Hindawi Publishing Corporation
Volume 2010, Article ID 571783, 12 pages
Inductionof Metastatic Gastric Cancerby
PeroxisomeProliferator-Activated Receptorδ Activation
ClaireB. Pollock,1Olga Rodriguez,1Philip L.Martin,2ChrisAlbanese,1Xin Li,3
LevyKopelovich,4and Robert I.Glazer1
1Department of Oncology, Lombardi Comprehensive Cancer Center, Washington, DC 20057, USA
2Center for Advanced Preclinical Research, SAIC/NCI-Frederick, Frederick, MD 21702, USA
3Department of Biostatistics, Bioinformatics, and Biomathematics, Lombardi Comprehensive Cancer Center, Washington,
DC 20057, USA
4Chemoprevention Agent Development and Research Group, Division of Cancer Prevention, National Cancer Institute,
Bethesda, MD 20814, USA
Correspondence should be addressed to Robert I. Glazer, firstname.lastname@example.org
Received 19 August 2010; Accepted 16 November 2010
Academic Editor: John P. Vanden Heuvel
Copyright © 2010 Claire B. Pollock et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Peroxisome proliferator-activated receptorδ (PPARδ) regulates a multiplicity of physiological processes associated with glucose
and lipid metabolism, inflammation, and proliferation. One or more of these processes likely create risk factors associated with the
ability of PPARδ agonists to promote tumorigenesis in some organs. In the present study, we describe a new gastric tumor mouse
model that is dependent on the potent and highly selective PPARδ agonist GW501516 following carcinogen administration. The
progression of gastric tumorigenesis was rapid as determined by magnetic resonance imaging and resulted in highly metastatic
squamous cell carcinomas of the forestomach within two months. Tumorigenesis was associated with gene expression signatures
indicative of cell adhesion, invasion, inflammation, and metabolism. Increased PPARδ expression in tumors correlated with
increased PDK1, Akt, β-catenin, and S100A9 expression. The rapid development of metastatic gastric tumors in this model will be
useful for evaluating preventive and therapeutic interventions in this disease.
Gastric cancer is the second leading cause of cancer-related
metastatic nature at the time of diagnosis [1, 2]. Among the
many risk factors for gastric cancer are diet, smoking, and
inflammation associated with H. pylori infection [3–5] that
are likely exacerbated in patients with proinflammatory gene
PPARs are ligand-dependent nuclear receptors that
regulate expression of a multiplicity of genes associated
with metabolic disorders, such as type II diabetes and
lipodystrophies [7, 8]. PPARs consist of the α, γ and δ
isotypes that regulate not only glucose and lipid metabolism,
but also proliferation, inflammation, and angiogenesis [9–
13]. PPARδ expression is increased in breast, colon, and
head and neck cancers [9, 14–17] and is associated with a
more aggressive phenotype in breast cancer cells . The
in mammary carcinogenesis  and colon tumorigenesis
[15, 20, 21], whereas disruption of PPARδ expression blocks
mammary  and colon tumorigenesis [23, 24]. PPARδ is
regulated by risk factors implicated in cancer progression,
including K-Ras , Wnt , and Pges/Cox2 [22, 27],
and is associated with activation of angiogenesis [20, 28–30].
PPARδ regulates proinflammatory signaling through NFκB
acid metabolism that serve as PPAR ligands [14, 29, 32].
Notwithstanding these results, there are several reports
showing that azoxymethane-induced colon carcinogenesis is
increased in PPARδ nullizygous mice [33, 34] and by the
selective PPARδ ligand GW0742  (reviewed in ).
account for some of these disparities have been discussed
tive agonist, GW501516, rapidly induces highly metastatic
gastric tumors following carcinogen administration, which
expressed a markedly increased inflammatory gene expres-
sion signature. These findings suggest a crucial role for
PPARδ in the progression of this disease.
2.1. Materials. GW501516 was synthesized as previously
described  and was provided by the Chemoprevention
Branch, National Cancer Institute, NIH, Bethesda, MD.
