Published OnlineFirst May 18, 2011; DOI: 10.1158/1940-6207.

CAPR-11-0023

Cancer
Prevention
Research Article Research

Rapamycin Partially Mimics the Anticancer Effects of Calorie

Restriction in a Murine Model of Pancreatic Cancer
Laura M. Lashinger1,2, Lauren M. Malone2, Graham W. Brown2, Elizabeth A. Daniels2, Jason A. Goldberg2,
Glen Otto3, Susan M. Fischer1, and Stephen D. Hursting1,2

Abstract
Etiologic factors for pancreatic cancer, the 4th deadliest malignant neoplasm in the United States,
include obesity and abnormal glucose metabolism. Calorie restriction (CR) and rapamycin each affect
energy metabolism and cell survival pathways via inhibition of mammalian target of rapamycin (mTOR)
signaling. By using a Panc02 murine pancreatic cancer cell transplant model in 45 male C57BL/6 mice,
we tested the hypothesis that rapamycin mimics the effects of CR on pancreatic tumor growth. A
chronic regimen of CR, relative to an ad libitum-fed control diet, produced global metabolic effects such
as reduced body weight (20.6
1.6 g vs. 29.3
2.3 g; P < 0.0001), improved glucose responsiveness,
and decreased circulating levels of insulin-like growth factor (IGF)-1 (126
8 ng/mL vs. 199
11 ng/mL;
P 0.0006) and leptin (1.14
0.2 ng/mL vs. 5.05
1.2 ng/mL; P 0.01). In contrast, rapamycin
treatment (2.5 mg/kg intraperitoneal every other day, initiated in mice following 20 weeks of ad libitum
control diet consumption), relative to control diet, produced no significant change in body weight, IGF-1
or leptin levels, but decreased glucose responsiveness. Pancreatic tumor volume was significantly reduced
in the CR group (221
107 mm3; P < 0.001) and, to a lesser extent, the rapamycin group (374
206 mm3;
P 0.04) relative to controls (550
147 mm3), and this differential inhibition correlated with expression
of the proliferation marker Ki-67. Both CR and rapamycin decreased phosphorylation of mTOR, p70/S6K,
and S6 ribosomal protein, but only CR decreased phosphorylation of Akt, GSK-3b, extracellular signal
regulated kinase/mitogen-activated protein kinase, and STAT3TYR705. These findings suggest that rapamy-
cin partially mimics the anticancer effects of CR on tumor growth in a murine model of pancreatic cancer.
Cancer Prev Res; 4(7); 104151. 2011 AACR.

Introduction creatic cancer prevention and control. Calorie restriction

Effective prevention and treatment strategies are urgently
needed for pancreatic cancer, the 4th leading cause of
cancer-related death in both men and women in the United
States (1). Only 10% to 15% of pancreatic cancer patients
have localized disease amenable to curative resection, and
the overall 5-year survival rate in affected patients is less
than 5% (1, 2). Interrelated etiologic factors in pancreatic
cancer include obesity, abnormal glucose metabolism, and
positive energy balance (defined as caloric intake exceeding
energy expenditure; refs. 35). Components of energy
metabolism pathways thus may be useful targets for pan-
Authors' Affiliations: 1Department of Molecular Carcinogenesis, Univer-
sity of Texas M.D. Anderson Cancer Center, Smithville; 2Departments of
Nutritional Sciences and 3Molecular Genetics and Microbiology, Univer-
sity of Texas at Austin, Austin, Texas
Corresponding Author: Stephen D. Hursting, Department of Nutritional
Sciences, Dell Pediatric Research Institute, University of Texas at Austin,
1400 Barbara Jordan Blvd., Mail Code R1800, Austin, TX 78723. Phone:
512-475-3020; Fax: 512-475-4945. E-mail: shursting@mail.utexas.edu
doi: 10.1158/1940-6207.CAPR-11-0023
2011 American Association for Cancer Research.
(CR) and the drug rapamycin are dietary and pharmaco-
logic interventions, respectively, that act on pathways
related to energy metabolism and inhibit various tumor
types (6, 7). To our knowledge, the effects of these inter-
ventions in pancreatic cancer have not been previously
compared.
CR, typically involving a 20% to 40% reduction in total
energy intake but isonutrient for vitamins, minerals, fatty
acids, and amino acids relative to an ad libitum-fed control
regimen, prevents or reverses obesity, improves insulin
sensitivity, and inhibits the development and/or progres-
sion of many types of cancer (7). In animal models, the
anticancer effects of CR are strongly associated with reduc-
tions in circulating levels of the nutrient-responsive mito-
gen insulinlike growth factor (IGF)-1 (8, 9). For example,
CR suppresses the development and pathological severity
of murine COX-2driven pancreatitis and pancreatic can-
cer through a reduction of circulating IGF-1 levels (10).
Epidemiologic evidence shows that (i) pancreatic cancer
patients, relative to healthy controls, have higher levels of
serum IGF-1 and pancreatic expression of the IGF-1 recep-
tor (IGF-1R), and (ii) pancreatic cancer patients with ele-
vated circulating IGF-1, relative to patients with normal

