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; andeDepartment 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)
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 muta-
tions, 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 (ApcMin) using Sleeping Beauty (SB)
transposon-mediated mutagenesis. ApcMinSB-mutagenized mice
developed three times as many polyps as mice with the ApcMin
allele alone. Analysis of transposon common insertion sites (CIS)
identified the Apc locus as a major target of SB-induced mutagen-
esis, 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 ApcMinphenotype.
Five CIS genes tested for their role in proliferation caused a signif-
icant 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 cooperat-
ing cancer genes; this approach will aid in the development of
combinatorial therapies targeting this deadly disease.
cancer gene discovery|transgenic mice
stability (CIN) or microsatellite instability (MSI). The majority
of CRC (∼80–90%) have a CIN phenotype; the remaining cases
are characterized by MSI (1). CRC displaying CIN frequently
harbor allelic losses or mutations in adenomatous polyposis coli
(APC), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
(KRAS), SMAD family member 4 (SMAD4), and tumor protein
p53 (TP53), whereas MSI-type CRC usually have a mutation in
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-
formation. For CIN CRC, APC plays the key gate-keeping role,
and its loss underlies the great majority of CIN CRC and >80%
of all CRC. Although both classes of CRC are characterized by
high-frequency mutations, such as those in APC, it is evident that
many more low-frequency mutations are required for CRC de-
velopment, and the majority of these low-frequency mutations
are unknown (3).
To identify these low-frequency mutations, we performed
a forward genetic screen in mice using the Sleeping Beauty (SB)
DNA transposon as a mutagen in intestinal epithelial cells. To
focus on mutations that contribute to the CIN phenotype, we
uman colorectal cancers (CRC) generally can be divided into
two classes based on whether they display chromosomal in-
conducted the screen in mice carrying the ApcMinallele. ApcMin
mice harbor a T→A nonsense mutation in the Apc gene (4, 5)
that results in a truncated protein product that is unable to bind
β-catenin and promote its degradation, thus leading to abnormal
levels of β-catenin protein and up-regulation of β-catenin target
genes such as cyclin D1 (Ccnd1) and myelocytomatosis oncogene
(C-Myc). The Min mutation corresponds to a mutational hotspot
in the human APC ortholog, and these mutations similarly result
in dysregulation of the Wnt/β-catenin signaling pathway. There is
strong evidence that β-catenin dysregulation is a common trans-
formative event in tumorigenesis in the ApcMinmouse and in both
the inherited form of APC-deficient CRC (familial adenomatous
is an informative genetic model for APC-deficient intestinal
cancer. ApcMinmice on the C57BL/6J background strain rarely
survive beyond 120 d and can develop >100 tumors throughout
the small and large intestine, with the phenotype dependent on
diet, mouse strain, and other environmental factors (7, 8).
As in human CRC patients, loss of heterozygosity (LOH)
leading to inactivation of both alleles of Apc is necessary for
tumorigenesis to commence in ApcMinmice (9, 10). However, in
contrast to LOH events in many human CRC, LOH in ApcMin
tumors occurs predominantly by homologous somatic recom-
bination (11). In this study we screened for mutations that co-
operate with the ApcMinmutation by randomly mutating genes
through selective activation of SB transposition in intestinal cells
of ApcMinmice. The results of our screen support the importance
of the loss of the second allele of Apc, because the great major-
ity of tumors analyzed contained a transposon insertion in Apc,
in particular in tumors in which there was maintenance of het-
erozygosity (MOH) for the Min allele. In addition to Apc, we
identified 32 other genes and loci that probably facilitate the
development of intestinal cancer in an ApcMinmodel. The func-
tion of these additional mutations could be to remove the re-
quirement for Apc LOH, or they may function in some other
manner. The majority of these genes have not been associated
with CRC previously. To confirm that these genes play a causal
Author contributions: T.K.S., P.M.S., D.A.L., and R.T.C. designed research; T.K.S., P.M.S.,
B.M.M., L.Z., B.L.N.T., M.G.O., and R.T.C. performed research; A.J.D. contributed new
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.
analyzed data; and T.K.S. wrote the paper.
