© 2008 Japanese Cancer Association
Cancer Sci| November 2008| vol. 99| no. 11| 2113–2119
Blackwell Publishing Asia
Expression of Pla2g2a prevents carcinogenesis in
R. J. A. Fijneman,1,2 J. R. Peham,1 M. A. van de Wiel,1,3,4 G. A. Meijer,1 I. Matise,5 A. Velcich6 and R. T. Cormier7,8
1Department of Pathology, 2Department of Medical Oncology, 3Department of Biostatistics, 4Department Of Mathematics, VU University Medical Centre,
Amsterdam, The Netherlands; 5University of Minnesota College of Veterinary Medicine, St. Paul, MN 55108; 6Albert Einstein Cancer Center, Bronx, NY 10467;
7University of Minnesota Medical School, Duluth, MN 55812, USA
(Received May 13, 2008/Revised June 16, 2008/Accepted June 30, 2008/Online publication October 15, 2008)
Goblet cell depletion and down-regulation of MUC2 expression are
observed in a significant percentage of human non-mucinous
colorectal adenocarcinomas. Direct evidence for the role of MUC2 in
gastrointestinal tumor formation was demonstrated by a knockout
of Muc2 in mice that resulted in the development of adenocarcinomas
in the small and large intestine. The secretory phospholipase Pla2g2a
is a protein that confers resistance to ApcMin/+-induced intestinal
tumorigenesis. Like Muc2, in the large intestine Pla2g2a is exclusively
expressed by the goblet cells and Pla2g2a’s tumor resistance is also
strongest in the large intestine. Possible genetic interactions between
Muc2 and Pla2g2a were examined by creating C57BL/6-Muc2–/–Pla2g2a
transgenic mice. Expression of a Pla2g2a transgene reduced tumorige-
nesis in the large intestine by 90% in male Muc2–/– mice and by nearly
100% in female Muc2–/– mice. Expression of Pla2g2a also inhibited
tumor progression. Microarray gene expression studies revealed
Pla2g2a target genes that modulate intestinal energy metabolism,
differentiation, inflammation, immune responses and proliferation.
Overall, results of the present study demonstrate an Apc-independent
role for Pla2g2a in tumor resistance and indicate that Pla2g2a plays
an important role, along with Muc2, in protection of the intestinal
mucosa. (Cancer Sci 2008; 99: 2113–2119)
tion to the epithelial mucosa.(1,2) In the intestinal epithelium of
mice and humans, the major secretory mucin is MUC2, which
is expressed in the goblet cell population.(3,4) MUC2 is a large,
heavily glycosylated, protein (5200 amino acids) that constitutes
the major structural component of mucus, thus contributing to
barrier functions. The role of MUC2 in intestinal tumorigenesis
appears to be complex. Goblet cell depletion and down-regulation
of MUC2 expression(5–7) or MUC2 protein alteration(8) occur in
a significant percentage of human non-mucinous colorectal aden-
ocarcinomas.(9) MUC2 is robustly expressed in normal colon tissue(3)
and its expression is observed in early stages of adenomagenesis
but progressive loss of MUC2 function occurs in the transition to
carcinogenesis.(5,9–12) In contrast, up-regulation of MUC2 is occa-
sionally observed in mucinous adenocarcinomas that are also
characterized by microsatellite instability.(10,12–17) In addition, mucins
secreted by colon cancer cells may contribute to the metastatic
process,(12) and are reported to increase the production of prostaglandin
E2 (PGE2) and cyclooxygenase-2 (COX-2) in macrophages.(18)
Velcich and colleagues generated a germline knockout of
Muc2(19) in mice on a C57BL/6J-129/SvOla mixed genetic back-
ground that resulted in the absence of recognizable goblet cells
along the entire length of the intestine, although the expression
of some goblet cell differentiation markers was retained. Muc2
knockout mice developed small intestinal tumors by 6 months of
age and eventually adenocarcinomas in the duodenum and rectum.