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 Published OnlineFirst May 18, 2011; DOI: 10.1158/1940-6207.CAPR-11-0023
Lashinger et al.
levels, have a worse prognosis (11, 12). IGF-1 exerts its
effects on cellular growth and metabolism, at least partially
through activation of the phosphatidylinositol 3-kinase
(PI3K)/Akt/mammalian target of rapamycin (mTOR) path-
way (13, 14).
Despite the substantial evidence highlighting the bene-
ficial effects of CR, maintaining this lifestyle in humans is
extremely challenging (7). This has prompted an intense
search for CR mimetics, which are pharmacologic agents
that possess anticancer effects similar to CR without
restricting energy intake (7). One putative CR mimetic is
rapamycin (7). Unlike CR, rapamycin and its analogs affect
neither body weight nor energy balance (15). However,
rapamycin and CR each suppress signaling through mTOR
and extend lifespan in both invertebrates and mammalian
species (7, 1618). The lifespan extension in mammals is
primarily due to reduced tumor development (17). mTOR
acts as a sensor that integrates growth factor signals, nutri-
ent availability, and energy status with translational control
of new proteins (19). Pharmacologic inhibition of mTOR
by rapamycin halts DNA synthesis and proliferation of
pancreatic cancer cells in vitro through regulation of ribo-
somal S6 kinase and other downstream targets (20, 21).
Rapamycin treatment in various xenograft models of pan-
creatic cancer in immunodeficient mice inhibits tumor
burden by impeding cell proliferation and angiogenesis
while promoting apoptosis (22, 23). In addition, recent
studies using murine prostatic, lung, and mammary cancer
models support a role for rapamycin and its analogs in
cancer chemoprevention (2427).
In the present study, we tested the hypothesis that
rapamycin mimics the effects of CR on pancreatic tumor
growth using a Panc02 pancreatic tumor cell transplant
model. We found that (i) chronic CR decreases pancreatic
tumor growth in association with a reduction in prosurvi-
val signaling; (ii) rapamycin also reduces tumor growth
through targeted inhibition of the mTOR pathway; and (iii)
rapamycin-induced mTOR inhibition, although effective,
does not completely mimic the effects of CR on tumor
growth inhibition, metabolism, or cell signaling in a pan-
creatic cancer cell transplant model.
Materials and Methods
Mice
The Institutional Animal Care and Use Committee of the
University of Texas, Austin, TX, approved all mouse experi-
ments. Male C57BL/6 mice were received from Jackson
Laboratories (Bar Harbor, ME) between 4 to 6 weeks of age,
singly housed in a semibarrier facility at the Animal
Resource Center, University of Texas at Austin, and fed a
chow diet for a 2-week acclimation period before study
initiation.
Interventions
Mice were randomized to receive 1 of 2 diets for 30
weeks: (i) control diet (Research Diets, Inc.; #D12450B)
consumed ad libitum, n 27; or (ii) CR diet (Research
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for Cancer Research.
Diets; #D03020702) consumed in daily aliquots to provide
70% of the energy and 100% of all nutrients (except
carbohydrates) relative to the control group, n 18. Food
intake and body weights were recorded weekly until
Panc02 tumors became palpable (27 weeks of study).
At 16 weeks of study, 5 mice from each diet group were
fasted for 12 hours then anesthetized by CO2 inhalation.
They then underwent cardiac puncture for blood collection
and subsequently were killed by cervical dislocation. After
coagulating at room temperature (RT) for 30 minutes,
blood samples were centrifuged at 9,300 g for 5 minutes.
Serum was separated, then snap-frozen and stored at
80 C until assayed for hormones. Tissues were collected
and flash-frozen for subsequent molecular and biochem-
ical studies.
At 20 weeks of study, the remaining mice in the control
group (n 22) were randomized to receive (every other
day) an intraperitoneal (i.p.) injection of vehicle (0.1%
dimethyl sulfoxide in 0.9% saline; n 11) or 2.5 mg/kg
rapamycin (n 11). The remaining mice in the CR group
(n 13) also began receiving vehicle via i.p. injection every
other day. This dose was chosen based on reports in the
literature showing effective tumor inhibition with rapamy-
cin between 1 to 10 mg/kg daily or every other day, with the
lower doses showing comparable antitumor effects without
the toxicity of the higher doses (15, 28). For our study, a
moderate dose was selected because of the extended treat-
ment period.
Tumor cell injection and tumor monitoring
Mouse pancreatic cancer cells (Panc02, generously pro-
vided by Dr. K. Hance and Dr. J. Schlom, NCI; ref. 29) were
cultured under an atmosphere of 5% CO2 in a 37 C
incubator with McCoys media (HyClone) supplemented
with 10% fetal bovine serum (HyClone), penicillin/strep-
tomycin, glutamine, nonessential amino acids, sodium
pyruvate, and HEPES. Cells were trypsinized, washed in
Hanks Buffered Saline Solution (HBSS), centrifuged, and
resuspended in HBSS for injection. Karyotyping and species
identification of Panc02 cells were verified by the Mole-
cular Cytogenetics Core Facility at The U.T. M.D. Anderson
Cancer Center (Houston, TX).
At 22 weeks of study, mice were s.c. injected into the right
flank with 1  106 Panc02 cells. One mouse from the
control and CR groups each and 2 mice from the control