Conflict of interest statement: D.A.L. is a cofounder of, and has an equity interest in,
Discovery Genomics Inc. (DGI), a biotechnology company that is pursuing SB technology
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.
All other authors state no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence may be addressed. E-mail: email@example.com or rcormier@d.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| April 5, 2011
| vol. 108
| no. 14
role in tumor development, we used siRNA to knock down
message levels of nine of the candidate genes in human colon
cancer cell lines and demonstrated that five of these genes af-
fected the growth rate of these cells.
Design of a Forward Genetic Screen for CRC Genes. In a previous
study we demonstrated that SB transposon-mediated mutagen-
esis in the intestinal tract of C57BL/6J Apc+/+mice resulted in
polyp formation (12). By mapping transposon insertions in DNA
extracted from these tumors, we were able to identify 77 genetic
loci which probably harbored genes that, when mutated, con-
tributed to tumor development. Because APC loss is rate limiting
in the development of most human CRC (13), we reasoned that
SB mutagenesis in a mouse already harboring a mutation in Apc
might generate more tumors with a shorter latency and reveal
mutations that cooperate with Apc during tumor development.
To identify these genes, we performed a forward genetic screen
using SB transposon-mediated mutagenesis in ApcMinmice. The
screen consisted of a cohort of ApcMinSB transgenic test mice
along with three groups of ApcMincontrol mice. The ApcMinSB
test mice harbored three transgenes required for targeting SB
mutagenesis to the gastrointestinal tract (Fig. S1). The first
transgene was a concatamer of oncogenic transposons (T2/Onc)
that were resident on chromosome 1 (14). To enhance the mu-
tagenic potential of the transposon, T2/Onc contains a strong
viral promoter, splice acceptors in both orientations, and a bi-
directional polyA signal. The second transgene was a condition-
ally expressed knockin SB11 transposase allele downstream of
the Rosa26 promoter (Rosa26-LsL-SB11) (15, 16). Because of
the presence of a floxed stop cassette, the transposase allele is
not expressed unless Cre recombinase protein is present. The
third transgene was Cre recombinase driven by the gastrointes-
tinal tract-specific Villin promoter (Vil-Cre) (17). We have shown
previously that these three transgenes effectively limit SB mu-
tagenesis to the intestinal tract (12). All mice were heterozygous
for the ApcMinallele, and all the transgenes were fully congenic
on the C57BL/6J genetic background. The first control group
contained Rosa26-LsL-SB11 and either Vil-Cre or T2/Onc; the
second control group contained T2/Onc and/or Vil-Cre but not
Rosa26-LsL-SB11; and the third control group harbored only the
ApcMinallele. Mice were killed when moribund or at 120 d.
Intestinal Tumorigenesis Is Enhanced Significantly in ApcMinSB Test
Mice. ApcMinmice that harbored all three transgenes (Rosa26-
LsL-SB11, T2/Onc, and Vil-Cre) developed an average of 360
polyps (test mice, Table 1). In contrast, mice carrying the ApcMin
allele alone developed an average of 112 polyps (control group 3,
Table 1), a result that is consistent with the phenotype of ApcMin
mice in our colony (18). Surprisingly, we also observed an en-
hanced rate of polyp development in control group 1 that carried
the Rosa-26-LsL-SB11 allele but not the complete combination
of alleles required for transposition. This control group de-
veloped an average of 182 polyps (control group 1, Table 1), a
result that was unexpected based on previous screens. It is pos-
sible that the increased polyp number in these animals is caused
by one or more modifiers linked to the Rosa26-LsL-SB11 trans-
gene, because strain-specific modifiers are known to exist (19).
