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: 9.67). 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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    • "The position and orientation of SB insertions can be used to infer the mode of action (MOA) of insertion (e.g. [28]). Most of the CISs have insertions distributed throughout the genes they identify, and in both orientations, suggesting loss of function (Loss). "
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    ABSTRACT: Background: Medulloblastomas, the most frequent malignant brain tumours affecting children, comprise at least 4 distinct clinicogenetic subgroups. Aberrant sonic hedgehog (SHH) signalling is observed in approximately 25% of tumours and defines one subgroup. Although alterations in SHH pathway genes (e.g. PTCH1, SUFU) are observed in many of these tumours, high throughput genomic analyses have identified few other recurring mutations. Here, we have mutagenised the Ptch+/- murine tumour model using the Sleeping Beauty transposon system to identify additional genes and pathways involved in SHH subgroup medulloblastoma development. Results: Mutagenesis significantly increased medulloblastoma frequency and identified 17 candidate cancer genes, including orthologs of genes somatically mutated (PTEN, CREBBP) or associated with poor outcome (PTEN, MYT1L) in the human disease. Strikingly, these candidate genes were enriched for transcription factors (p=2x10-5), the majority of which (6/7; Crebbp, Myt1L, Nfia, Nfib, Tead1 and Tgif2) were linked within a single regulatory network enriched for genes associated with a differentiated neuronal phenotype. Furthermore, activity of this network varied significantly between the human subgroups, was associated with metastatic disease, and predicted poor survival specifically within the SHH subgroup of tumours. Igf2, previously implicated in medulloblastoma, was the most differentially expressed gene in murine tumours with network perturbation, and network activity in both mouse and human tumours was characterised by enrichment for multiple gene-sets indicating increased cell proliferation, IGF signalling, MYC target upregulation, and decreased neuronal differentiation. Conclusions: Collectively, our data support a model of medulloblastoma development in SB-mutagenised Ptch+/- mice which involves disruption of a novel transcription factor network leading to Igf2 upregulation, proliferation of GNPs, and tumour formation. Moreover, our results identify rational therapeutic targets for SHH subgroup tumours, alongside prognostic biomarkers for the identification of poor-risk SHH patients.
    07/2013; 1(1):35. DOI:10.1186/2051-5960-1-35
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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 · 3.20 Impact Factor
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