plus rapamycin (CONRapa) group died before tumor
development within 2 weeks of Panc02 injection, and were
thus censored from subsequent analyses. Once palpable,
tumors were measured with calipers weekly until approxi-
mately half of tumors from any group were 1.5 cm in
diameter. Tumor volume was approximated using the
formula for an ellipsoid (4/3pr12r2). At study termination,
all remaining mice were fasted for 12 hours then anesthe-
tized by CO2 inhalation. They then underwent cardiac
puncture for blood collection and subsequently were killed
by cervical dislocation. Blood samples were processed as
detailed earlier. Pancreatic tumors were harvested and
either snap-frozen in liquid nitrogen and stored at
Cancer Prevention Research
 Published OnlineFirst May 18, 2011; DOI: 10.1158/1940-6207.CAPR-11-0023
80 C, or fixed with 10% neutral buffered formalin over-
night and then switched to 70% ethanol, paraffin
embedded, and used for histologic and immunohisto-
chemical analyses as described before.
Glucose tolerance test
A glucose tolerance test (GTT) was done, as previously
described (30), on randomly selected mice from the con-
trol and CR groups at 14 weeks of study (n 12 per group).
At 22 weeks of study, following 2 weeks of rapamycin or
vehicle treatment and before Panc02 cell injection, a sec-
ond GTT was done on a subset of 10 mice from each diet/
drug group.
Energy balancerelated serum hormones
Serum IGF-1 was measured using radioimmunoassay
according to manufacturers instructions (DSL-2900 Kit,
DSL/Beckman Coulter Laboratories, Webster, TX). Serum
insulin and leptin were measured using Lincoplex bead
based assays (Millipore Corporation) on a BioRad Bioplex
analyzer (BioRad) according to manufacturers directions.
Analyses were conducted on samples obtained at 16 weeks,
before Panc02 cell injection (to assess diet-induced changes
in the absence of potentially confounding tumor effects on
circulating hormone levels; n 5 per group) and at study
termination (to exclude the possibility that rapamycin
affected circulating levels within the control group; control,
n 6; rapamycin-treated control, n 3).
Histopathology and immunohistochemistry
Formalin-fixed pancreatic tumors were embedded in
paraffin and then cut into 4-mm-thick sections and pro-
cessed for either hematoxylin and eosin (H&E) or immu-
nohistochemical staining at the Histology Core
Laboratory at The U.T. M.D. Anderson Cancer Center,
Science Park Research Division (Smithville, TX). Antibo-
dies used for immunohistochemistry were optimized by
core personnel using both positive and negative controls
that were repeated with each analysis. Slides were depar-
affinized and hydrated sequentially in ethanol to water.
Antigen retrieval required microwaving slides for 10
minutes with 10 mmol/L citrate buffer. Endogenous
peroxidase activity was quenched with 3% hydrogen
peroxide for 10 minutes. Nonspecific binding was
blocked with Biocare blocking reagent (Biocare Medical)
for 30 minutes at RT, then sections were incubated with
primary antibody diluted in blocking buffer. The follow-
ing primary antibodies (source, dilution, and incubation
conditions presented parenthetically) were used: Ki-67
(Dako; 1:200, 4 C overnight); phospho (p)-IGF-1RTyr1131
and IGF-1R (Cell Signaling; 1:50, 4 C overnight);
p-AktSer473 (Santa Cruz Biotechnology; 1:50, 1 hour
RT); Akt (Cell Signaling; 1:100; 4 C overnight) p-GSK-
3bSer9 (Santa Cruz Biotechnology; 1:50, 1 hour RT); GSK-
3b (Millipore; 1:200; 1 hour RT); p-extracellular signal
regulated kinase (ERK)Thr202/Tyr204 (Cell Signaling; 1:100;
1 hour RT); ERK (Cell Signaling; 1:25; 1 hour RT);
p-STAT3Tyr705 (Cell Signaling; 1:50; 4 C overnight);
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Rapamycin and Calorie Restriction Inhibit Pancreatic Cancer
p-STAT3Ser727 (Cell Signaling; 1:50, 4 C overnight);
STAT3 (Cell Signaling; 1:100; 1 hour RT); p-mTORSer2448
(Cell Signaling; 1:50, 4 C overnight); mTOR (Cell Signal-
ing; 1:100; 1 hour RT); p-S6 ribosomal protein(Ser235/236)
(Cell Signaling; 1:50, 1 hour RT); S6 (Cell Signaling;
1:100; 4 C overnight); and cyclin D1 (Santa Cruz Bio-
technology; 1:500, 2 hours RT). Slides were washed twice
in PBS, incubated with horseradish peroxidase (HRP)
labeled a-rabbit secondary antibody (Dako) for 30 min-
utes at RT at a concentration of 1:200 with the following
exceptions: (a) Ki-67 [rabbit anti-rat immunoglobulin
(Ig) G; 1:200, 30 minutes RT; Vector]; (b) p-STAT3 and
p-mTOR (rabbit HRP-polymer; 30 minutes RT; Biocare);
and (c) cyclin D1 (rabbit anti-mouse F(ab)0 ; 1:250, 15
minutes RT; Accurate Chemical). Slides were then washed
5 times with PBS. Diaminobenzidine was used to develop
the antibody staining followed with a hematoxylin coun-
terstain. Images were captured using a light microscope
equipped with a digital camera (Leica Camera, Inc.).
Immunohistochemically stained tumor sections were
assigned a score based on the following criteria: 1 mild
(majority of tumor section exhibited weakly positive
staining pattern or less than 50% of cells in field stained
an equivalent intensity to control tumors), 2 moderate
(majority of tumor section exhibited moderate-to-dark
brown stain or 50% to 75% of cells in field stained an
equivalent intensity to control tumors), or 3 marked
(majority of tumor section exhibited dark brown stain or