2.2. Carcinogenesis. DMBA (Sigma-Aldrich, St Louis, MO)
was dissolved in cottonseed oil at a concentration of
10mg/ml, and six week-old female FVB mice were admin-
istered 4 weekly doses of 1mg DMBA by gavage. Animals
were fed a standard diet or a diet supplemented with 0.005%
(w/w) GW501516 beginning one day after the last dose of
DMBA as previously described . Mice were euthanized
when gastric tumors reached ≥2cm3as determined by MRI
imaging or if mice became moribund. All protocols were
approved by the Georgetown University Animal Care and
2.3. Magnetic Resonance Imaging. MRI was performed on
a 7.0 Tesla Bruker horizontal spectrometer/imager with
a 20cm bore equipped with 100gauss/cm microimaging
gradients and run by Paravision 4.0 software in the Preclini-
cal Imaging Research Laboratory, Lombardi Comprehensive
Cancer Center. Micewereanesthetized using 1.5% isoflurane
and 30% nitrous oxide, positioned in a custom-made
stereotaxic animal holder with temperature and respiration
control, and imaged in a 72mm birdcage radiofrequency
volume coil as previously described . The imaging
protocol used was a T2-weighted RARE (rapid acquisition
with refocused echos) two-dimensional sequence with the
following parameters: Matrix: 256, TR: 5822msec, TE:
36msec, number of averages: 4, RARE factor: 8, FOV:
4.0cm, number of slices: 4, slice thickness: 0.5mm, and with
respiratory gating to account for breathing movement.
ach and tumors were excised, and formalin-fixed, paraffin-
embedded sections were prepared for H&E staining and
IHC. Antigen retrieval was carried out by incubation of
tissue sections in 10mM sodium citrate buffer (pH 6.0) for
20min at a subboiling temperature in an electric steamer
as previously described [19, 39]. Endogenous peroxidase
and incubated for 30min with blocking solution (10%
goat serum in Tris-buffered saline), followed by incubation
overnight at 4◦C with the appropriate primary antibody
diluted in blocking solution. Biotin-conjugated secondary
antibodies were diluted in TBS containing 0.1% Tween-20
and incubated for 30min at room temperature using the
ABC Vectastain (Vector Laboratories) detection system and
diaminobenzidine (Pierce), and slides were counterstained
with Harris-modified hematoxylin (Fisher Scientific) and
dehydrated and mounted in Permount (Fisher Scientific).
The following antibodies and their dilutions were used: anti-
CK14 (1:200, ms-115-P1, Neomarkers), anti-CK18 (1:200,
sc-7197, Santa Cruz Biotechnology), PDK1 (1:50, sc-17765,
Santa Cruz Biotechnology), anti-pS473 Akt (1:200, 40515,
Cell Signaling Biotechnology), anti-β-catenin (1:50, sc-
7963, Santa-Cruz Biotechnology) and anti-S100a9 (1:50, sc-
65580, Santa Cruz Biotechnology).
2.5. Gene Microarray Analysis. Three groups of tissues were
analyzed: (1) gastric tumors, (2) forestomach (nonglan-
dular) from GW501516-treated mice, and (3) forestomach
from DMBA-treated mice. Tissue was excised, washed in
at −20◦C until RNA extraction. Tissue was snap-frozen in
liquid nitrogen, pulverized in a mortar and pestle, and RNA
extracted using an RNeasy Mini Kit (Qiagen) according to
the manufacturer’s protocol. RNA purity was assessed by
an A260/A280 ratio of ≥1.9, and by the integrity of 18S
and 28S rRNA using an Agilent microfluidic chip. Array
analysis was carried out on cRNA prepared from equal
amounts of RNA (1μg) pooled from 5 mice per group
as previously described [19, 40]. Biotin-labeled cRNA was
fragmented at 94◦C for 35min and hybridized overnight
to an Affymetrix mouse 430A 2.0 GeneChip representing
approximately 14,000 annotated mouse genes. GeneChips
were scanned with an Agilent Gene Array scanner, and grid
alignment and raw data generation with the Affymetrix
GeneChip Operating software 1.1. A noise value (Q) based
on the variance of low-intensity probe cells was used to
calculate a minimum threshold for each GeneChip. Samples
were averaged and data refined by eliminating genes with
signal intensities <300 in both comparison groups, and
heat maps were generated from ≥3-fold changes in gene
