genetics, Cell Biology and Development, Center for Genome Engineering, Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 03/2011; 108(14):5765-70. DOI: 10.1073/pnas.1018012108
Source: PubMed

ABSTRACT It is proposed that a progressive series of mutations and epigenetic events leads to human colorectal cancer (CRC) and metastasis. Furthermore, data from resequencing of the coding regions of human CRC suggests that a relatively large number of mutations occur in individual human CRC, most at low frequency. The functional role of these low-frequency mutations in CRC, and specifically how they may cooperate with high-frequency mutations, is not well understood. One of the most common rate-limiting mutations in human CRC occurs in the adenomatous polyposis coli (APC) gene. To identify mutations that cooperate with mutant APC, we performed a forward genetic screen in mice carrying a mutant allele of Apc (Apc(Min)) using Sleeping Beauty (SB) transposon-mediated mutagenesis. Apc(Min) SB-mutagenized mice developed three times as many polyps as mice with the Apc(Min) allele alone. Analysis of transposon common insertion sites (CIS) identified the Apc locus as a major target of SB-induced mutagenesis, suggesting that SB insertions provide an efficient route to biallelic Apc inactivation. We also identified an additional 32 CIS genes/loci that may represent modifiers of the Apc(Min) phenotype. Five CIS genes tested for their role in proliferation caused a significant change in cell viability when message levels were reduced in human CRC cells. These findings demonstrate the utility of using transposon mutagenesis to identify low-frequency and cooperating cancer genes; this approach will aid in the development of combinatorial therapies targeting this deadly disease.

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    • "To demonstrate that TE insertion or TE-mediated rearrangement causes human cancer is difficult because i) sequencing repetitive elements remain a challenge and ii) experimental demonstration is impossible in humans. However, recent animal model studies with refined bioinformatic analyses of insertional mutagenesis screens [145] [146] [147] [148], and improved human genome analysis such as the recent work of Lee et al. [101], offer solid arguments in favor of the causative role of TEs in cancer. Conversely, the SETMAR example illustrates the case of a positive antitumor effect due to TE domestication. "
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    ABSTRACT: Transposable elements are present in almost all genomes including that of humans. These mobile DNA sequences are capable of invading genomes and their impact on genome evolution is substantial as they contribute to the genetic diversity of organisms. The mobility of transposable elements can cause deleterious mutations, gene disruption and chromosome rearrangements that may lead to several pathologies including cancer. This mini-review aims to give a brief overview of the relationship that transposons and retrotransposons may have in the genetic cause of human cancer onset, or conversely creating protection against cancer. Finally, the cause of TE mobility may also be the cancer cell environment itself.
    Biochimica et Biophysica Acta 09/2012; 1835(1):28-35. DOI:10.1016/j.bbcan.2012.09.001 · 4.66 Impact Factor
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    • "Alternatively, spatial and temporal patterns of gene expression can be visualized at cellular resolution by tagging each active gene using random transposon insertions. Here, a marker tag enables both the cloning of the active gene and visualization of its activity (Starr et al. 2011). "
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    ABSTRACT: The gene-trap lacZ reporter insertion, ROSA11, in the Cbx5 mouse gene illuminates the regulatory complexity of this locus in Apc(Min) (/+) mice. The insertion site of the β-Geo gene-trap element lies in the 24-kb intron proximal to the coding region of Cbx5. Transcript analysis indicates that two promoters for Cbx5 flank this insertion site. Heterozygotes for the insertion express lacZ widely in fetal tissues but show limited expression in adult tissues. In the intestine, strong expression is limited to proliferative zones of crypts and tumors. Homozygotes for ROSA11, found at a lower than Mendelian frequency, express reduced levels of the coding region transcript in normal tissues, using a downstream promoter. Analysis via real-time polymerase chain reaction indicates that the upstream promoter is the dominant promoter in normal epithelium and tumors. Bioinformatic analysis of the Cbx5 locus indicates that WNT and its target transcription factor MYC can establish a feedback loop that may play a role in regulating the self-renewal of the normal intestinal epithelium and its tumors.
    G3-Genes Genomes Genetics 05/2012; 2(5):569-78. DOI:10.1534/g3.112.002436 · 2.51 Impact Factor
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    ABSTRACT: The recent development of the Sleeping Beauty (SB) system has led to the development of novel mouse models of cancer. Unlike spontaneous models, SB causes cancer through the action of mutagenic transposons that are mobilized in the genomes of somatic cells to induce mutations in cancer genes. While previous methods have successfully identified many transposon-tagged mutations in SB-induced tumors, limitations in DNA sequencing technology have prevented a comprehensive analysis of large tumor cohorts. Here we describe a novel method for producing genetic profiles of SB-induced tumors using Illumina sequencing. This method has dramatically increased the number of transposon-induced mutations identified in each tumor sample to reveal a level of genetic complexity much greater than previously appreciated. In addition, Illumina sequencing has allowed us to more precisely determine the depth of sequencing required to obtain a reproducible signature of transposon-induced mutations within tumor samples. The use of Illumina sequencing to characterize SB-induced tumors should significantly reduce sampling error that undoubtedly occurs using previous sequencing methods. As a consequence, the improved accuracy and precision provided by this method will allow candidate cancer genes to be identified with greater confidence. Overall, this method will facilitate ongoing efforts to decipher the genetic complexity of the human cancer genome by providing more accurate comparative information from Sleeping Beauty models of cancer.
    PLoS ONE 09/2011; 6(9):e24668. DOI:10.1371/journal.pone.0024668 · 3.23 Impact Factor
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