A Sleeping Beauty transposon-mediated screen identifies murine susceptibility 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 (4, 5) Furthermore, data from resequencing of the coding regions of that results in a truncated 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 MEDICAL SCIENCES 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 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 (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 Early Edition | 1of6 Downloaded by guest on October 1, 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. Min using SB transposon-mediated mutagenesis in Apc mice. The Although polyp number was greatly increased by SB muta- Min screen consisted of a cohort of Apc SB transgenic test mice genesis, there was no evidence of local or systemic metastasis in Min Min along with three groups of Apc control mice. The Apc SB experimental or control mice. We performed histopathologic test mice harbored three transgenes required for targeting SB analysis of tumors collected from 10 animals. These analyses mutagenesis to the gastrointestinal tract (Fig. S1). The first identified numerous microadenomas and adenomas in the small transgene was a concatamer of oncogenic transposons (T2/Onc) intestine and a much smaller number of these lesions in the large that were resident on 1 (14). To enhance the mu- intestines. No adenocarcinomas were identified, perhaps because Min tagenic potential of the transposon, T2/Onc contains a strong of the short lifespan of Apc SB test mice. Immunohistochem- viral promoter, splice acceptors in both orientations, and a bi- istry for β-catenin was performed on 24 adenomas from seven directional polyA signal. The second transgene was a condition- animals. There was increased expression of β-catenin in all tumors ally expressed knockin SB11 transposase allele downstream of compared with the adjacent normal mucosa epithelium (Fig. 1). the Rosa26 promoter (Rosa26-LsL-SB11) (15, 16). Because of the presence of a floxed stop cassette, the transposase allele is Analysis of Common Insertion Sites Identifies 30 Candidate Cancer Genes. To identify genes that contribute to tumor initiation and not expressed unless Cre recombinase protein is present. The Min third transgene was Cre recombinase driven by the gastrointes- development in Apc mice, we analyzed transposon insertions in tinal tract-specific Villin promoter (Vil-Cre) (17). We have shown 96 polyps, representing all regions of the intestines, from 12 mice previously that these three transgenes effectively limit SB mu- to find common insertion sites (CIS). A CIS is defined by ana- tagenesis to the intestinal tract (12). All mice were heterozygous lyzing transposon insertions in many tumors and identifying ge- Min for the Apc allele, and all the transgenes were fully congenic nomic loci that contain transposon insertions at a higher rate than on the C57BL/6J genetic background. The first control group would be expected by chance (SI Materials and Methods). The contained Rosa26-LsL-SB11 and either Vil-Cre or T2/Onc; the presence of a CIS indicates that a transposon-mediated mutation second control group contained T2/Onc and/or Vil-Cre but not in that locus probably has contributed to tumor development. By Rosa26-LsL-SB11; and the third control group harbored only the analyzing the genes within the CIS, one can identify candidate Min Apc allele. Mice were killed when moribund or at 120 d. cancer genes. To map transposon insertions, we isolated DNA from the 96 Min Intestinal Tumorigenesis Is Enhanced Significantly in Apc SB Test tumors, digested the DNA with restriction enzymes, and per- Min Mice. Apc mice that harbored all three transgenes (Rosa26- formed ligation-mediated PCR (LM-PCR) to amplify trans- LsL-SB11, T2/Onc, and Vil-Cre) developed an average of 360 poson-genomic fragments specifically (20). Barcodes and fusion Min polyps (test mice, Table 1). In contrast, mice carrying the Apc sequences were attached to the LM-PCR primers to enable allele alone developed an average of 112 polyps (control group 3, pooling of the amplicons, which then were sequenced using the Min Table 1), a result that is consistent with the phenotype of Apc Roche GS FLX pyrosequencing machine. Six separate sequencing mice in our colony (18). Surprisingly, we also observed an en- runs produced 347,993 sequence reads, 93% of which (324,898) hanced rate of polyp development in control group 1 that carried contained a barcode, the transposon sequence, and sufficient

Table 1. Polyp number and age of death for transgenic mice Average no. polyps per mouse†

‡ Group* Number per group Large intestine Small intestine Total Date of death

Test 38 14.7 345 360 85 Control 1 57 4.2 178 182 110 Control 2 9 2.4 113 115 120+ Control 3 100 2 110 112 120+