pervised hierarchical cluster analysis as previously described
PCR). Total RNA was extracted as described above, and
equal amounts of RNA (1μg) were pooled from each group
(five samples per group) and reverse transcribed with the
Omniscript RT kit (Qiagen) in a total volume of 20μL
as previously described [19, 39]. PCR was performed in
triplicate using an ABI-Prism 7700 (Applied Biosystems,
Foster City, CA) with SYBRGreen I detection (Qiagen)
according to the manufacturer’s protocol. Amplification
material available at doi:10.1155/2010/571783) was con-
firmed by ethidium bromide staining of the PCR products
on an agarose gel. The expression of each target gene was
normalized to GAPDH and is presented as the ratio of
Figure 1: PPARδ agonist GW501516 induces gastric tumorigenesis after DMBA treatment. (a) In vivo axial T2-weighted abdominal MR
Images showing gastric tumor progression at the indicated time points; d, number of days animals were administered the GW501516-
supplemented diet. Tumor growth initiated in the forestomach (arrow) at 19 days, rapid tumor growth between 27 and 50 days, and tumor
invasion through the stomach (arrow) at 56 days. (b) Gross morphology of tumors at indicated time points. Forestomach (∗), gastric tumors
(T) and metastases (arrow) were evident at 56 days, and intraperitoneal metastases along the abdominal wall (white arrows) occurred at 70
(a) (b) (c) (d)(e)
Figure 2: Pathophysiology of gastric tumorigenesis. (A) Histological changes in the forestomach after GW501516 and/or DMBA treatment.
treated mice, and (e) metastases of the abdominal wall. Lower panel: higher magnification (200x) of the boxes in areas in the upper panel.
Insets,magnification 400x. (a) Morphology of the normalforestomach wall. (b) Squamous epithelial hyperplasia, where the basal membrane
is well defined. (c) Squamous cell carcinoma. (d) Invasive squamous cell carcinoma showing disruption of the basement membrane. (e)
Metastatic squamous carcinoma showing invasion into the abdominal wall. (B) Histological changes in the stomach and esophagus after
treatment with either GW501516 or DMBA. H&E sections of the forestomach wall (a, b, c) and esophagus (d, e, f) six months after
administration of GW501516 or five months after treatment with DMBA. Both GW501516 and DMBA were associated with increased
keratinization (∗) of squamous epithelium (arrow). Magnification 400x. (C) Cytokeratin 14 (CK14) and cytokeratin18 (CK18) expression
in gastric tissue and tumors. Squamous epithelium of the forestomach (a), gastric mucosa (b), and gastric tumors (c, d). Tumors are
CK14+/CK18−indicating a squamous epithelial origin. Magnification 200x, insets, 400x.
4.97.2 9.411.6 14
7.6 8.9 10.314.8
mRNA fold change
mRNA fold change
mRNA fold change
Figure 3: Differential gene expression in GW501516-treated stomach and gastric tumors. Gene microarray analysis was carried out
with pooled RNA samples prepared from either five tissue samples of forestomach from untreated and GW501516 treated mice or six
gastric tumors. Heat maps represent unsupervised hierarchical clustering of ≥3-fold changes in signal intensity normalized to untreated
forestomach. (a) Heatmap of GW501516-treated versus stomach representing 42 genes. (b) Tumor versus stomach representing 811 genes.
(c) qRT-PCR analysis of the relative changes in gene expression in the stomach after GW501516 treatment (GW501516), and in tumors
(Tumor). GW501516 treated: array, cross-hatch; qRT-PCR, diagonal; Tumors: array, black; qRT-PCR, white.
6 PPAR Research
Table 1: Gastric tumor occurrence after treatment with DMBA and
No. animals with tumors
the target gene to GADPH expression calculated using the
formula, 2−ΔCt, where ΔCt = CtTarget−Ct18s.
3.1. PPARδ Agonist GW501516 Rapidly Promotes Gastric
Tumorigenesis. Mice maintained on a diet supplemented
with PPARδ agonist GW501516 following carcinogen
administration resulted in the rapid development of gastric
tumors in 12/15 animals, whereas treatment with either
GW501516 or DMBA alone was not tumorigenic (Table 1).