Muc2–/– tumors showed no alterations in the β-catenin pathway;
thus Muc2 deficiency likely represents an Apc-independent tumor
pathway. Despite the different pathways of tumorigenesis, inactivation
ucins are the principal components of intestinal mucus, a
viscous substance that provides protection and lubrica-
of p21WAF1/CIP1 similarly affects tumor formation in both the Muc2
and mutant Apc mouse models of intestinal carcinogenesis.(20)
The secretory phospholipase Pla2g2a is a modulator of tum-
origenesis that has been shown to confer resistance to intestinal
cancer in the mouse. The Pla2g2a gene is part of the Mom1
(Modifier of Min-1) complex of genes on distal mouse chromo-
some four that confers resistance to tumorigenesis in the ApcMin/+
mouse.(21,22) Pla2g2a is naturally mutant in three strains of mice
(including C57BL/6 J and 129/SvOla) that develop a severe
ApcMin/+ phenotype and is wildtype in six strains that demon-
strate resistance to intestinal tumorigenesis.(23,24) Genetic evidence
for the function of Pla2g2a as a tumor suppressor was demon-
strated by the reduction in tumors observed in ApcMin/+ mice
carrying a wildtype Pla2g2a transgene derived from the AKR
strain.(25) The Pla2g2a transgene resistance phenotype is strongest
in the large intestine and like Muc2, Pla2g2a is exclusively
expressed by the goblet cell population.(22)
Pla2g2a and Muc2 share several common genetic and bio-
chemical pathways. Cytokines such as interleukin-1β (IL-1β),(26)
growth factors such as epidermal growth factor (EGF)(27) and
prostanoids such as PGE2 that lie upstream (IL-1β) and down-
stream (EGF, COX-2, prostaglandins) of Pla2g2a are potent
activators of MUC2. MUC2 and Pla2g2a also share a common
signaling pathway that involves protein kinase C (PKC), mitogen-
activated protein kinase (MAPK), cyclic adenosine monophos-
phate (cAMP), tumor necrosis factor alpha (TNF-α), extracellular
signal regulated kinase 1 and 2 (ERK 1, ERK 2) and nuclear
factor B (NF-κB). For example, phorbol myristate acetate (PMA)
induces expression of both MUC2 and Pla2g2a and Muc2 is
induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) through
PKC and by cAMP through protein kinase A (PKA) in HT29
colon cancer cells.(28,29) Further, arachidonic acid, a long-chain fatty
acid that results from Pla2g2a hydrolysis of membrane glycero-
phospolipids, has been shown to induce secretion of mucins.(30)
Because of the commonalities shared by Pla2g2a and Muc2
and as the genetic background used by Velcich and coworkers
in the original Muc2 knockout study was naturally mutant for
Pla2g2a (both C57BL/6 J and 129/SvOla), we sought to directly
test for genetic interactions between MUC2 and Pla2g2a by gen-
erating C57BL/6J-MUC2–/– mice that also expressed a wildtype
Materials and Methods
Mice. C57BL/6 J mice were obtained from the Jackson
Laboratory (Bar Harbor, ME, USA). Muc2 knockout mice(19)
were obtained from Dr Anna Velcich, Albert Einstein School of
Medicine (Bronx, NY, USA). Pla2g2a transgenic mice(22) were
obtained from a colony maintained at the University of Minnesota
Medical School (Duluth, MN, USA). All mice were housed and
8To whom correspondence should be addressed. E-mail: email@example.com
Abbreviations: APC, adenomatous polyposis coli; B6, C57BL/6; MUC2, Mucin 2
2114 doi: 10.1111/j.1349-7006.2008.00924.x
© 2008 Japanese Cancer Association
bred at the University of Minnesota Medical School Duluth
Animal Services Facility (AALAC accredited) and all mouse
experiments followed a protocol approved by the University of
Minnesota Institutional Animal Care and Use Committee. Mice
were housed in microisolator cages that were changed in a
laminar flow hood following a customary protocol to prevent
pathogen transfer. Sentinel mice caged on the same racks as
experimental mice tested negative for a panel of common
pathogens during the entire period of the study. All mice were
fed food (Purina 5001 Rodent Chow) and tap water ad libitum.
All mice were monitored daily for signs of distress and
sacrificed when either distressed or moribund or at greater than
180 days of age up to 15 months. Test mice were clustered at
mean ages of 30 weeks or 60 weeks of age.
Experimental crosses. Muc2 mutant mice that were predom-
inantly congenic on the C57BL/6 genetic background were
backcrossed to C57BL/6 J for an additional six generations to
ensure that both the Muc2 mutation and the Pla2g2a transgene
were on an isogenic C57BL/6 J background. A three-generation
cross established breeding pairs of Muc2–/– mice that were either
positive or negative for the Pla2g2a transgene. For gene expression
analyses, C57BL/6J-Muc2–/– mice both positive and negative for
the Pla2g2a transgene and of both sexes were sacrificed at 100
days of age and the distal colons were removed and processed
in Qiagen RNA Later (Qiagen, Valencia, CA, USA).
Genotyping. DNA was isolated from tail snips using a Qiagen
DNA Easy kit (Qiagen, Valencia, CA, USA). The genotype of the
Pla2g2a locus was determined by PCR assay as previously
described.(22) The Muc2 genotype was determined by a published
PCR protocol.(19) All PCR was performed on an Applied Biosystems
GeneAmp 9700 Thermal Cycler (Applied Biosystems, Foster City,
Tumor statistical analysis. Two-sided P-values for tumor counts
were determined by use of the non-parametric Wilcoxon Rank
Sum Test comparing sex- and age-matched classes produced in
the same genetic crosses.
Microarray and Data Analysis; RNA Extraction; Quantitative Real
Time PCR; Tumor Analysis; Immunohistochemistry; Histopathology. See
Expression of Pla2g2a significantly reduced tumor multiplicity and
incidence in the large intestine of Muc2 mutant mice. To test for
interactions between Pla2g2a and Muc2 in Muc2-induced intestinal
cancer, we introgressed a Pla2g2a transgene into the B6-Muc2–/–
strain, eventually creating Muc2–/– mice that were either positive
or negative for the Pla2g2a transgene. We found that expression
of Pla2g2a strongly inhibited tumorigenesis in the Muc2–/– mouse
(Table 1). In male Muc2–/– mice, Pla2g2a reduced tumor number
in the large intestine by 88% in mice at 30 weeks of age
(P = 0.03) and by 91% in mice at 60 weeks of age (P = 0.0001).