Control animals carrying the ApcMinallele, T2/Onc, and/or Vil-
Cre, but not Rosa26-LsL-SB11 (control group 2, Table 1) devel-
oped the same number of polyps as the control mice carrying the
ApcMinallele alone. Although the Rosa26-LsL-SB11 allele alone
contributes to polyp formation, the effect of active SB trans-
test mice. In addition, the tumor burden was so extensive that
ApcMinSB test mice became moribund earlier than any of the
three control groups (Table 1). Indeed, in a subset of ApcMinSB
test mice the tumor load was very severe, with some animals de-
veloping as many as 700 tumors.
Although polyp number was greatly increased by SB muta-
genesis, there was no evidence of local or systemic metastasis in
experimental or control mice. We performed histopathologic
analysis of tumors collected from 10 animals. These analyses
identified numerous microadenomas and adenomas in the small
intestine and a much smaller number of these lesions in the large
intestines. No adenocarcinomas were identified, perhaps because
of the short lifespan of ApcMinSB test mice. Immunohistochem-
istry for β-catenin was performed on 24 adenomas from seven
compared with the adjacent normal mucosa epithelium (Fig. 1).
Analysis of Common Insertion Sites Identifies 30 Candidate Cancer
Genes. To identify genes that contribute to tumor initiation and
96 polyps, representing all regions of the intestines, from 12 mice
to find common insertion sites (CIS). A CIS is defined by ana-
lyzing transposon insertions in many tumors and identifying ge-
nomic loci that contain transposon insertions at a higher rate than
would be expected by chance (SI Materials and Methods). The
presence of a CIS indicates that a transposon-mediated mutation
in that locus probably has contributed to tumor development. By
analyzing the genes within the CIS, one can identify candidate
To map transposon insertions, we isolated DNA from the 96
tumors, digested the DNA with restriction enzymes, and per-
formed ligation-mediated PCR (LM-PCR) to amplify trans-
poson-genomic fragments specifically (20). Barcodes and fusion
sequences were attached to the LM-PCR primers to enable
pooling of the amplicons, which then were sequenced using the
runs produced 347,993 sequence reads, 93% of which (324,898)
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 intestineSmall intestineTotal Date of death‡
*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 ApcMinonly.
†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.
| www.pnas.org/cgi/doi/10.1073/pnas.1018012108 Starr et al.
genomic sequence (>16 bp) for BLAST analysis. We were able to
map more than half of these sequences (53%) unambiguously to
the mouse genome. Of the 173,101 mapped sequences, 100,171
(67%) were redundant, leaving 72,930 nonredundant mapped
insertions. Roughly half of the nonredundant insertions mapped
to the same chromosome as the donor transposon concatamer
(Chr 1), as expected because of the phenomenon of local hopping
seen in other SB screens (12, 14, 21). To eliminate statistical bias
in the dataset, these sequences were eliminated along with a
smaller number of insertions that probably represent PCR arti-
facts (SI Materials and Methods). The remaining 30,088 insertions
(Dataset S1) were analyzed to determine CIS. We used Monte
Carlo simulations to find insertion rates in a given genomic win-
dow size that would not be expected to occur by chance (12). For
example, based on a random assignment of 30,088 insertions to
the mouse genome, one would not expect to find five or more
insertions within a 12-kb window. Using these Monte Carlo-
defined parameters, we identified 37 CIS. Two of these CIS were
to these two CIS originated from a single mouse, indicating the
tumors may be clonally related. Two more CIS were removed
because they also were identified in a control dataset of tail-snip
DNA from mice harboring unselected SB insertions and may
represent hotspots for SB insertions (12) Because this control
dataset was generated from tail snips, it is possible that other
hotspots exist in other types of cells. After removal of these pos-
sible artifacts, 33 CIS remained (Table 2).