greater than 75% of cells in field stained an equivalent
intensity to control tumors). The quality of staining
determined whether the intensity of stain or percentage
of stained cells in field was quantified. For example, the
antibodies against p-IGF1-R and p-ERK produced weak
but consistent signal intensity; therefore, the percentage
of stained cells in field was assessed. Sample size was
determined based on the quality of tissue fixation/stain-
ing. More tumors were used to analyze phosphorylated
IGF-1R, Akt, GSK-3b, ERK, STAT3, mTOR, and S6 ribo-
somal protein antibodies, and total cyclin D1 antibody
(for each antibody: control, n 4; rapamycin-treated
control, n 7; CR, n 5) than Ki-67 (n 3/group)
because the latter yielded a more easily quantifiable
nuclear staining pattern. Images were captured at 40
magnification (with the exception of cyclin D1 which was
captured at 20) in 3 to 4 fields in a tumor section from
each tumor analyzed.
Western blotting
Pancreatic tumors were homogenized and lysed in radio-
immunoprecipitation assay buffer (Sigma) with protease
inhibitor tablet (Roche Applied Sciences) and phosphatase
inhibitor cocktails I and II (Sigma). Protein lysates (5080
mg) were resolved by SDS-PAGE using 6%, 10%, or 12%
gels, transferred to polyvinylidene difluoride membranes
(Bio-Rad), and blocked using LI-COR Blocking Buffer
(LI-COR Biotechnologies). Membranes were incubated
overnight at 4 C with primary antibody (from Cell Signal-
ing unless otherwise stated) diluted in blocking buffer and
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 Published OnlineFirst May 18, 2011; DOI: 10.1158/1940-6207.CAPR-11-0023
Lashinger et al.
specific for: p-AktSer473 (1:500); Akt (1:1,000); actin
(1:1,000); p-GSK-3bSer9 (Santa Cruz, 1:1,000); GSK-3b
(Millipore, 1:1,000); p-ERKThr202/Tyr204 (1:1,000); ERK
(1:1,000); p-mTORSer2448 (1:500); mTOR (1:1,000);
p-p70/S6KThr389 (1:500); p70/S6K (1:1,000); and myosin
IIa (1:1,000). Actin was used as a loading control for all
antibodies except mTOR (for which myosin IIa was used
because the 6% polyacrylamide gel used to resolve mTOR
did not retain actin). After 3 washes (5 minutes each) in
0.1% Tween-20/PBS (PBS-T), membranes were incubated
for 1 hour at RT in species-specific secondary antibody (LI-
COR Biotechnologies) diluted in LI-COR blocking buffer
(1:5,000). Following 3 washes in PBS-T, membranes were
scanned using the Odyssey infrared fluorescent imaging
system. Densitometry was performed using LI-COR Soft-
ware (LI-COR Biotechnologies). Relative expression of
phosphorylated proteins was calculated by using 3 tumors
per group.
Statistical analyses
Data are presented as mean
SD, except serum hormone
levels and Ki-67 which are presented as mean
SEM.
Statistical analyses were conducted using SPSS (Apache
Software Foundation). Temporal differences between
groups with respect to body weight and caloric intake were
assessed using repeated measures analysis; final measure-
ments were compared using independent t tests. Pretumor
serum hormone levels between the control and CR groups
were compared using independent t tests. Pairwise com-
parisons of glucose tolerance and tumor burden as a
function of time and treatment group were performed
using a linear mixed effects model. Final tumor volume
measurements were compared using independent t tests.
Differences between groups in immunohistochemical
staining of (i) Ki-67 were determined by 1-way ANOVA
followed by Tukeys post hoc test of significance and (ii) all
other antibodies were determined by Fisher exact test.
Differences in Western blot densitometry between each
test group (rapamycin-treated controls and CR), relative
to the control group, were compared by independent t tests.
Results were considered significant if P < 0.05.
Results
Effects of CR on body weight, glucose tolerance, and
serum hormone levels
Male C57BL/6 mice were fed either a control diet known
to result in an overweight phenotype or a 30% CR diet (7)
for 30 weeks (including 22 weeks of diet before Panc02 cell
injection). Relative to controls, the CR mice had signifi-
cantly reduced body weights beginning as early as 3 weeks
on study (P < 0.01; Fig. 1A and B). Diet-dependent effects
on body weight continued throughout the study and at 27
weeks, mean body weights were 20.6
1.6 g in CR mice
(n 12; P < 0.0001) and 29.3
2.3 g in controls (n 10).
The CR group, relative to control, displayed enhanced
glucose tolerance as assessed by GTT. At 14 weeks of study
(Fig. 1C), blood glucose concentrations following glucose
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bolus injection in CR mice peaked at 15 minutes and
averaged 332
78 mg/dL, whereas the control group
peaked at 30 minutes and averaged 510
73 mg/dL (n
12 per group). After the peaks were achieved, blood
glucose concentrations remained consistently lower in the
CR mice (P < 0.0001 for between-group comparison at the
120-minute measurement). Comparable diet-dependent
effects on glucose tolerance were noted at 22 weeks of
study, at which time the CR and control mice had been
receiving, for 2 weeks, vehicle by i.p. injection every other
day (Fig. 1E; n 10 each).
In a subset of mice fed for 16 weeks and then bled (n 5
per group), the CR mice had significantly lower serum
levels of IGF-1 (126
8 ng/mL; P 0.0006) and leptin
(1.14
0.17 ng/mL; P 0.01) than control mice (199

