A Sleeping Beauty Transposon-Mediated Screen Identifies Murine Susceptibility Genes for Adenomatous Polyposis Coli (Apc)-Dependent Intestinal Tumorigenesis
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A Sleeping Beauty transposon-mediated screen identifies murine susceptibility genes for adenomatous polyposis coli (Apc)-dependent intestinal tumorigenesis Timothy K. Starra,1, Patricia M. Scottb, Benjamin M. Marshb, Lei Zhaob, Bich L. N. Thanb, M. Gerard O’Sullivana,c, Aaron L. Sarverd, Adam J. Dupuye, David A. Largaespadaa, and Robert T. Cormierb,1 aDepartment of Genetics, Cell Biology and Development, Center for Genome Engineering, Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455; bDepartment of Biochemistry and Molecular Biology, University of Minnesota Medical School, Duluth, MN 55812; cDepartment of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108; dDepartment of Biostatistics and Informatics, Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455; and eDepartment of Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242 Edited* by William F. Dove, University of Wisconsin, Madison, WI, and approved March 2, 2011 (received for review December 1, 2010) Min Min It is proposed that a progressive series of mutations and epigenetic conducted the screen in mice carrying the Apc allele. Apc events leads to human colorectal cancer (CRC) and metastasis. mice harbor a T→A nonsense mutation in the Apc gene (4, 5) Furthermore, data from resequencing of the coding regions of that results in a truncated protein product that is unable to bind human CRC suggests that a relatively large number of mutations β-catenin and promote its degradation, thus leading to abnormal occur in individual human CRC, most at low frequency. The levels of β-catenin protein and up-regulation of β-catenin target functional role of these low-frequency mutations in CRC, and genes such as cyclin D1 (Ccnd1) and myelocytomatosis oncogene specifically how they may cooperate with high-frequency muta- (C-Myc). The Min mutation corresponds to a mutational hotspot tions, is not well understood. One of the most common rate- in the human APC ortholog, and these mutations similarly result limiting mutations in human CRC occurs in the adenomatous in dysregulation of the Wnt/β-catenin signaling pathway. There is polyposis coli (APC) gene. To identify mutations that cooperate strong evidence that β-catenin dysregulation is a common trans- Min with mutant APC, we performed a forward genetic screen in mice formative event in tumorigenesis in the Apc mouse and in both Min carrying a mutant allele of Apc (Apc ) using Sleeping Beauty (SB) the inherited form of APC-deficient CRC (familial adenomatous Min Min transposon-mediated mutagenesis. Apc SB-mutagenized mice polyposis, FAP) and in sporadic CRC (6). Thus, the Apc mouse developed three times as many polyps as mice with the ApcMin is an informative genetic model for APC-deficient intestinal Min allele alone. Analysis of transposon common insertion sites (CIS) cancer. Apc mice on the C57BL/6J background strain rarely identified the Apc locus as a major target of SB-induced mutagen- survive beyond 120 d and can develop >100 tumors throughout esis, suggesting that SB insertions provide an efficient route to the small and large intestine, with the phenotype dependent on biallelic Apc inactivation. We also identified an additional 32 CIS diet, mouse strain, and other environmental factors (7, 8). Min genes/loci that may represent modifiers of the Apc phenotype. As in human CRC patients, loss of heterozygosity (LOH) Five CIS genes tested for their role in proliferation caused a signif- leading to inactivation of both alleles of Apc is necessary for Min icant change in cell viability when message levels were reduced in tumorigenesis to commence in Apc mice (9, 10). However, in Min human CRC cells. These findings demonstrate the utility of using contrast to LOH events in many human CRC, LOH in Apc transposon mutagenesis to identify low-frequency and cooperat- tumors occurs predominantly by homologous somatic recom- ing cancer genes; this approach will aid in the development of bination (11). In this study we screened for mutations that co- Min combinatorial therapies targeting this deadly disease. operate with the Apc mutation by randomly mutating genes through selective activation of SB transposition in intestinal cells Min cancer gene discovery | transgenic mice of Apc mice. The results of our screen support the importance of the loss of the second allele of Apc, because the great major- uman colorectal cancers (CRC) generally can be divided into ity of tumors analyzed contained a transposon insertion in Apc, Htwo classes based on whether they display chromosomal in- in particular in tumors in which there was maintenance of het- stability (CIN) or microsatellite