Expression of Pla2g2a also produced a corresponding reduction
(by ~ 2/3) in tumor incidence in the large intestine of male
Muc2–/– mice. Almost all of the large intestinal tumors of both
genotypes were found clustered midway between the cecum and
distal rectum, generally in the distal colon. Colonic tumors that
did appear in the Pla2g2a transgenic mice also tended to be
much smaller (generally less than 3 mm in diameter, even at
60 weeks) than tumors from Muc2–/– mice (which were often as
large as 6 mm or more in diameter) but there were too few
samples to conduct a meaningful size comparison. In female
Muc2–/– mice expression of Pla2g2a nearly completely abolished
tumorigenesis in the large intestine.
Duodenal and ileal tumors in C57BL/6J-Muc2-deficient mice. In
the duodenum of male Muc2–/– mice, expression of Pla2g2a
caused a 50% reduction in tumors in mice at 60 weeks of age.
Duodenal tumor incidence was only half of that observed in the
Muc2–/– class. In female Muc2–/– mice, expression of Pla2g2a
completely abolished tumorigenesis in the duodenum (Table 1).
A novel type of lesion found in Muc2–/– mice on the C57BL/6 J
background is a sessile adenoma that predominantly arises in
the distal ileum, sometimes close to the ileocecal junction.
These tumors are broad, very low lesions and they are difficult
to identify and separate in whole-mount tissue analysis (see
Fig. 1d). The incidence and multiplicity of these tumors was
modestly higher in Muc2–/– mice than in Muc2–/– Pla2g2a
transgenic mice and the ileal tumors were more numerous in
female mice than males. Histopathological analysis revealed that
most of these tumors were tubular adenomas with high-grade
dysplasia. Our pathologist (I.M) did identify one likely ileal
adenocarcinoma and several more potential adenocarcinomas
based on observing glandular structures within the submucosa
where the greatest difficulty was distinguishing glandular herniation
from true invasion. These ileal tumors were not reported in
Muc2–/– mice on the C57BL/6 J × 129/SvOla mixed background,
thus the appearance of ileal tumors may be due to factors in the
C57BL/6 J genetic background. Finally, we also found a few
Table 1. Expression of Pla2g2a prevents tumorigenesis in Muc2–/– mice
Ileum tumor incidence
3.6 ± 2.9
2.6 ± 1.8
5.4 ± 3.1
0.9 ± 1.2
0.3 ± 0.6
1.6 ± 1.1
2.3 ± 3.4
2.5 ± 3.5
1.9 ± 3.4
0.4 ± 0.6
0.3 ± 0.4*
0.5 ± 0.8**
0.5 ± 0.5
0.3 ± 0.5
0.8 ± 1.0***
0.2 ± 1.2
0.5 ± 1.7
1.5 ± 1.7
2.6 ± 1.6
0.8 ± 1.4
0.3 ± 0.6
0.4 ± 1.7
0.2 ± 0.7
1.8 ± 2.3
0.9 ± 2.8
2.3 ± 2.3
0.1 ± 0.6
0.2 ± 0.8
1.2 ± 2.3
0.3 ± 0.6
3.0 ± 1.5
*P = 0.03; **P = 0.0001; ***P = 0.21.
P-values compare age and sex matched classes. N = number of mice.
Fijneman et al.
Cancer Sci| November 2008| vol. 99| no. 11| 2115
© 2008 Japanese Cancer Association
adenomas in the medial region of the small intestine of C57BL/
Tumorigenesis in Muc2-deficient mice is affected by genetic
background. Previous work showed that Muc2 nullizygosity
caused small intestinal tumors in some mice as early as 26 weeks
of age (6 months).(19) In that study, mice were on a mixed genetic
background (C57BL/6J ×129/SvOla; F2 to F5 backcross to
C57BL/6J). In the current study, all mice were on a fully
isogenic C57BL/6J background. Notably, on the C57BL/6 J
background, Muc2-deficient mice developed at least 4-fold more
tumors overall. These increases were observed in both the small
and large intestine but the difference was strongest in the large
intestine. On the mixed genetic background, Muc2 knockout
mice developed no tumors in the large intestine at 26 weeks and
only 0.26 tumors at 52 weeks of age.(19) On the C57BL/6 J
background, Muc2 knockout mice (combined male and female
classes) developed a mean average of 2.6 tumors in the large
intestine at 30 weeks of age and 3.3 tumors at 60 weeks of age;
thus, overall, Muc2 knockout mice on the C57BL/6 J background
developed greater than 10-fold more large intestinal tumors than
on the C57BL/6 J × 129/SvOla mixed background. In addition,
genetic background also affected the location of tumors. The
rare tumors in the large intestine of older mice on the mixed
background were most common in the distal rectum but on the
C57BL/6 J background tumors were more often found in the
medial large intestine/distal colon. However, we did still observe
some tumors in the distal rectum of the C57BL/6J-Muc2–/– mice.