11 ng/mL and 5.05
1.18 ng/mL, respectively; Fig. 1D).
No between-group difference in serum insulin levels was
detected (P 0.15).
Effects of rapamycin on body weight, glucose
tolerance, and serum hormones
Beginning at 20 weeks of study, the control mice
received their assigned diet plus i.p. injections every other
day of either 2.5 mg/kg rapamycin [for CONRapa
group, n 9] or vehicle (CON, n 10). The CR group
(n 12) also received vehicle. Moderate reduction of
caloric intake in the control group was noted between
weeks 22 and 24 relative to the CONRapa group
(Fig. 1A). However, this decrease did not approach sta-
tistical significance, nor did it significantly affect body
weight (Fig. 1B). This modest differential between the 2
groups subsided by week 25. Between weeks 20 to 27 of
study, body weights remained stable within each diet/
drug group and at 27 weeks were comparable between the
control mice (29.3
2.3 g) and rapamycin-treated mice
(32.8
2.1 g; Fig. 1A). As previously mentioned, the CR
mice were leaner than the others.
The average peak glucose levels in the rapamycin-treated
mice (530
130 mg/dL) occurred 60 minutes after the
glucose bolus was administered, whereas control mice
receiving vehicle peaked at 30 minutes and only reached,
on average, 441
80 mg/dL (Fig. 1E). Moreover, blood
glucose levels at the final time point (120 minutes after
glucose bolus) were significantly higher in rapamycin-treated
mice (462
169 mg/dL; P 0.02) relative to the control plus
vehicle group (309
87 mg/dL). Consistent with the GTT
results at 14 weeks, CR mice, as compared with the others,
exhibited the lowest levels of glucose at all points including
the final assessment (151
24 mg/dL; P < 0.0001).
To exclude the possibility that rapamycin impacts circu-
lating energy balancerelated hormones relative to the con-
trol plus vehicle group, serum collected at study termination
was assessed for levels of IGF-1, insulin, and leptin (Fig 1F).
No statistical differences (all P > 0.15) were detected
between rapamycin-treated mice (n 3) and controls
(n 6), respectively, in circulating levels of IGF-1 (176