To follow the onset and progression of tumorigenesis more
precisely, five mice were monitored by MRI (Figure 1(a)).
Tumors were visible as early as 19 days after beginning the
GW501516 diet and appeared to initiate in the forestomach
(Figure 1(a)). By 50 days, tumor had filled the stomach
lumen, and by 56 days it had extravasated through the
gastric wall (Figure 1(a)). Gross inspection of the stomach
confirmed that the tumor was confined within the stomach
at day 20 but had invaded through the stomach wall forming
local metastases by day 56 (Figure 1(b)). Mice showed
signs of morbidity between days 63 and 70 (mean survival
67 days), where metastases were present throughout the
mesentery and adjacent serosal organ surfaces including the
abdominal wall (Figure 1(b)).
Primary tumors and metastases were uniformly squa-
mous cell carcinomas with varying degrees of keratinization
(Figure 2(A)). Animals fed the GW501516 diet for six
months without prior DMBA treatment did not exhibit
hyperplasia or dysplasia (Figure 2(B)), and DMBA treat-
ment alone produced squamous cell hyperplasia of the
forestomach without signs of dysplasia (Figures 2(A) and
2(B)). No changes occurred in the gastric mucosa resulting
from DMBA and GW501516 treatment alone (data not
shown), and esophageal squamous epithelium was unaf-
fected by DMBA treatment (Figure 2(B)). Gastric tumors
were positive for the squamous basal cell marker CK14, and
negative for the columnar epithelial cell marker CK18 
3.2. Differential Gene Expression in Gastric Tumors and Stom-
ach. Tumors manifested a marked inflammatory phenotype
denoted by increased expression of chemokines Cxcl-1,-2,
-5,-9,-14 and Ccl-2,-3,-8, S100a8, S100a9, and S100a3 and
interleukins IL-1β, IL-6, and IL-24 (Table 2, Figure 3(b), and
Table S2). In addition to these changes, increased expression
of prostaglandin metabolism genes Ptgs2/Cox2 and Ptges
and reduced expression of PPARγ and PPARα were noted.
To determine if these changes were tumor specific, gene
expression was assessed in stomach tissue after treatment
with either GW501516 for seven days (Figure 3(a), Table S3)
or DMBA for four weeks (Table S4). GW501516 increased
expression of only five genes ≥3-fold, Angptl4, Cyp2b10,
Cfd/Adipsin, Adipoq and Chi3l4 and markedly reduced
expression of Gast, Ccla3, Glycam1, Spp1, Serpina1a, Cela1,
Cldn2, and Fabp2 (Table S3). DMBA increased expression of
S100a8, S100a9, and Ccl8 4–10-fold and reduced expression
of the same subset of genes as GW501516 (Table S4). Thus,
changes in Gast, Ccla3, Glycam1, Spp1, Serpina1a, Cela1,
Cldn2, and Fabp2 are a result of GW501516 treatment and
are not tumor specific. On the other hand, DMBA produced
and S100A9 although it was an order of magnitude less than
in tumors. The increase in Krt6a by DMBA is consistent
with increased keratinization in the stomach (Figure 2(B))
analysis generally confirmed the changes in expression of
several genes, including Cldn8, Cxcl1, Cxcl5, Foxg1, S100a8,
Angptl4, Cyp2b10, Vegfα and Spp1, Gast, Dkk1, Bmp3,
Bmp4, PPARα, and PPARγ (Figure 3(c)).
ated with its signaling were assessed in tumors and forestom-
ach after GW501516 treatment (Figure 4). GW501516
increased nuclear localization of PPARδ in gastric squamous
epithelium and tumors, in contrast to its diffuse cytoplasmic
staining in untreated gastric tissue. GW501516 also elicited
strong pS473Akt and pT308Akt staining in basal cells and
in the submucosal layer, as well as in tumor and stromal
tissue, which correlated with more intense PDK1 expression.