We assigned a candidate gene to each CIS if the majority of
the insertions were in or near a single gene (Table 2). Four of the
33 CIS did not have an annotated gene within 40 kb and were not
assigned a candidate gene. Another CIS contained two over-
lapping genes, SET domain-containing 5 (Setd5) and lipoma
HMGIC fusion partner-like 4 (Lhfpl4), and all insertions in this
CIS were in both genes. Notably, this CIS is located adjacent to
the Rosa26 locus where the conditional SB11 knockin is located,
and eight of nine insertions in this CIS are oriented with the in-
ternal promoter in the direction that would cause overexpression
of the transgene. Rather than tagging an endogenous cancer
gene, this CIS could represent selective pressure for increased
mutagenesis via overexpression of SB11 transposase. Whether
the transposon insertion caused a gain- or loss-of-function mu-
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
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
the wild-type allele in ApcMinmice. Previous studies have dem-
onstrated that loss of the Apc+allele is an early event that occurs
in almost every adenoma in ApcMinmice (9, 10). In addition, in-
activation of the wild-type Apc allele is caused predominantly by
homologous somatic recombination events, leading to the re-
placement of the Apc wild-type allele with a second ApcMinallele
(22). We reasoned that in our transgenic model LOH could be
accomplished by an inactivating transposon insertion, as opposed
to duplication of the Min allele. To test this hypothesis, we per-
formed PCR on DNA from tumors to amplify the region sur-
rounding the Min mutation (T2860A). By sequencing the PCR
amplicon, LOH can be ascertained in ApcMinmice by measuring
the ratio of the T:A trace peak heights at the location of the Min
mutation. In heterozygous tissue the T:A ratio is between 0.8 and
1.2, which is considered MOH, but in tissue that has lost the wild-
type allele the ratio drops below 0.5 (Fig. S2). Ratios between 0.5–
0.8 and >1.2 are considered uninformative, most likely caused by
contamination from nontumor tissue. Of the 96 tumors tested, 47
gave informative results (Table S1). Of these 47 tumors, 32 had an
identified transposon insertion in the Apc locus, and 15 did not.
The majority (73%) of tumors lacking a transposon insertion in
Apc had T:A ratios <0.5, indicating LOH probably caused by loss
of the entire allele. In support of our hypothesis, 53% of the
tumors that had a transposon insertion in the Apc locus had T:A
ratiosbetween0.8 and 1.2,indicating maintenance ofthe wild-type
Apc locus at the site of the Min mutation. This result suggests that
in these tumors the wild-type Apc allele is inactivated by the
transposon more frequently than by duplication of the Min allele.
Set of CIS Identified in ApcMinMice Differs Significantly from Those
Found in Apc Wild-Type Mice. We compared the list of genes
identified in this study with the 77 genes identified in the screen
we performed on an Apc wild-type background (12). Surpris-
ingly, only four genes were identified in both studies: Apc, nu-
clear receptor binding SET domain protein 1 (Nsd1), Sfi1
homolog, spindle assembly associated (Sfi1), and WW domain
containing adaptor with coiled-coil (Wac). There are several
reasons that could explain why the overlap between the two
studies was low. First, the total number of genes that could
contribute to tumor formation may be large enough that the size
of these two studies is not sufficient to saturate the candidate
genes. Second, because we use a statistical method to identify
cancer genes, it is likely that transposon insertions contributed to
carcinogenesis in some of the tumors, but the insertions did not
occur at a rate high enough to qualify as a CIS. In support of this
hypothesis, 70% of the loci identified as CIS in this study (23 of
33) also had one or more insertions in the same locus in the
previous study (12). Third, the overlap may be small because
selection pressure for specific genetic mutations in cells that al-
ready have an Apc mutation is biased toward a different set of
cancer genes than in cells with a different initial mutation.
Fourth, because of technical limitations, our method of ampli-
for β-catenin (B and C). (B) There is increased staining for β-catenin (arrow) in
the adenoma. (C) Higher-power magnification of a different section show-
ing increased cytoplasmic and nuclear (arrows) staining for β-catenin in tu-
mor cells compared with adjacent normal tissue seen in lower right and
bottom of picture. (Scale bars: A and B, 500 μm; C, 50 μm.)