14 vs. 147
21 ng/mL), insulin (1.9
0.3 vs. 1.2

0.3 ng/mL), or leptin (2.0
0.6 vs. 1.9
0.2 ng/mL).
Cancer Prevention Research
 Published OnlineFirst May 18, 2011; DOI: 10.1158/1940-6207.CAPR-11-0023

Rapamycin and Calorie Restriction Inhibit Pancreatic Cancer

GTT #2/
Tumor
injection
A Rapa Tumor B
GTT #1 initiated palpable

100 40

Average body weight (g)

Average caloric intake

80 30
(kcal)

60
* 20 *
40
CON
CON + Rapa 10 CON
20 CON + Rapa
CR
CR
0 0
0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28
Weeks on diet Weeks on diet
C D Serum hormone levels prior to tumor injection (16 wk)
14 wk GTT
Blood glucose (mg/dL)

600 CON 250 0.5 7

Leptin levels (ng/mL)

Insulin levels (ng/mL)
IGF-1 levels (ng/mL)

500 CR 6
200 0.4
400 5
150 0.3
300 * 4
3
200 100 0.2

100 * 50 0.1
2
1 *
0 0 0.0 0
0 15 30 60 120 CON CR CON CR CON CR
Time after glucose injection
(min)
E F Serum hormone levels in control groups at study termination
22 Week GTT (2 wk after rapa initiated)
Blood glucose (mg/dL)

600 200 2.5

Insulin levels (ng/mL)

Leptin levels (ng/mL)

IGF-1 levels (ng/mL)

500 b
CON 150 2.0
400 CON + Rapa 2
a 1.5
300 CR 100
1.0
200 1
c 50
0.5
100
0 0 0.0 0
0 15 30 60 120 CON CON + Rapa CON CON + Rapa CON CON + Rapa

Time after glucose injection

(min)

Figure 1. Effects of CR versus rapamycin on food intake, body weight, glucose tolerance, and serum hormone levels. A and B, C57BL/6 male mice fed
a CR diet (n 12) for 27 weeks consumed fewer calories and weighed significantly less than mice fed a control diet (CON; n 10). Within the mice
receiving control diet, a 10-week rapamycin treatment (CONRapa; n 9), initiated at 20 weeks of study, had no significant effect on caloric intake or body
weight relative to the control mice administered vehicle (n 10). C, GTT performed on a subset of mice fed either a control or CR diet for 14 weeks
(n 12/group). D, pretumor serum levels of IGF-1, insulin, and leptin in a subset of mice fed either a control or CR diet for 16 weeks (n 5/group). E, GTT
performed at 22 weeks of study (immediately prior to Panc02 cell injection) on mice fed CR or control diet and administered vehicle or rapamycin beginning
at week 20 of the study (n 10/group). F, posttumor serum levels (at study termination) of IGF-1, insulin, and leptin in mice receiving the control diet vehicle
(CON, n 6) versus the control diet rapamycin (CONRapa; n 3). A to C and E, error bars represent SD. D and E, error bars represent SEM. A to D,
significant differences (P < 0.05) are denoted with *. E, different letters denote significant differences. F, no values were significantly different.

Effects of CR and rapamycin on Panc02 tumor growth slower than in control mice receiving vehicle. CR exerted a
and histology more dramatic inhibitory effect than rapamycin as evi-
To compare the anticancer effects of CR and rapamycin denced by a significant difference between their tumor
interventions, we injected mice from each diet/drug group growth (P 0.005; Fig. 2A). Final tumor volumes from
with Panc02 cells at week 22 and monitored tumor growth rapamycin-treated mice (374
206 mm3; P 0.04) and
during the next 8 weeks. Tumors in rapamycin-treated mice CR mice (221
107 mm3; P < 0.0001) were significantly
(P 0.009) and CR mice (P < 0.0001) grew significantly smaller, on average, than controls (550
147 mm3). The

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Lashinger et al.

A B

Tumor volume (mm3)

700 CON
600 CON + Rapa a Tumor incidence
500 CR
Week 5 Week 6 Week 7 Week 8
400 b
CON 30% 89% 100% 100%
300 CON + Rapa 22% 33% 88% 100%
c
200 CR 0% 17% 66% 100%
100
0
5 6 7 8
Weeks after injection
C CON CON + Rapa CR
D

Ki-67-positive cells/field
300
a
Ki67
200
b

100 c

0
CON CON + Rapa CR
H&E

Figure 2. Effect of CR versus rapamycin treatment on tumor growth and histology. A, weekly tumor measurements were taken using calipers beginning
when the first tumor became palapable (5 weeks) after Panc02 cells were s.c. injected into C57BL/6 mice receiving the control diet plus vehicle (CON, n 10),
control diet plus rapamycin (CON Rapa, n 9), or CR diet plus vehicle (n 12). Error bars represent SD. B, percentage of mice in each diet/drug
group with palpable tumor at weeks 5, 6, 7, and 8 after Panc02 cell injection. C, immunohistochemical staining for Ki-67 expression and H&E staining
of tumors. Scale bars for Ki-67, 100 mm and H&E, 400 mm. Arrows indicate adipocytes. D, quantification of Ki-67positive cells from representative
images captured at 40 magnification. Data are expressed as mean
SEM number of Ki-67positive cells/field in tumors (n 3). Values with different
letters are significantly different at P < 0.05.