*Groups: Test = ApcMin × Rosa26-LsL-SB11 × T2/Onc × Vil-Cre; Control 1 = littermates harboring either ApcMin × Rosa26-LsL-SB11 or ApcMin × Rosa26-LsL-SB11 × T2/Onc or ApcMin × Rosa26-LsL-SB11 × Vil-Cre; Control 2 = littermates harboring either ApcMin × T2/Onc or ApcMin × Vil-Cre or ApcMin × T2/Onc × Vil-Cre; Control 3 = contemporaneous mice harboring ApcMin only. †Average number of polyps per mouse by large intestine, small intestine, and total. ‡Control groups 2 and 3 were killed at 120 d whether they were moribund or not.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1018012108 Starr et al. Downloaded by guest on October 1, 2021 B the transposon insertion caused a gain- or loss-of-function mu- A tation sometimes can be predicted by analyzing the location and orientation of the insertions in all the tumors that comprise a single CIS. If all the tumors in a single CIS have transposon insertions in the same intron, and all the transposons are oriented in the direction of transcription, we predict the insertion causes a gain-of-function mutation. If the distribution of transposon insertions in all the tumors of a CIS is apparently random, and there is no bias in orientation, we predict a loss-of-function effect. Table 2 lists the predictions for the CIS. In total we identified 30 C genes and four genomic loci with no annotated genes that prob- ably contribute to intestinal tract cancer when mutated.