A pedunculated adenoma stained with H&E (A) or immunostained
Starr et al. PNAS
| April 5, 2011
| vol. 108
| no. 14
fying and sequencing transposon insertions does not identify all
transposon insertions, so a portion of driver mutations will not be
identified. For example, analysis of replicate sequencing runs
indicates that 20–40% of the PCR amplicons in a given library
are not sequenced in a given GS FLX sequencing run (SI
Materials and Methods and Table S2).
Relevance to Human Disease. To determine the relevance of these
findings to human cancers, we analyzed the regions of human
orthology to the CIS loci and the orthologous human genes. Of
the 30 candidate mouse genes associated with a CIS, 28 had hu-
man orthologs. We queried the literature for mutations and re-
current copy number changes in these genes in human CRC.
Three of the genes, APC, NSD1, and phosphodiesterase 4D-
interacting protein (PDE4DIP), are considered bona fide cancer
genes based on the cancer gene census maintained by the Well-
come Trust Sanger Institute (23). Eight genes, APC, activating
transcription factor-2 (ATF2), atlastin GTPase 2 (ATL2), casein
kinase 1, alpha 1 (CSNK1A1), integrin, alpha M (ITGAM), pro-
grammed cell death 6-interacting protein (PDCD6IP), and WAC,
have documented mutations in human cancers cataloged in the
COSMIC database (24).
We found strong concordance between the CIS mouse loci
and orthologous regions in the human genome showing recur-
rent chromosomal losses and gains in human CRC (25–32). Of
the 33 identified CIS, 31 can be mapped to an orthologous hu-
man locus. Of these 31 candidate cancer loci, 24 are found in
regions that commonly are lost or gained in human CRC (Table
S3), including several of the CIS that are orthologous to human
chromosomal arms 18q, 17p, 5q, and 4q. Interestingly, one CIS
that has no annotated genes nearby (CIS No gene 16) is in an
orthologous region (3q21–24) that is associated with CRC based
on genome-wide linkage analyses (33, 34). To determine the
significance of this overlap, we performed the analysis using
randomly generated CIS lists and a single dataset of regions that
are lost recurrently in human CRC (27). Roughly 250 genomic
regions in this dataset were lost in >5% of the human samples
tested, and 22 of the 31 CIS were located within these regions. In
10,000 simulations using equivalent-sized randomly generated
CIS lists, we find an overlap of this magnitude <0.3% of the time.
These results suggest that our SB screen may be capable of
pinpointing the affected genes in these regions.
Candidate Genes Regulate Proliferation of Human CRC Cell Lines. We
tested nine genes [CCR4-NOT transcription complex, subunit 1
(CNOT1), PDE4DIP, PDCD6IP, ATF2, SFI1, formin-binding
protein 1-like (FNBP1L), myosin VB (MYO5B), sorting nexin 24
(SNX24), and stromal antigen 1 (STAG1)] for their effect on
Table 2. List of 33 CIS
Candidate gene ChromosomeStart address*End address*
No Gene 16
No Gene 18
No Gene 4
No Gene Y
Setd5 or Lhfpl4
*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.
| www.pnas.org/cgi/doi/10.1073/pnas.1018012108Starr et al.
proliferation of the human CRC cell line SW480 by knocking
down message levels using siRNA. We used the SW480 line
because it has an APC gene-truncation mutation similar to the
ApcMinmutation (35). Cells were transfected two times at 48-h
intervals with siRNA targeting the human genes. Knockdown
efficiency was at least 50% for all nine genes as measured by
quantitative real-time PCR. Cell proliferation was measured
using a tetrazolium-based colorimetric assay on days two and six
after the second transfection. Depletion of five (CNOT1,
PDE4DIP, PDCD6IP, ATF2, and SFI1) of the nine genes tested
resulted in a significant decrease in cell viability compared with
a control siRNA of at least 33% at day six after transfection
Using a transposon-based forward genetic screen in mice, we
identified 33 genomic loci that probably cooperate with a germ-
line mutation in the Apc gene to cause intestinal tumorigenesis.