The corresponding tumor incidence was also much higher in
Muc2–/– mice on the C57BL/6 J strain. For example, in the
combined sex groups, overall, 76% of mice on the C57BL/6 J
background developed tumors in the large intestine versus only
11% of mice on the mixed background.(19)
Sex also significantly affected the phenotype of Muc2 knockout
mice in our study. Male mice developed more tumors in both the
large intestine and duodenum. For example, male knockout mice
(30 and 60 weeks of age combined) developed a mean of 3.6
large intestinal tumors and 0.9 duodenal tumors compared with
1.5 and 0.3 tumors in female mice, respectively (Table 1).
Histopathology of Muc2-deficient tumors. Similar to the previous
report by Velcich et al.(19) in the current study C57BL/6J-Muc2–/–
mice developed adenocarcinomas in the large intestine. Adenoc-
arcinomas were scored only if there was clear invasion of
submucosa or muscularis and an effort was made to distinguish
these characteristics from gland herniation and tissue invasion
(see Fig. 1a,b). Adenocarcinomas ranged from well to moderately
differentiated. However, unlike the previous study we found
adenocarcinomas only in the medial large intestine/distal colon
and not in the distal rectum. We observed a frequency of
adenocarcinomas in the large intestine of 30% (6/20) compared
with 15% (3/19) in mice from the mixed genetic background.(19)
The remaining 70% of tumors in the large intestine were described
as tubular adenomas with high grade dysplasia. Interestingly, we
observed no adenocarcinomas in the duodenum of C57BL/6J-
Muc2–/– (0/18) in mice at 60 weeks of age, unlike the report of
duodenal adenocarcinomas in the mice on the mixed genetic
background.(19) Moreover, duodenal tumors were extremely rare
in the C57BL/6 J class at 30 weeks of age, again, different than
that reported in the mice on the mixed background. The duodenal
adenomas that developed at 60 weeks in C57BL/6 J mice
generally were described as sessile tubular adenomas, with
high-grade dysplasia and extensive Brunner’s gland hyperplasia
(see Fig. 1c).
Pla2g2a may prevent cancer progression. Muc2–/– mice that ex-
pressed the Pla2g2a transgene failed to develop a single tumor
normal tissues from the duodenum, ileum and colon obtained from Muc2–/– mice that were either positive or negative for the Pla2g2a transgene.
Multiple sections were obtained from 18 duodenal tumors, 20 large intestinal tumors and 11 ileal tumors from both male and female Muc2–/– mice
and six duodenal tumors, seven colon tumors and four ileal tumors from male Muc2–/– Pla2g2a transgenic mice. In Muc2–/–mice we observed
adenocarcinomas in six of 20 tumors from the large intestine (virtually all from the distal colon); the remaining 14 tumors were described as tubular
adenomas with high grade dysplasia. No adenocarcinomas were observed in 18 duodenal tumors. The duodenal tumors were generally described
as tubular adenomas, sessile, with Brunner’s gland hyperplasia. In the ileum, one of 11 tumors was described as an adenocarcinoma with the
remaining tumors described as tubular adenomas, sessile with high grade dysplasia. Figure 1 shows representative images from the
histopathological analysis of Muc2–/– tumors. (a) Adenocarcinoma from the distal colon (40×). (b) The same tumor with arrows indicating invasive
areas (200×). (c) Duodenal adenoma with the arrow pointing to the extensive Brunner’s gland hyperplasia (40×). (d) Adenoma from the ileum (40×).
Pla2g2a transgenic mice did not develop adenocarcinomas and adenomas were generally identical to those seen in Muc2–/– mice and generally
were described as tubular adenomas.
Histopathology of tumors from Muc2–/– mice. Histopathological analysis was performed on formaldehyde-fixed tumors and adjacent
© 2008 Japanese Cancer Association
that was classified as invasive (0/17, see Table 2). The rare
lesions in the colon of Pla2g2a transgenic mice at 60 weeks of
age were described as either tubular adenomas or as structures
characterized by thickening of colonic mucosa suggestive of
adenoma or diffuse, mild, crypt hyperplasia. None of the colonic
lesions in the transgenic mice showed any evidence of invasion.
Tumors from the duodenum of Pla2g2a transgenic mice were
identical in histopathological description to tumors from non-
transgenics: tubular adenomas with Brunner’s gland hyperplasia.