Transposon Insertions Implicated in LOH of the Wild-Type Allele of Apc. The most commonly mutated gene in this study was Apc (in 72 of 96 tumors), reflecting the strong selective pressure for loss of Min the wild-type allele in Apc mice. Previous studies have dem- + onstrated that loss of the Apc allele is an early event that occurs Min in almost every adenoma in Apc mice (9, 10). In addition, in- activation of the wild-type Apc allele is caused predominantly by homologous somatic recombination events, leading to the re- Fig. 1. A pedunculated adenoma stained with H&E (A) or immunostained Apc ApcMin for β-catenin (B and C). (B) There is increased staining for β-catenin (arrow) in placement of the wild-type allele with a second allele the adenoma. (C) Higher-power magnification of a different section show- (22). We reasoned that in our transgenic model LOH could be ing increased cytoplasmic and nuclear (arrows) staining for β-catenin in tu- accomplished by an inactivating transposon insertion, as opposed mor cells compared with adjacent normal tissue seen in lower right and to duplication of the Min allele. To test this hypothesis, we per- bottom of picture. (Scale bars: A and B, 500 μm; C,50μm.) formed PCR on DNA from tumors to amplify the region sur- rounding the Min mutation (T2860A). By sequencing the PCR Min amplicon, LOH can be ascertained in Apc mice by measuring genomic sequence (>16 bp) for BLAST analysis. We were able to the ratio of the T:A trace peak heights at the location of the Min MEDICAL SCIENCES map more than half of these sequences (53%) unambiguously to mutation. In heterozygous tissue the T:A ratio is between 0.8 and the mouse genome. Of the 173,101 mapped sequences, 100,171 1.2, which is considered MOH, but in tissue that has lost the wild- (67%) were redundant, leaving 72,930 nonredundant mapped type allele the ratio drops below 0.5 (Fig. S2). Ratios between 0.5– insertions. Roughly half of the nonredundant insertions mapped 0.8 and >1.2 are considered uninformative, most likely caused by to the same chromosome as the donor transposon concatamer contamination from nontumor tissue. Of the 96 tumors tested, 47 (Chr 1), as expected because of the phenomenon of local hopping gave informative results (Table S1). Of these 47 tumors, 32 had an seen in other SB screens (12, 14, 21). To eliminate statistical bias identified transposon insertion in the Apc locus, and 15 did not. in the dataset, these sequences were eliminated along with a The majority (73%) of tumors lacking a transposon insertion in smaller number of insertions that probably represent PCR arti- Apc had T:A ratios <0.5, indicating LOH probably caused by loss facts (SI Materials and Methods). The remaining 30,088 insertions of the entire allele. In support of our hypothesis, 53% of the (Dataset S1) were analyzed to determine CIS. We used Monte tumors that had a transposon insertion in the Apc locus had T:A Carlo simulations to find insertion rates in a given genomic win- ratios between 0.8 and 1.2, indicating maintenance of the wild-type dow size that would not be expected to occur by chance (12). For Apc locus at the site of the Min mutation. This result suggests that example, based on a random assignment of 30,088 insertions to in these tumors the wild-type Apc allele is inactivated by the the mouse genome, one would not expect to find five or more transposon more frequently than by duplication of the Min allele. insertions within a 12-kb window. Using these Monte Carlo- Min defined parameters, we identified 37 CIS. Two of these CIS were Set of CIS Identified in Apc Mice Differs Significantly from Those removed from further analysis because all the tumors contributing Found in Apc Wild-Type Mice. We compared the list of genes to these two CIS originated from a single mouse, indicating the identified in this study with the 77 genes identified in the screen tumors may be clonally related. Two more CIS were removed we performed on an Apc wild-type background (12). Surpris- because they also were identified in a control dataset of tail-snip ingly, only four genes were identified in both studies: Apc, nu- DNA from mice harboring unselected SB insertions and may clear receptor binding SET domain protein 1 (Nsd1), Sfi1 represent hotspots for SB insertions (12) Because this control homolog, spindle assembly associated (Sfi1), and WW domain dataset was generated from tail snips, it is possible that other containing adaptor with coiled-coil (Wac). There are several hotspots exist in other types of cells. After removal of these pos- reasons that could explain why the overlap between the two sible artifacts, 33 CIS remained (Table 2). studies was low. First, the total number of genes that could We assigned a candidate gene to each CIS if the majority of contribute to tumor formation may be large enough that the size the insertions were in or near a single gene (Table 2). Four of the of these two studies is not sufficient to saturate the candidate 33 CIS did not have an annotated gene within 40 kb and were not genes. Second, because we use a statistical method to identify assigned a candidate gene. Another CIS contained two over- cancer genes, it is likely that transposon insertions contributed to lapping genes, SET domain-containing 5 (Setd5) and lipoma carcinogenesis in some of the tumors, but the insertions did not HMGIC fusion partner-like 4 (Lhfpl4), and all insertions in this occur at a rate high enough to qualify as a CIS. In support of this CIS were in both genes. Notably, this CIS is located adjacent to hypothesis, 70% of the loci identified as CIS in this study (23 of the Rosa26 locus where the conditional SB11 knockin is located, 33) also had one or more insertions in the same locus in the and eight of nine insertions in this CIS are oriented with the in- previous study (12). Third, the overlap may be small because ternal promoter in the direction that would cause overexpression selection pressure for specific genetic mutations in cells that al- of the transgene. Rather than tagging an endogenous cancer ready have an Apc mutation is biased toward a different set of gene, this CIS could represent selective pressure for increased cancer genes than in cells with a different initial mutation. mutagenesis via overexpression of SB11 transposase. Whether Fourth, because of technical limitations, our method of ampli-

Starr et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 Table 2. List of 33 CIS Number of Tumors with Predicted effect † ‡ Candidate gene Chromosome Start address* End address* insertions insertions on gene§

4930422G04Rik 3 127241424 127431287 23 12 NP AC115907.7 3 127697338 127730035 8 8 NP AC131780.5 9 3001410 3030207 7 7 NP Adamts6 13 105093822 105104050 5 3 NP Ap1ar 3 127454370 127633377 17 10 NP Apc 18 34324389 34514767 185 72 Loss Atf2 2 73708689 73773260 9 9 NP Atl2 17 80239637 80306988 10 7 Loss Cnot1 8 98254541 98300340 8 8 Loss Csnk1a1 18 61726208 61750047 7 6 Loss Elac1 18 73903365 73913215 5 3 Gain Emcn 3 136918543 137092521 17 9 NP Esco1 18 10578932 10773953 15 11 Loss Fnbp1l 3 122217801 122319064 11 11 Loss Itgam 7 135180978 135240752 11 10 NP Myo5b 18 74613029 74796289 18 17 Gain No Gene 16 16 29019897 29031672 5 3 NP No Gene 18 18 26169184 26282443 12 7 NP No Gene 4 4 131170124 131238909 12 8 NP No Gene Y Y 2781406 2897989 16 15 NP Nsd1 13 55352993 55372133 6 6 Gain Pdcd6ip 9 113560446 113723693 14 14 Loss Pde4dip 3 97593369 97718572 14 11 NP Pigl 11 62203495 62369763 16 16 NP Setd5 or Lhfpl4 6 113039133 113100176 9 9 NP Sfi1 11 3004743 3179859 21 19 Loss Snx24 18 53440600 53638364 19 12 Loss Srfbp1 18 52654288 52675270 6 5 NP Stag1 9 100583003 100698850 12 12 Loss Tmem132b 5 126077980 126153976 11 4 Loss Wac 18 7855248 8046126 17 15 Loss Zfp397 18 24110107 24157877 8 7 NP Zfp609 9 65561432 65607064 8 8 Loss