The most frequently mutated locus was the Apc locus, a result
that supports the hypothesis that there is strong selective pres-
sure to lose the wild-type copy during tumor formation. SB
insertions in Apc were found in 72 of 96 tumors (75%), sug-
gesting that 75% of the tumors in ApcMinSB test mice undergo
LOH at the Apc locus via SB insertional mutagenesis. This hy-
pothesis is supported by sequencing of the region spanning the
1-bp Min mutation, which indicated that the majority of tumors
containing SB insertions at the Apc locus maintained heterozy-
gosity at the location of the T:A Min mutation. In contrast, the
majority of tumors lacking a transposon insertion in Apc showed
loss of the wild-type sequence at the Min mutation site. These
results suggest that the majority of tumors underwent biallelic
loss of Apc activity through transposon insertion or somatic re-
combination. However, it also is possible that in some cases
activity was lost through other mechanisms of Apc inactivation or
through transposon insertion substituting for loss of the wild-
Although LOH at the Apc locus is the rate-limiting event in
tumor initiation in the ApcMinmouse and in familial and sporadic
APC-deficient CRC, loss of APC probably is insufficient for the
survival and growth of transformed cells into adenomas and,
eventually, adenocarcinomas. SB-mutagenized animals showed
increased polyp number but no evidence of adenocarcinoma or
metastasis. Thus, it is likely that the CIS candidate genes identi-
fied in this SB screen contribute in a diverse fashion to initiation,
establishment, and survival of adenomas. Moreover, depending
on the complexity of the mechanism or pathway, the CIS candi-
dates discovered in our screen, like the relatively large number of
genes reported to be mutant in individual human CRC (3), might
be expected to occur at a low frequency if mutations at any one of
multiple genes in a complex pathway can contribute equally
Aside from Apc, only a few of the remaining 28 known genes
that we identified as CIS in our screen have been implicated di-
rectly in CRC development, although >90% are located in ge-
nomic regions that are lost or gained in human CRC. To
eliminate false positives, we removed CIS that also were identi-
fied in a control dataset of unselected transposon insertions
mapped in tail snips. However, it is possible that other tissue-
specific hotspots could result in false positives. Nevertheless, the
known functions of several of the CIS candidate genes make them
plausible candidates for drivers of human CRC. For example,
CNOT1 is a member of the Ccr4-Not complex, which is im-
plicated in mRNA decay and transcriptional repression. In hu-
man cells, CNOT1 has been reported to be a repressor of nuclear
receptor-mediated transcription (36). One target of CNOT1 re-
pression appears to be estrogen receptor alpha (ERα), via
interactions between CNOT1 and the ligand-binding domain of
ERα. Inhibition of CNOT1 caused an increase in the expression
of ERα target genes in breast cancer cells, and ERα has been
showntobea tumorsuppressorgeneintheintestinal tract(37);in
particular, knockout of ERα in ApcMinmice caused a significant
increase in intestinal tumorigenesis (38).
Another gene identified in this study, Pdcd6ip (also known as
ALG-2 interacting protein X, Alix) is involved in membrane
trafficking and apoptosis (39). Pdcd6ip produces a protein that
binds to the protein product of Pdcd6, a proapoptotic gene in-
volved in T-cell receptor–, Fas-, and glucocorticoid-induced cell
death (40). Pdcd6ip also can block down-regulation of the EGF
receptor (EGFR), thereby having a positive effect on growth
factor signaling (41). These contradictory roles could explain why
loss of Pdcd6ip in the mouse tumors promoted growth (via the
loss of the proapoptotic function) but the loss of Pdcd6ip in
SW480 cells caused decreased proliferation (via increased down-
regulation of EGFR). Further functional studies are required to
elucidate the role of this adaptor protein.
In summary, our approach identified 30 genes that probably
modify tumorigenesis in the ApcMinmodel of human CRC.
Further functional analysis of these CIS candidate genes may
provide insights into the etiology and treatment of human CRC,
especially those cancers arising downstream of APC deficiency.
Materials and Methods
Detailed protocols are given in SI Materials and Methods.