Pla2g2a protein is expressed and secreted by cells of the goblet
cell region of the large intestine in B6-Muc2–/– Pla2g2a transgenic
mice. As Muc2 deficiency prevents the development of cells of
goblet cell morphology, we wished to determine whether the
expression pattern of Pla2g2a might be altered in the absence
of Muc2. We found abundant expression of Pla2g2a in the
epithelial cells and extracellular lumen of the large intestine,
indicating that secretion of Pla2g2a is not dependent on Muc2
activity (see Fig. 2). The expression and secretion of Pla2g2a
was generally not inhibited by the complete loss of goblet-like
cells in Muc2–/– intestine, although immunohistochemical analysis
did reveal a patchy pattern to Pla2g2a protein expression in
comparison with the robust Pla2g2a protein expression found
in Pla2g2a transgenic mice that are wildtype for Muc2 (see
Gene expression regulated by Pla2g2a in colon of Muc2-deficient
mice. Deficiency for Muc2 results in profound changes in gene
expression in the colon of C57BL/6 mice. An extensive study of
Muc2 knockout mice describing changes in gene expression in
different regions of the gastrointestinal tract and at different ages
is being published separately (A. Velcich, unpublished data).
Here, using the Agilent 4 × 44K microarray platform, we took a
snapshot of gene expression changes in the distal colon at 100
days of age, comparing C57BL/6J-Muc2–/– mice with C57BL/
6J-Muc2+/+-Pla2g2a-Tg and C57BL/6J-Muc2–/–-Pla2g2a-Tg
mice. First, comparing C57BL/6J-Muc2–/– mice with C57BL/6J-
Muc2+/+-Pla2g2a-Tg mice we observed that deficiency for both
Muc2 and Pla2g2a caused the up-regulation of groups of genes
that are involved in the cell cycle (Cdc7, Cdca5, Ccna2, Cdca2,
Cdca1, Cdkn3, Ccnf, Cdca3, Cdk2, Ccnb1), inflammation
(S100a11, Il1rl1, Tnfrs1b, Il1b, S100a16) and oncogenicity
(Has1, Nrg1, Fgfr4, Stc1, Mmp10, Baiap2, Afp, Bysl, Ptgs2,
Mybl2, Egfr, Bmp7, Kras, Nras). Genes significantly down-
regulated in this comparison were involved in some of these
same processes and included Igfbp6, Plp1, Grb14, Reck, Ssbp2,
Glis2, Lefty1, Tgfb1l1, Efna3, Gtl2, Adam11, Sst, Ptger3 and Ppara.
A select list of genes that are differentially expressed in the
C57BL/6J-Muc2–/–/C57BL/6J-Pla2g2a-Tg comparison is provided
in Suppl. Table S1.
We then compared the gene expression profiles of C57BL/6J-
Muc2–/– mice with C57BL/6J-Muc2–/–-Pla2g2a-Tg mice. Overall,
224 genes and transcripts were found to be significantly
changed (a complete list of these genes, grouped by functional
class, is included in Suppl. Table S2). First, we found that
expression of Pla2g2a on the Muc2 knockout background signif-
icantly modulated a number of genes involved in intestinal lipid
and energy metabolism. Genes in this category that were up-
regulated included Akr1c18, Aldh1l2, Mocos and Aqp5. Among
the down-regulated genes were two members of the chymo-
trypsin family, Ctrb1 and Ctrl, plus Enpp1, Cel, Cpa2 and Pnlip.
Pla2g2a also modulated a number of genes involved in cell sig-
naling and nuclear transactivation. Genes up-regulated in this
group included Mier2, Lhx1, Mlx1pL, Rgs3, Runx1 and Spp1.
Genes down regulated in this category included Egfr, Reg2, Id4,
Gli3 and Tgfb2. Eight Pla2g2a target genes have been reported
to be Notch pathway targets including Adam 11, Lhx1, Spp1,
Table 2. Tumor progression in Muc2–/– mice
Class Large intestineDuodenumIleum
Tumors were obtained from 15 Muc2–/– mice (both males and females)
and six Muc2–/–-Pla2g2a transgenic mice (males only for colon and
duodenum tumors). All tumors from Muc2–/– mice were from the
60-week age class. Tumors from the Muc2–/– Pla2g2a transgenic mice
include all of the colon and duodenum tumors that arose in these mice
(at both 30 and 60 weeks). *Single asterisk indicates that several more
ileal lesions were potential adenocarcinomas but a definitive
classification could not be made.
of Muc2–/– Pla2g2a transgenic mice. Expression
patterns of Pla2g2a protein were determined by
immunohistochemistry using a polyclonal rabbit
antimouse antibody at 1:90 000 dilution and
visualized with avidin–biotin complex reagents.
Formaldehyde-fixed tissues were obtained from
the duodenum, jejunum and ileum and several
regions of the colon. In the small intestine
Pla2g2a was expressed normally, with strong
staining seen at the bottom of crypts in the
Paneth cell compartment (data not shown). Figure 2
depicts a representative tissue section from the
large intestine. (a) Abundant secreted Pla2g2a
protein found in the columnar epithelial cells and
lumen of crypts is clearly lacking in cells of goblet
cell morphology (200×). Only columnar epithelial
cells can be seen lining the crypt. Arrows point
to regions of Pla2g2a expression. (b) A higher
resolution image of Pla2g2a staining in C57BL/6J-
Muc2–/– Plag2g2a transgenic mice (400×). Arrows
point to Pla2g2a expression. (c) Pla2g2a immuno-
staining from tissue from a C57BL/6J Pla2g2a
transgenic mouse that is wildtype for Muc2 (40×).