*Genomic address based on National Center for Biotechnology Information Mouse genome Build 37. † Number of nonredundant SB transposon insertions within the locus. ‡ Number of independent tumors with an insertion within the locus. §Predicted effect is based on an analysis of the location and orientation of SB transposon insertions in all tumors in a single CIS (see text for discussion). Gain, gain of function; Loss, loss of function; NP, no prediction.

fying and sequencing transposon insertions does not identify all rent chromosomal losses and gains in human CRC (25–32). Of transposon insertions, so a portion of driver mutations will not be the 33 identified CIS, 31 can be mapped to an orthologous hu- identified. For example, analysis of replicate sequencing runs man locus. Of these 31 candidate cancer loci, 24 are found in indicates that 20–40% of the PCR amplicons in a given library regions that commonly are lost or gained in human CRC (Table are not sequenced in a given GS FLX sequencing run (SI S3), including several of the CIS that are orthologous to human Materials and Methods and Table S2). chromosomal arms 18q, 17p, 5q, and 4q. Interestingly, one CIS that has no annotated genes nearby (CIS No gene 16) is in an Relevance to Human Disease. To determine the relevance of these orthologous region (3q21–24) that is associated with CRC based findings to human cancers, we analyzed the regions of human on genome-wide linkage analyses (33, 34). To determine the orthology to the CIS loci and the orthologous human genes. Of significance of this overlap, we performed the analysis using the 30 candidate mouse genes associated with a CIS, 28 had hu- randomly generated CIS lists and a single dataset of regions that man orthologs. We queried the literature for mutations and re- are lost recurrently in human CRC (27). Roughly 250 genomic current copy number changes in these genes in human CRC. regions in this dataset were lost in >5% of the human samples Three of the genes, APC, NSD1, and phosphodiesterase 4D- tested, and 22 of the 31 CIS were located within these regions. In interacting protein (PDE4DIP), are considered bona fide cancer 10,000 simulations using equivalent-sized randomly generated genes based on the cancer gene census maintained by the Well- CIS lists, we find an overlap of this magnitude <0.3% of the time. come Trust Sanger Institute (23). Eight genes, APC, activating These results suggest that our SB screen may be capable of transcription factor-2 (ATF2), atlastin GTPase 2 (ATL2), casein pinpointing the affected genes in these regions. kinase 1, alpha 1 (CSNK1A1), integrin, alpha M (ITGAM), pro- grammed cell death 6-interacting protein (PDCD6IP), and WAC, Candidate Genes Regulate Proliferation of Human CRC Cell Lines. We have documented mutations in human cancers cataloged in the tested nine genes [CCR4-NOT transcription complex, subunit 1 COSMIC database (24). (CNOT1), PDE4DIP, PDCD6IP, ATF2, SFI1, formin-binding We found strong concordance between the CIS mouse loci protein 1-like (FNBP1L), myosin VB (MYO5B), sorting nexin 24 and orthologous regions in the showing recur- (SNX24), and stromal antigen 1 (STAG1)] for their effect on