Mice. Mice containing ApcMin, Rosa26-LsL-SB11, Villin-Cre, and T2/Onc were
reared using Institutional Animal Care and Use Committee-approved pro-
tocols. All mice were on an isogenic C57BL/6J background. Mice were
monitored daily and killed and necropsied when moribund or after 120 d.
Histopathology and Immunohistochemistry. Formalin-fixed tissues were em-
bedded in paraffin, and standard techniques were used to stain tissue sec-
tions with H&E. Standard immunohistochemistry techniques were used to
Linker-Mediated PCR. Linkers [described previously (42)] were ligated to
NlaIII- (right-side) or BfaI- (left side) digested genomic DNA using T4 DNA
ligase. A secondary digest (XhoI, right side; BamHI, left side) was performed
to destroy concatamer-generated products. Primary and secondary PCR was
performed using primers specific for linker and SB transposon sequences
along with Fusion and barcode sequences. PCR amplicons were sequenced
using the GS FLX (Roche).
Sequence Analysis. Sequences were analyzed for the presence of the barcode,
inverted repeat/direct repeat (IR/DR) sequences required for transposition,
and linker sequences. Genomic sequence was blasted against the mouse
genomeusingBLASTNat 95%stringency andrequiring asingle match.Of the
324,898 sequences analyzed, 53% could be uniquely mapped to the mouse
genome. Sequences were removed if they were redundant, on the donor
concatamer resident chromosome (Chr 1), in the En2 gene (because the En2
sequence is present in the transposon), and when a single TA dinucleotide
contained multiple insertions from several tumors from multiple mice (be-
sequences were used to identify CIS. A CIS was defined by Monte Carlo
simulations using a random dataset of 30,088 insertions.
Apc LOH Analysis. To measure LOH for the ApcMinmutation, DNA was isolated
from individual polyps, and PCR was performed using primers that flank the
mutation (sense primer: CGGAGTAAGCAGAGACACAA; antisense primer:
GGGAGGTATGAATGGCTGAT). The PCR product was purified using Qiagen
96 MinElute vacuum purification plates per the manufacturer’s protocol and
was sequenced using the sense primer as the sequencing primer. Trace peak
heights at the location of the mutation were measured for each tumor, and
the ratio of the T peak to the A peak was calculated.
Comparisons with Human Data. Eight publicly available studies measuring
DNA copy number in CRC compared with normal tissue were analyzed.
Mutations in human tumors were examined using the Catalog of Somatic
Mutations in Cancer database (24), and cancer gene status was based on the
Census of Human Cancer Genes maintained by the Wellcome Trust Sanger
Starr et al.PNAS
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| vol. 108
| no. 14
Knockdown of CIS Candidate Genes using siRNA in SW480 Cells. SW480 cells
were obtained from ATCC (catalog no. CCL-228) and were cultured under
recommended conditions. Transient siRNA transfection was used to deplete
expression of Min CIS genes.
Cell Viability Assay. Viability of siRNA-treated SW480 cells was determined
using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay (Cell Viability Kit 1; Roche Applied Sciences).
ACKNOWLEDGMENTS. Pauline Jackson, Jerica Burchard, and Annette Rod
provided technical assistance for the gene expression analyses of CIS genes.
We thank the following University of Minnesota Masonic Cancer Center
Cores: Biostatistics and Informatics Shared Resource, Comparative Pathol-
ogy, and Mouse Genetics Laboratory. We also thank the Minnesota
Supercomputing Institute and the BioMedical Genomics Center. Research
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
Grants R01CA113636-01A1 (to D.A.L.) and R01 CA134759-01A1 (to D.A.L.
and R.T.C.). A University of Minnesota Academic Health Center Faculty
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
Boman Women’s Fellowship program. B.L.T. and L.Z. were recipients of
a research support award from the University of Minnesota Duluth chapter
of Sigma Xi.
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| www.pnas.org/cgi/doi/10.1073/pnas.1018012108Starr et al.