β-Catenin was expressed in the nuclei of basal squamous
epithelial cells and was not altered by GW501516 treatment,
whereas tumors expressed increased β-catenin at cellular
junctions. S100a9 was absent in untreated gastric epithelium
but was expressed in endothelial and epithelial cells from
GW501516-treated mice. Tumors expressed S100a9 in a
diffuse pattern, with strong expression in blood vessels and
adjacent epithelial cells.
The present study describes a new model of metastatic
gastric cancer that is dependent on the tumor promoting
activity of PPARδ agonist GW510516 following carcinogen
administration. In contrast to a previous study reporting
a low percentage of squamous cell carcinomas of the
forestomach by DMBA , our DMBA regimen produced
only forestomach hyperplasia without signs of dysplasia up
to five months after treatment (Figure 2(B)). This suggests a
high sensitivity of mouse forestomach squamous epithelium
to dysplasia, and the predilection of GW501516 to promote
tumors of this histotype . This model differs from
N-methyl-N-nitrosourea-induced gastric tumors in wild-
type and APCMintransgenic mice [44, 45] in that it is
dependent on both DMBA-induced mutagenesis and the
tumor-promoting effects of GW501516. A feature of this
model is its short latency of approximately three weeks
in comparison to 10 to 20 weeks for NMU-treated wild-
type and APCMinmice. An important histopathological
distinction, and perhaps disadvantage of the GW501516
pT308 Akt, PDK1, β-catenin, and S100a9. Magnification: untreated and GW501516 treated, 400x; tumor, PPARδ, PDK1, and β-catenin,
epithelium, increased nuclear localization after GW501516 treatment, and strong nuclear expression in tumor and stromal cells (inset).
pS473Akt and pT308Akt expressed weak diffuse reactivity in untreated gastric tissue, increased staining in basal cells and the submucosal
cell layer after GW501516 treatment, and strong reactivity in tumor and stromal cells. PDK1 exhibited diffuse cytoplasmic localization
throughout the untreated squamous epithelium and was unchanged after GW501516 treatment, whereas PDK1 was increased in tumors
similarly to pS473Akt and pT308Akt. Nuclear β-catenin was present in basal cells of untreated squamous epithelium and was unchanged
tissue and was increased in blood vessels and squamous epithelium (inset), whereas tumors exhibited increased diffuse cytoplasmic staining.
Table 2: Differentially expressed genes in gastric tumors.
Gene NameFold Change
S100 calcium-binding protein A8 (calgranulin A)
S100 calcium-binding protein A9 (calgranulin B)
chemokine (C-X-C motif) ligand 2
chemokine (C-X-C motif) ligand 2
chemokine (C-X-C motif) ligand 5
chemokine (C-X-C motif) ligand 1
chemokine (C-X-C motif) ligand 9
chemokine (C-C motif) ligand 2
chemokine (C-C motif) ligand 3
chemokine (C-C motif) ligand 8
interleukin 1 beta
matrix metallopeptidase 10
matrix metallopeptidase 12
matrix metallopeptidase 13
matrix metallopeptidase 3
matrix metallopeptidase 9
peroxisome proliferator activated receptor alpha
peroxisome proliferator activated receptor gamma
prostaglandin E synthase
prostaglandin-endoperoxide synthase 2
tumor model, is that it produces squamous cell carcinomas
from the nonglandular forestomach rather than adeno-
carcinomas from the glandular tissue that comprises the
majority of human gastric cancer . Since this model was
dependent on the selective PPARδ agonist GW501516 ,
it is important to note that the dose of GW501516 used
in the present and previous studies  is equivalent to
daily oral doses of 3–10mg/kg that were previously shown to
specifically enhance PPARδ-dependent fatty acid oxidation
in mice . In addition, PPARδ agonist GW7042, which
is almost identical to GW501516 in structure, potency,
and specificity, was inactive in inducing gene expression
in PPARδ knockout mice , suggesting that the tumor
promoting effects of GW501516 and GW7042 are not due
to off-target effects.