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1018012108 Starr et al. Downloaded by guest on October 1, 2021 proliferation of the human CRC cell line SW480 by knocking of ERα target genes in breast cancer cells, and ERα has been down message levels using siRNA. We used the SW480 line shown to be a tumor suppressor gene in the intestinal tract (37); in Min because it has an APC gene-truncation mutation similar to the particular, knockout of ERα in Apc mice caused a significant Min Apc mutation (35). Cells were transfected two times at 48-h increase in intestinal tumorigenesis (38). intervals with siRNA targeting the human genes. Knockdown Another gene identified in this study, Pdcd6ip (also known as efficiency was at least 50% for all nine genes as measured by ALG-2 interacting protein X, Alix) is involved in membrane quantitative real-time PCR. Cell proliferation was measured trafficking and apoptosis (39). Pdcd6ip produces a protein that using a tetrazolium-based colorimetric assay on days two and six binds to the protein product of Pdcd6, a proapoptotic gene in- after the second transfection. Depletion of five (CNOT1, volved in T-cell receptor–, Fas-, and glucocorticoid-induced cell PDE4DIP, PDCD6IP, ATF2, and SFI1) of the nine genes tested death (40). Pdcd6ip also can block down-regulation of the EGF resulted in a significant decrease in cell viability compared with receptor (EGFR), thereby having a positive effect on growth a control siRNA of at least 33% at day six after transfection factor signaling (41). These contradictory roles could explain why (Table S4). loss of Pdcd6ip in the mouse tumors promoted growth (via the loss of the proapoptotic function) but the loss of Pdcd6ip in Discussion SW480 cells caused decreased proliferation (via increased down- Using a transposon-based forward genetic screen in mice, we regulation of EGFR). Further functional studies are required to identified 33 genomic loci that probably cooperate with a germ- elucidate the role of this adaptor protein. line mutation in the Apc gene to cause intestinal tumorigenesis. In summary, our approach identified 30 genes that probably Min The most frequently mutated locus was the Apc locus, a result modify tumorigenesis in the Apc model of human CRC. that supports the hypothesis that there is strong selective pres- Further functional analysis of these CIS candidate genes may sure to lose the wild-type copy during tumor formation. SB provide insights into the etiology and treatment of human CRC, insertions in Apc were found in 72 of 96 tumors (75%), sug- especially those cancers arising downstream of APC deficiency. Min gesting that 75% of the tumors in Apc SB test mice undergo LOH at the Apc locus via SB insertional mutagenesis. This hy- Materials and Methods pothesis is supported by sequencing of the region spanning the Detailed protocols are given in SI Materials and Methods. 1-bp Min mutation, which indicated that the majority of tumors containing SB insertions at the Apc locus maintained heterozy- Mice. Mice containing ApcMin, Rosa26-LsL-SB11, Villin-Cre, and T2/Onc were gosity at the location of the T:A Min mutation. In contrast, the reared using Institutional Animal Care and Use Committee-approved pro- majority of tumors lacking a transposon insertion in Apc showed tocols. All mice were on an isogenic C57BL/6J background. Mice were MEDICAL SCIENCES loss of the wild-type sequence at the Min mutation site. These monitored daily and killed and necropsied when moribund or after 120 d. results suggest that the majority of tumors underwent biallelic fi loss of Apc activity through transposon insertion or somatic re- Histopathology and Immunohistochemistry. Formalin- xed tissues were em- bedded in paraffin, and standard techniques were used to stain tissue sec- combination. However, it also is possible that in some cases Apc tions with H&E. Standard immunohistochemistry techniques were used to activity was lost through other mechanisms of inactivation or detect β-catenin. through transposon insertion substituting for loss of the wild- type allele. Linker-Mediated PCR. Linkers [described previously (42)] were ligated to Although LOH at the Apc locus is the rate-limiting event in NlaIII- (right-side) or BfaI- (left side) digested genomic DNA using T4 DNA Min tumor initiation in the Apc mouse and in familial and sporadic ligase. A secondary digest (XhoI, right side; BamHI, left side) was performed APC-deficient CRC, loss of APC probably is insufficient for the to destroy concatamer-generated products. Primary and secondary PCR was survival and growth of transformed cells into adenomas and, performed using primers specific for linker and SB transposon sequences eventually, adenocarcinomas. SB-mutagenized animals showed along with Fusion and barcode sequences. PCR amplicons were sequenced increased polyp number but no evidence of adenocarcinoma or using the GS FLX (Roche). metastasis. Thus, it is likely that the CIS candidate genes identi- fi SB Sequence Analysis. Sequences were analyzed for the presence of the barcode, ed in this screen contribute in a diverse fashion to initiation, inverted repeat/direct repeat (IR/DR) sequences required for transposition, establishment, and survival of adenomas. Moreover, depending and linker sequences. Genomic sequence was blasted against the mouse on the complexity of the mechanism or pathway, the CIS candi- genome using BLASTN at 95% stringency and requiring a single match. Of the dates discovered in our screen, like the relatively large number of 324,898 sequences analyzed, 53% could be uniquely mapped to the mouse genes reported to be mutant in individual human CRC (3), might genome. Sequences were removed if they were redundant, on the donor be expected to occur at a low frequency if mutations at any one of concatamer resident chromosome (Chr 1), in the En2 gene (because the En2 multiple genes in a complex pathway can contribute equally sequence is present in the transposon), and when a single TA dinucleotide to tumorigenesis. contained multiple insertions from several tumors from multiple mice (be- Aside from Apc, only a few of the remaining 28 known genes cause of the possibility of a PCR artifact). The remaining 30,088 nonredundant fi sequences were used to identify CIS. A CIS was defined by Monte Carlo that we identi ed as CIS in our screen have been implicated di- simulations using a random dataset of 30,088 insertions. rectly in CRC development, although >90% are located in ge- nomic regions that are lost or gained in human CRC. To Apc LOH Analysis. To measure LOH for the ApcMin mutation, DNA was isolated eliminate false positives, we removed CIS that also were identi- from individual polyps, and PCR was performed using primers that flank the fied in a control dataset of unselected transposon insertions mutation (sense primer: CGGAGTAAGCAGAGACACAA; antisense primer: mapped in tail snips. However, it is possible that other tissue- GGGAGGTATGAATGGCTGAT). The PCR product was purified using Qiagen specific hotspots could result in false positives. Nevertheless, the 96 MinElute vacuum purification plates per the manufacturer’s protocol and known functions of several of the CIS candidate genes make them was sequenced using the sense primer as the sequencing primer. Trace peak plausible candidates for drivers of human CRC. For example, heights at the location of the mutation were measured for each tumor, and CNOT1 is a member of the Ccr4-Not complex, which is im- the ratio of the T peak to the A peak was calculated. plicated in mRNA decay and transcriptional repression. In hu- Comparisons with Human Data. Eight publicly available studies measuring man cells, CNOT1 has been reported to be a repressor of nuclear DNA copy number in CRC compared with normal tissue were analyzed. receptor-mediated transcription (36). One target of CNOT1 re- Mutations in human tumors were examined using the Catalog of Somatic pression appears to be estrogen receptor alpha (ERα), via Mutations in Cancer database (24), and cancer gene status was based on the interactions between CNOT1 and the ligand-binding domain of Census of Human Cancer Genes maintained by the Wellcome Trust Sanger ERα. Inhibition of CNOT1 caused an increase in the expression Institute (23).