Pla2g2a is clearly detected in the goblet cells but
not the enterocytes at the crypt borders. (d)
Immunostained tissue from a C57BL/6J mouse
(200×). No Pla2g2a protein is detected.
Immunohistochemistry for Pla2g2a in colon
Fijneman et al.
Cancer Sci| November 2008| vol. 99| no. 11| 2117
© 2008 Japanese Cancer Association
Runx1 and Megf11 (up-regulated), and Ela2a, Dtx1, Gli3, Myl9
(down regulated). Finally, genes involved in inflammatory and
immune responses were differentially expressed in Pla2g2a
positive Muc2–/– colon. Down regulated genes in this category
included Clps and Cd248 and up-regulated genes included
Defb11, Ccr3, Ccl7, Ccl24 and Tm7sf4. Pla2g2a target genes
were also involved in apoptosis and oxidative stress (e.g. Nudt15),
protein degradation (e.g. Syvn1), intracellular trafficking (e.g.
Praf2), cell structure and adhesion (e.g. Lmod1), transporters
(e.g. Slc25a3) and proteases (e.g. Adam11). Finally, via subtraction
of genes that were differentially expressed in the comparison
C57BL/6J-Pla2g2a-Tg/C57BL/6 J (data not shown) from the genes
differentially expressed in the comparison C57BL/6J-Muc2–/–-
Pla2g2a-Tg/C57BL/6J-Muc2–/–, we identified a set of 99 Pla2g2a
target genes that were unique to the Muc2–/– background. These
Pla2g2a-Muc2 interactor genes include Nfkbil1, Reg2, Limk1,
Hoxb9, Ctrb1 and Ela2a. A complete list of these genes is pro-
vided in Suppl. Table S3. A subset of the most significant genes
with a focus on genes involved in intestinal energy metabolism
and inflammation were confirmed by quantitative reverse tran-
scriptase–polymerase chain reaction (qRT-PCR) (Table 3).
Muc2 plays a key role in protecting the intestinal mucosa and
maintaining intestinal homeostasis. In the mouse, the targeted
inactivation of Muc2 caused dysregulation of homeostasis as
demonstrated by increased intestinal cell proliferation, decreased
apoptosis, loss of goblet-like cells in the large intestine and
increased migration of intestinal epithelial cells, eventually
resulting in tumors that progressed to invasive adenocarcinomas.
Importantly, these tumors arose via an Apc/β-catenin-independent
pathway.(19) Like Muc2, in the large intestine the phospholipase
Pla2g2a is exclusively expressed by the goblet cell population
and its secreted proteins accumulate in the mucinous gel in the
crypt lumen. Both these goblet cell specific proteins affect tumor
formation in the large intestine as previous studies showed that
expression of Pla2g2a significantly reduced tumor multiplicity
in the large intestine in the ApcMin/+ mouse model. These data
suggest that Pla2g2a and Muc2 may function in common biological
pathways, raising the expectation that Pla2g2a would not be able
to (strongly) suppress tumorigenesis in Muc2-deficient mice. As
the natural Pla2g2a gene is mutant in the two strains of mice
(C57BL/6 J and 129/Sv) that have been previously tested for
Muc2 deficiency, we examined whether expression of functional
Pla2g2a as a transgene in C57BL/6 J mice provided resistance
to Muc2-deficient intestinal tumorigenesis. We here report that
Pla2g2a significantly reduced tumor multiplicity, incidence and
possibly tumor progression in the large intestine of Muc2–/–
mice. Moreover, these results are the first evidence that Pla2g2a
can act to suppress tumors independent of the Apc/Wnt signaling
pathway. Therefore our data suggest that Pla2g2a protects
against tumorigenesis via modulation of biochemical pathways
that are common in both the Apc and Muc2 mouse models. The
ability of Pla2g2a to rescue the Muc2 mutant phenotype was
somewhat surprising given that goblet cell morphology is absent
in Muc2–/– mice. Immunohistochemical analysis (Fig. 2) shows
expression of Pla2g2a in the columnar epithelial cells and lumen
of colonic crypts that contain no goblet-like cells, reinforcing the
concept that in Muc2–/– mice the goblet cell lineage is not abrogated.
A second finding of our study is that the phenotype of Muc2
deficiency on the C57BL/6 J genetic background differs from
the previous report using mice on a mixed (C57BL/6 J×129SvOla)
background. C57BL/6J-Muc2–/– mice developed more tumors in
both the large intestine and duodenum than mice on the mixed
background and the tumors in the large intestine appeared much
earlier in C57BL/6J-Muc2–/– mice. The region of the large intestine
affected also varied by strain as C57BL/6J-Muc2–/– mice devel-
oped tumors in a cluster in the middle of the large intestine
whereas tumors in the C57BL/6 J × 129/SvOla-Muc2–/– mice
were generally restricted to the distal rectum. Another strain
difference is that C57BL/6J-Muc2–/– mice did not develop aden-
ocarcinomas in the duodenum (0/18), unlike the numerous invasive
tumors found in Muc2–/– mice on the mixed background. Finally,
C57BL/6J-Muc2–/– mice developed sessile adenomas and adeno-
carcinomas in the ileum that were not observed in the C57BL/
6J ×129/SvOla-Muc2–/– mice. It is likely then, that the C57BL/
6J and 129/SvOla strains carry modifier genes that modulate the
severity of the Muc2 deficiency.