Tumors induced by GW501516 exhibited a distinct
inflammatory gene expression signature comprised predom-
inantly of chemokine, MMP, and S100 genes (Table S2). This
was unexpected in view of the lack of a similar signature
after treatment with GW501516 (Table S3), and the fact
that GW501516 induces an anti-inflammatory response in
macrophages  and protects the heart against oxidative
stress [51, 52]. Gene ontology analysis of gene expression in
the gastric tumors indicates that PPARδ, MMP12, MMP13,
Cxcl1, Cxcl5, S100A8, and S100A9 share both common and
disparate pathway interactions that likely contributed to the
tumorigenic phenotype (Figure 5). PPARδ is associated with
activation of genes related to proliferation (EGFR, Akt1) and
adhesion (Itgb2), whereas S100A9 is associated with angio-
genesis (Fgf2) and inflammation (Ager). Cxcl1 activates
proliferation (Mapk3, Mapk14, and Akt1), angiogenesis
(Fgf2), and invasion (MMP2, MMP9), and Cxcl5 activates
This scheme reiterates the ability of S100A8 and S100A9
to act as ligands for Ager (advanced glycation end-product
receptor), which mediates acute and chronic inflammation,
tumor development, and metastasis [53, 54]. This paradigm
is also consistent with GW501516-induced activation of
Ptges and Ptgs2/Cox-2 expression [55, 56], which initiate
the production of prostacyclins  and arachidonic acid
2 inhibitors reduce inflammation-related gastrointestinal
carcinogenesis , and overexpression or deletion of Ptgs2
increases or suppresses tumorigenesis, respectively [59, 60];
this suggests cooperativity between PPARδ and inflamma-
tory signaling pathways in gastric tumorigenesis. The ability
of PPARδ to have an anti-inflammatory effect in normal
cells [51, 52] and a proinflammatory effect in tumors is
S100A8 and S100A9 expression in the presence of activated
Ras  but acts as a repressor of inflammation-induced
Figure 5: Signaling networks associated with PPARδ and inflammatory and invasive gene expression. Common signaling pathways were
analyzed for six genes with the greatest changes in expression in gastric tumors (highlighted in blue) versus forestomach using Ariadne
Pathway Studio 7.1.
PPARδ expression in normal cells [64, 65]. The increased
expression of the PPARδ target gene, Agptl4, the TGFβ-
activated genes Runx1 and Runx2, and S100A8 and S100A9
in the gastric tumors indeed suggests a duality of function of
both PPARδ and TGFβ signaling in gastric tumorigenesis.
and gastric tumors , and GW501516 elicited increased
PPARδ nuclear staining and elevated pAkt in gastric epithe-
lium and tumors. PPARδ-dependant activation of Akt is
required for the growth-promoting and antiapoptotic effects
of PPARδ [66–68], as shown by the delayed wound-
healing response of PPARδ-deficient keratinocytes [57, 69].
Enhanced Krt6a and Krt16 expression in tumors further
suggests that PPARδ plays an important role in gastric
squamous cell differentiation and tissue renewal.
Tumors also exhibited reduced PPARγ and PPARα
expression that may have resulted, in part, from the negative
regulation of PPARγ by PPARδ . PPARγ suppresses the
growth and invasion of human colon  and gastric [72,
73] and esophageal carcinoma cells , and both PPARγ
 and PPARα have anti-inflammatory actions . Thus,
reduction of PPARα and PPARγ expression may be an
additional mechanism for facilitating the proinflammatory
and tumor-promoting effects of GW501516.
In summary, we describe a rapidly developing metastatic
gastric cancer model dependent on the tumor-promoting
effects of GW501516 following carcinogen treatment, which
suggests a proinflammatory switch in PPARδ function. This
animal model will therefore be useful to delineate the role of
PPARδ in tumor initiation and progression and as a possible
target for early intervention.
This work was supported by Grant no. R01 CA111482 and
Contract no. N01 CN43309 from the National Institutes
of Health, Bethesda, MD. This investigation was conducted
using the Animal Research, Histopathology and Tissue,
Genomics and Epigenomics, and Preclinical Imaging Shared
Resources supported by Research Facilities Improvement
Grant no. C06 RR14567 from the National Center for
Research Facilities, and by Cancer Center Support Grant
no. 1P30-CA-51008 from the National Cancer Institute. The
authors thank Yi Chien Lee for his help with the MRI.
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