instability (MSI). The majority erozygosity (MOH) for the Min allele. In addition to Apc,we fi of CRC (∼80–90%) have a CIN phenotype; the remaining cases identi ed 32 other genes and loci that probably facilitate the ApcMin are characterized by MSI (1). CRC displaying CIN frequently development of intestinal cancer in an model. The func- harbor allelic losses or mutations in adenomatous polyposis coli tion of these additional mutations could be to remove the re- Apc (APC), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog quirement for LOH, or they may function in some other MEDICAL SCIENCES (KRAS), SMAD family member 4 (SMAD4), and tumor protein manner. The majority of these genes have not been associated fi p53 (TP53), whereas MSI-type CRC usually have a mutation in with CRC previously. To con rm that these genes play a causal one of six DNA mismatch repair genes (2). In both CIN and MSI CRC complete functional loss of a gatekeeper tumor suppressor gene typically is the rate-limiting event in intestinal cell trans- Author contributions: T.K.S., P.M.S., D.A.L., and R.T.C. designed research; T.K.S., P.M.S., APC B.M.M., L.Z., B.L.N.T., M.G.O., and R.T.C. performed research; A.J.D. contributed new formation. For CIN CRC, plays the key gate-keeping role, reagents/analytic tools; T.K.S., P.M.S., B.M.M., L.Z., B.L.N.T., M.G.O., A.L.S., and R.T.C. and its loss underlies the great majority of CIN CRC and >80% analyzed data; and T.K.S. wrote the paper. of all CRC. Although both classes of CRC are characterized by Conflict of interest statement: D.A.L. is a cofounder of, and has an equity interest in, high-frequency mutations, such as those in APC, it is evident that Discovery Genomics Inc. (DGI), a biotechnology company that is pursuing SB technology many more low-frequency mutations are required for CRC de- for human gene therapy. No resources or personnel from DGI were involved in this work. The University of Minnesota has filed a patent related to the work described in this paper. velopment, and the majority of these low-frequency mutations All other authors state no conflict of interest. are unknown (3). *This Direct Submission article had a prearranged editor. To identify these low-frequency mutations, we performed 1To whom correspondence may be addressed. E-mail: [email protected] or rcormier@d. a forward genetic screen in mice using the Sleeping Beauty (SB) umn.edu. DNA transposon as a mutagen in intestinal epithelial cells. To This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. focus on mutations that contribute to the CIN phenotype, we 1073/pnas.1018012108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1018012108 PNAS | April 5, 2011 | vol. 108 | no. 14 | 5765–5770 Downloaded by guest on September 27, 2021 role in tumor development, we used siRNA to knock down the Rosa-26-LsL-SB11 allele but not the complete combination message levels of nine of the candidate genes in human colon of alleles required for transposition. This control group de- cancer cell lines and demonstrated that five of these genes af- veloped an average of 182 polyps (control group 1, Table 1), a fected the growth rate of these cells. result that was unexpected based on previous screens. It is pos- sible that the increased polyp number in these animals is caused Results by one or more modifiers linked to the Rosa26-LsL-SB11 trans- Design of a Forward Genetic Screen for CRC Genes. In a previous gene, because strain-specific modifiers are known to exist (19). Min study we demonstrated that SB transposon-mediated mutagen- Control animals carrying the Apc allele, T2/Onc, and/or Vil- +/+ esis in the intestinal tract of C57BL/6J Apc mice resulted in Cre, but not Rosa26-LsL-SB11 (control group 2, Table 1) devel- polyp formation (12). By mapping transposon insertions in DNA oped the same number of polyps as the control mice carrying the Min extracted from these tumors, we were able to identify 77 genetic Apc allele alone. Although the Rosa26-LsL-SB11 allele alone loci which probably harbored genes that, when mutated, con- contributes to polyp formation, the effect of active SB trans- tributed to tumor development. Because APC loss is rate limiting position was much greater, resulting in twice as many polyps in the in the development of most human CRC (13), we reasoned that test mice. In addition, the tumor burden was so extensive that Min SB mutagenesis in a mouse already harboring a mutation in Apc Apc SB test mice became moribund earlier than any of the Min might generate more tumors with a shorter latency and reveal three control groups (Table 1). Indeed, in a subset of Apc SB mutations that cooperate with Apc during tumor development. test mice the tumor load was very severe, with some animals de- To identify these genes, we performed a forward genetic screen veloping as many as 700 tumors.