Another result that may be linked to genetic background is
that sex affected the Muc2 phenotype. Overall, male mice devel-
oped more tumors in both the duodenum and large intestine than
female mice. This difference was also seen in mice carrying the
Pla2g2a transgene where the rare tumors that were observed
were only found in male mice as only one female Pla2g2a
transgenic mouse developed a tumor in the colon and none
developed tumors in the duodenum. Interestingly, we did observe
more ileal tumors in female mice of both genotypes although the
difference was not statistically significant. Sex differences in the
phenotype of the ApcMin/+ mouse model have been shown in
several studies. For example, it was recently reported that male
ApcMin/+ mice developed more colon tumors than female mice
but fewer tumors in the small intestine, especially in the ileum.(31)
Consistent with this finding there is growing evidence that estrogen
is a protective factor in the mouse large intestine, as ovariect-
omized ApcMin/+ mice develop more tumors than littermate
controls.(32) Our new data indicates that female protection against
tumorigenesis in the large intestine extends beyond the Apc/β-
catenin pathway. Finally, we also note that tumor multiplicity at
60 weeks of age was generally equal to or larger than at 30 weeks
of age, conforming to expectations of an increased tumor load
with age (Table 1). However, colon tumor multiplicity in female
Muc2 knockout mice formed an exception, as tumor number
appeared to be higher in mice at 30 weeks of age than at
60 weeks of age. Because tumor multiplicity is not expected to
decrease over time, we assume this observation has occurred ‘by
chance’ owing to random (stochastic) sampling of relatively small
groups of mice, and may disappear when the number of mice
Table 3.qRT-PCR analysis of genes significantly changed in microarray studies
9.8 × 10–10
4.1 × 10–10
4.8 × 10–6
1.6 × 10–6
2.8 × 10–8
1.6 × 10–8
4.9 × 10–6
6.4 × 10–6
1 × 10–4
5.7 × 10–6
qRT-PCR, quantitative reverse transcriptase–polymerase chain reaction.
© 2008 Japanese Cancer Association
within this group would be increased. Moreover, there is no
published evidence of estrogen fluctuations in mice between 30
and 60 weeks (and estrogen levels begin to decline at ~ 16 months
in female mice) thus differences in estrogen levels are not likely
to underlie the difference in phenotypes in these mice.
The evidence that Pla2g2a prevents tumors arising from both
Muc2 and Apc deficiency suggests that Pla2g2a affects very
fundamental processes in tumorigenesis, at least to some extent
independently from biochemical pathways affected by Muc2 or
Apc. One possibility is that Pla2g2a acts outside the cell to protect
the intestinal mucosa from environmental insult that can lead to
inflammation and tumor promotion, for instance, in the absence
of Muc2. Secreted Pla2g2a molecules form a part of the muci-
nous extracellular gel in the colonic crypt lumen where they
may exert potent bactericidal activity.(33) This could modulate
biochemical signaling pathways. To find out what genes are
affected by Pla2g2a in Muc2-deficient mice, we performed micro-
array gene expression analyses. Our snapshot of gene expression
in the distal colon at 100 days of age revealed that loss of Muc2
and Pla2g2a (C57BL/6J-Muc2–/–/C57BL/6J-Muc2+/+-Pla2g2a-Tg)
resulted in the up-regulation of a number of pro-inflammatory
genes (Il1rl1, Il1b, s100a11, Capg, s100a16), and up-regulation
of a number of genes associated with oncogenicity in human
and rodent cancers (Ptgs2, Mmp10, Has1, Afp, Fgfr4, Birc5,
Itga3, Nrg1, Reg2, Trim29, Baiap2). When we then compared
C57BL/6J-Muc2–/–-Pla2g2a-Tg mice with C57BL/6J-Muc2–/–
littermate mice (C57BL/6J-Muc2–/–-Pla2g2a-Tg/C57BL/6J-Muc2–/–,
a gain of Pla2g2a in the Muc2–/– background) we found the reversal
of some gene expression changes observed in the C57BL/6J-
Muc2–/–/C57BL/6J-Muc2+/+-Pla2g2a-Tg comparison. Egfr, Ctrb1,
Ela2a, Reg2, Ctrl, Dner, Cel, Cpa2,Yap1, Rnase1, Klf16, Limk1,
Ssbp2, Aldh2, Akt1s1, Sst and Fbxl19 are genes that were either
up- or down-regulated by loss of Muc2 and Pla2g2a but which
were then significantly changed in the opposite direction upon
the gain of expression of Pla2g2a in the Muc2 knockout mice.
Most of these reversal genes are Pla2g2a–Muc2 interactor genes
that are specific to Muc2 deficiency, i.e., they are genes that are
not differentially expressed in a C57BL/6J–Muc2+/+–Pla2g2a–
Tg/C57BL/6J–Muc2+/+ comparison (unpublished data). Notably,
the proto-oncogene c-Myc, a gene that is significantly up-regu-
lated by Muc2 deficiency,(19) is not altered by expression of Pla2g2a.