Starr et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 Knockdown of CIS Candidate Genes using siRNA in SW480 Cells. SW480 cells Cores: Biostatistics and Informatics Shared Resource, Comparative Pathol- were obtained from ATCC (catalog no. CCL-228) and were cultured under ogy, and Mouse Genetics Laboratory. We also thank the Minnesota recommended conditions. Transient siRNA transfection was used to deplete Supercomputing Institute and the BioMedical Genomics Center. Research expression of Min CIS genes. was funded by American Cancer Society postdoctoral fellowship PF-06-282- 01-MGO (to T.K.S.), a National Cancer Institute Pathway to Independence Award 1K99CA151672-01 (to T.K.S.), and by National Institutes of Health Cell Viability Assay. Viability of siRNA-treated SW480 cells was determined Grants R01CA113636-01A1 (to D.A.L.) and R01 CA134759-01A1 (to D.A.L. using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and R.T.C.). A University of Minnesota Academic Health Center Faculty assay (Cell Viability Kit 1; Roche Applied Sciences). Development Grant provided additional funding (to D.A.L. and R.T.C.). B.L.T.’s research is supported in part by a fellowship from the Annette ACKNOWLEDGMENTS. Pauline Jackson, Jerica Burchard, and Annette Rod Boman Women’s Fellowship program. B.L.T. and L.Z. were recipients of provided technical assistance for the gene expression analyses of CIS genes. a research support award from the University of Minnesota Duluth chapter We thank the following University of Minnesota Masonic Cancer Center of Sigma Xi.

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