Article

A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma.

Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Nature Biotechnology (Impact Factor: 39.08). 03/2009; 27(3):264-74. DOI: 10.1038/nbt.1526
Source: PubMed

ABSTRACT We describe a system that permits conditional mobilization of a Sleeping Beauty (SB) transposase allele by Cre recombinase to induce cancer specifically in a tissue of interest. To demonstrate its potential for developing tissue-specific models of cancer in mice, we limit SB transposition to the liver by placing Cre expression under the control of an albumin enhancer/promoter sequence and screen for hepatocellular carcinoma (HCC)-associated genes. From 8,060 nonredundant insertions cloned from 68 tumor nodules and comparative analysis with data from human HCC samples, we identify 19 loci strongly implicated in causing HCC. These encode genes, such as EGFR and MET, previously associated with HCC and others, such as UBE2H, that are potential new targets for treating this neoplasm. Our system, which could be modified to drive transposon-based insertional mutagenesis wherever tissue-specific Cre expression is possible, promises to enhance understanding of cancer genomes and identify new targets for therapeutic development.

Download full-text

Full-text

Available from: Ilze Matise, Jun 30, 2015
0 Followers
 · 
179 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Breast cancer is the most common type of cancer in women. A substantial fraction of breast cancers have acquired mutations that lead to activation of the phosphoinositide 3-kinase (PI3K) signaling pathway, which plays a central role in cellular processes that are essential in cancer, such as cell survival, growth, division and motility. Oncogenic mutations in the PI3K pathway generally involve either activating mutation of the gene encoding PI3K (PIK3CA) or AKT (AKT1), or loss or reduced expression of PTEN. Several kinases involved in PI3K signaling are being explored as a therapeutic targets for pharmacological inhibition. Despite the availability of a range of inhibitors, acquired resistance may limit the efficacy of single-agent therapy. In this review we discuss the role of PI3K pathway mutations in human breast cancer and relevant genetically engineered mouse models (GEMMs), with special attention to the role of PI3K signaling in oncogenesis, in therapeutic response, and in resistance to therapy. Several sophisticated GEMMs have revealed the cause-and-effect relationships between PI3K pathway mutations and mammary oncogenesis. These GEMMs enable us to study the biology of tumors induced by activated PI3K signaling, as well as preclinical response and resistance to PI3K pathway inhibitors.
    Molecular oncology 02/2013; 7(2). DOI:10.1016/j.molonc.2013.02.003 · 5.94 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Nonmelanoma skin cancer (NMSC) is by far the most frequent type of cancer in humans. NMSC includes several types of malignancies with different clinical outcomes, the most frequent being basal and squamous cell carcinomas. We have used the Sleeping Beauty transposon/transposase system to identify somatic mutations associated with NMSC. Transgenic mice bearing multiple copies of a mutagenic Sleeping Beauty transposon T2Onc2 and expressing the SB11 transposase under the transcriptional control of regulatory elements from the keratin K5 promoter were treated with TPA, either in wild-type or Ha-ras mutated backgrounds. After several weeks of treatment, mice with transposition developed more malignant tumors with decreased latency compared with control mice. Transposon/transposase animals also developed basal cell carcinomas. Genetic analysis of the transposon integration sites in the tumors identified several genes recurrently mutated in different tumor samples, which may represent novel candidate cancer genes. We observed alterations in the expression levels of some of these genes in human tumors. Our results show that inactivating mutations in Notch1 and Nsd1, among others, may have an important role in skin carcinogenesis.Journal of Investigative Dermatology advance online publication, 26 July 2012; doi:10.1038/jid.2012.245.
    Journal of Investigative Dermatology 07/2012; DOI:10.1038/jid.2012.245 · 6.37 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzyme recognition sites. Consequently, sequence coverage cannot be used to assess the clonality of individual insertions. We have developed a novel method, called shear-splink, for the semiquantitative high-throughput analysis of insertional mutations. Shear-splink employs random fragmentation of genomic DNA, which reduces unwanted amplification biases. Additionally, shear-splink enables us to assess clonality of individual insertions by determining the number of unique ligation points (LPs) between the adapter and genomic DNA. This parameter serves as a semiquantitative measure of the relative clonality of individual insertions within heterogeneous tumors. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV)-induced tumors showed that shear-splink enables the semiquantitative assessment of the clonality of MMTV insertions. Further, shear-splink analysis of 16 MMTV- and 127 Sleeping Beauty (SB)-induced tumors showed enrichment for cancer-relevant insertions by exclusion of irrelevant background insertions marked by single LPs, thereby facilitating the discovery of candidate cancer genes. To fully exploit the use of the shear-splink method, we set up the Insertional Mutagenesis Database (iMDB), offering a publicly available web-based application to analyze both retroviral- and transposon-based insertional mutagenesis data.
    Genome Research 08/2011; 21(12):2181-9. DOI:10.1101/gr.112763.110 · 13.85 Impact Factor