When Pla2g2a target genes in Muc2 knockout mice were
grouped into functional categories we found altered expression
of genes involved in intestinal lipid and energy metabolism, cell
signaling and transactivation and inflammation and immune
responses (see Suppl. Table S2). Our microarray data indicate
that cancers arise in Muc2–/– mice following early dysregulation
of many pro-inflammatory genes and genes involved in intestinal
energy metabolism. Pla2g2a and its target genes may prevent
the development of these cancers by modulating inflammation
and energy metabolism in the intestinal mucosa, via an effect in
either the epithelial or stromal compartments (or both). To fur-
ther investigate these specific pathways we employed qRT-PCR
to test Pla2g2a target genes involved in inflammatory and energy
processes that were identified in the array studies (Table 3). One
gene is Defb11 (Defensin B11, up-regulated 4-fold by qRT-PCR
in Pla2g2a + Muc2 knockout mice), a member of the defensin
family, an important component of the innate immune system.
Defensins have potent antimicrobial properties and have been
described as the first line of defense in the human colon mucosa,
and polymorphisms in defensin genes are linked to susceptibility
to Crohn disease.(34–39) Another Pla2g2a target gene is Reg2 (down-
regulated in the arrays and down-regulated 10-fold in qRT-PCR).
C57BL/6 mice (Pla2g2a negative) fed a high fat diet become
obese, develop type-2 diabetes and overexpress Reg2 in pancreatic
cells. Reg2 is pro-inflammatory and is significantly up-regulated
in rodent models of experimental colitis.(40,41) A third gene ana-
lyzed by qRT-PCR is Cel (carboxyl ester lipase, down-regulated
in the arrays and down-regulated by 2.9 fold in qRT-PCR), a
secreted lipase found in the gastrointestinal tract that is involved
in fat digestion and absorption, and insulin and cholesterol
metabolism, and Cel knockout mice are resistant to obesity in
mice caused by high fat diets.(42–44) Other Pla2g2a target intestinal
enzymes confirmed by qRT-PCR were Clps (pancreatic colipase,
down-regulated 4-fold), a type-2 diabetes susceptibility gene;(45)
Ela2a (a GI tract elastase down-regulated 5.9-fold)(46) and Ctrb1
(chymotrypsinogen B1, down-regulated 24 fold).(47) Although
the role of each of these Pla2g2a target genes in susceptibility to
intestinal neoplasia is unclear, considered together these highly
significant target genes demonstrate that Pla2g2a exerts a strong
influence on genes involved in intestinal energy metabolism and
inflammation that may work in unison to maintain intestinal
homeostasis. However, the exact role of Pla2g2a and its down-
stream effectors, such as PGE2, in inflammation and cancer in
the gastrointestinal tract remains controversial. For example, it
has been shown that pharmacological and genetic blockage of
COX-2 and/or prostaglandin receptors inhibits tumorigenesis in
Apc mutant mice. Yet, there is also substantial evidence that PGE2
can prevent inflammatory bowel disease in mouse models,(48,49)
suggesting that PGE2, acting downstream of Pla2g2a activity,
may also prevent gastrointestinal cancer by resolving chronic
In summary, we now know that Pla2g2a can significantly pre-
vent tumors independent of the Apc pathway and that its tumor
prevention functions may involve interactions with Muc2, possibly
in the extracellular environment, as well as Muc2-independent
mechanisms. More has also been learned about Muc2 because
genetic background and sex modulate the severity of its defi-
ciency phenotype. Finally, we have discovered gene expression
profiles associated with Muc2 deficiency, and Pla2g2a target
genes that likely underlie Pla2g2a’s tumor resistance. Ongoing
experiments are exploring the contributions of specific Pla2g2a
target genes to tumor susceptibility.
We wish to thank Dr Rita Mulherkar, Tata Institute, Mumbai, India for
generously providing the Pla2g2a antiserum and Dr Wan Cai Yang,
pathologist at the Albert Einstein College of Medicine, for additional
histopathological analysis of tumors. We also acknowledge M. Tijssen
and P. Eijk for their excellent technical assistance. Funding for this work
was provided by the University of Minnesota Academic Health Center
and the University of Minnesota Medical School (R.T.C.) and the1st
AEGON International Scholarship in Oncology (R.J.A.F.).
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Additional Supporting Information may be found in the online version of this article:
Select list of genes differentially expressed in C57BL/6J-Muc2–/– mice compared with C57BL/6J-Pla2g2a-Tg mice.
Genes differentially expressed in C57BL/6J-Muc2–/– Pla2g2a-Tg mice compared with C57BL/6J-Muc2–/– mice.
Differentially expressed genes in C57BL/6J-Muc2–/–-Pla2g2a-Tg/C57BL/6J-Muc2–/– colon that are unique from genes differentially
expressed in C57BL/6J-Pla2g2a-Tg/C57BL/6J comparison.
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