CR and rapamycin interventions each delayed tumor onset,
with only 17% and 33% of mice, respectively, having
palpable tumors 6 weeks after Panc02 cell injection com-
pared to 89% of the control group (Fig. 2B). Only at study
termination, did all mice in the CR and control diet plus
rapamycin groups have detectable tumors.
The influence of CR or rapamycin treatment on cell
proliferation was assessed in tumor tissues by immunohis-
tochemical staining against Ki-67 (Fig. 2C and D). CR
(74.8
9.9 positive cells/field; n 3; P 0.001) and
rapamycin treatment (141.9
11.0 positive cells/field;
n 3; P 0.013) each significantly reduced cell prolifera-
tion (based on Ki-67 immunopositivity) relative to con-
trols (231.7
21.3 positive cells/field; n 3). CR
produced a more substantial reduction in proliferation
(P 0.04) relative to rapamycin treatment.
The effect of CR and rapamycin intervention on tumor
histology was qualitatively assessed using H&E staining
(Fig. 2C). There was a considerable presence of adipocytes
in the control tumors that were not seen in the CR tumors.
Unlike CR, rapamycin treatment did not lessen the level of
adipocyte infiltration associated with the control diet.
Effects of CR and rapamycin on signaling
intermediates
Based on immunohistochemical analysis of signaling
intermediates, CR tumors relative to control tumors dis-
played reduced expression of phosphorylated Akt (P
0.03), ERK (P 0.05), STAT3Tyr705 (P 0.02), STAT3Ser727
(P 0.01), mTOR (P 0.02), S6 ribosomal protein (P
0.004), GSK-3b (P 0.06), IGF-1R (P 0.11), and
cyclin D1 (P 0.11; Fig. 3A and B). Rapamycin produced
a more selective signaling profile than CR in which only
p-STAT3Ser727 (P 0.02) and mTOR pathway components
were inhibited relative to control, including p-mTOR (P
0.02), p-S6 ribosomal protein (P 0.006; Fig. 3A and B),
but not p-IGF-1R (P 1), p-Akt (P 0.3), p-GSK-3b (P
1), p-ERK (P 0.54), or p-STAT3TYR705 (P 0.24). No
appreciable changes to total protein expression were noted
(data not shown).
Immunoblotting of various signaling intermediates con-
firmed the dampening of other survival signals in CR
tumors (Fig. 4A). CR (n 3) significantly reduced phos-
phorylation of Akt (P 0.006), GSK-3b (P 0.04), ERK
(P 0.004), mTOR (P 0.05), and p70/S6K (P 0.05)
relative to the control diet which, despite an occasionally
variable expression pattern, had higher levels of phos-
phorylated proteins when averaged over 3 tumors
(Fig. 4B). Rapamycin potently and consistently reduced
phosphorylation of mTOR (P 0.005) and p70/S6K (P
0.02) when compared with control tumors, and decreased
phosphorylation of p70/S6K more robustly than did CR
(P 0.008; Fig. 4B). Despite the potent inhibitory effects of
rapamycin on mTOR signaling intermediates, levels of
phosphorylated Akt, GSK-3b, and ERK were similar to
control tumors (Fig. 4A). Neither CR nor rapamycin had
a significant effect on total protein expression of Akt,
mTOR, p70/S6K, GSK-3b, or ERK (Fig. 4A).

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Rapamycin and Calorie Restriction Inhibit Pancreatic Cancer

A CON CON + Rapa CR

B

p-IGF1R

pIGF1R pAkt
a a a a a b
p-Akt 100 pIGF1R 100 pAkt staining

percentage

percentage
75 staining score: 75 score:

Score
3 3

Score
50 2 50 2
25 1 25 1
0 0
CON CON + Rapa CR CON CON + Rapa CR
p-GSK3 pGSK3 pERK
a a a P = 0.06 100 a a b
100 pERK staining
pGSK3 staining

percentage
percentage
75 score:
75 score:
3

Score
Score
3 50
50
2 2
25 1 25 1
0 0
p-ERK CON CON + Rapa CR CON CON + Rapa CR

pSTATTYR705 pSTATSER727
a a b 100 a b b
100 pSTATTYR705 pSTATSER727

percentage
percentage

75 staining score: 75 staining score:

Score
Score

3 3
50 50 2
2
p-STAT3Tyr705 25 1 25 1
0 0
CON CON + Rapa CR CON CON + Rapa CR

pmTOR pS6
100
a b b a b b
pmTOR 100 pS6 staining
percentage

staining score: score:

percentage
75 75
Score

p-STAT3Ser727 3 3

Score
50 2 50 2
25 1 1
25
0 0
CON CON + Rapa CR
CON CON + Rapa CR

Cyclin D1
100
a a a
p-mTOR Cyclin D1
percentage

75 staining score:
Score

3
50 2
25 1
0
CON CON + Rapa CR

p-S6

Cyclin D1

Figure 3. Effect of CR and rapamycin on signaling pathways in pancreatic tumors by immunohistochemical analysis. A, immunohistochemical analyses
of phosphorylated IGF1R, Akt, GSK-3b, ERK, STAT3, mTOR, and S6 ribosomal protein as well as total protein expression of cyclin D1. Scale bars,
100 mm except cyclin D1, 200 mm. B, quanitification of antibody staining patterns. Tumors were assigned a score that represented the majority of the
section: 1 (mild white), 2 (moderate gray), or 3 (marked black). The bar graph represents the percentage of tumors in that group associated with the
corresponding score assignment (CON, n 4; CONRapa, n 7; CR, n 5). Values with different letters are significantly different at P < 0.05.

Discussion anticancer effects of a chronic CR dietary regimen versus an

CR is effective at inhibiting cancer growth in many
model systems (7). Although we found a strong anticancer
effect of CR in a COX-2driven transgenic model of pan-
creatic neoplasia (10), we sought to establish and under-
stand the growth prohibitive effects of CR on transplanted
pancreatic tumors. In addition, we wanted to compare the
acute pharmacological intervention, rapamycin, which
share several downstream signaling targets but have not
been directly compared in a pancreatic cancer model. We
found in C57BL/6 mice that CR (relative to a control diet
fed ad libitum that results in overweight mice) reduced
Panc02 tumor burden. Furthermore, we established that
exposure to rapamycin (at a dose of 2.5 mg/kg i.p. every

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Lashinger et al.

A B
0.5

Relative phosphorylation
CON CON + Rapa CR CON
CON + Rapa
CR
0.4
p-Akt
0.3
Akt
0.2
*
Actin 0.1
* * Figure 4. Effect of CR and
0.0 rapamycin on signaling pathways
p-GSK3 pAkt pGSK3 pERK
in pancreatic tumors by
a immunoblot analysis. A,
GSK3 5 immunoblotting analyses of
b

phosphorylation
Relative mTOR
4 phosphorylated and total protein
b
Actin expression of Akt, GSK-3b, ERK,
3 mTOR, and p70/S6K. Data shown
2 are representative blots from at
p-Erk least 3 tumors for each treatment.
1 B, relative phosphorylation of
Erk p-Akt, p-GSK-3b, p-ERK, p-
0
CON CON + Rapa CR mTOR, and p-p70/S6K proteins,
Actin quantified by densitometry using
LI-COR Odyssey software. Data
0.4 a represent mean of 3 tumors/
p-mTOR
group; error bars represent SD.
Relative p70/S6K
phosphorylation

0.3 Significance denoted by * or

mTOR
different letters (P < 0.05).
Myosin 0.2 c
b
0.1
p-p70/S6k
0.0
p70/S6K CON CON + Rapa CR

Actin

other day, which effectively inhibited mTOR without toxi-
city) also significantly suppressed Panc02 tumor growth,
although to a lesser extent than CR. Central to both inter-
ventions is an ability to dampen signaling through the
mTOR pathway, a highly conserved protein at the crux of
intracellular energy sensing and external growth factor
signaling. However, the unique ability of CR to alter multi-
ple signaling pathways involved in proliferation and sur-
vival was more influential on pancreatic tumor growth
than the selective targeting of mTOR with rapamycin.
Our finding that chronic CR (30 weeks), relative to
control diet, inhibited Panc02 tumor growth in association
with improved insulin sensitivity and reduced circulating
IGF-1 and leptin, is consistent with established links
between altered metabolism and pancreatic cancer. Speci-
fically, there is a 2-fold higher risk for developing pancrea-
tic cancer in the context of altered glucose metabolism,
such as that occurring in diabetes (4, 31). Although fasting
levels of insulin were moderately but not statistically
decreased in the CR group (P 0.08), the functional
sensitivity of CR mice to insulin was dramatically improved
relative to the control group, as indicated by GTT. The
antidiabetic biguanide metformin improves insulin resis-
tance, reduces pancreatic cancer risk in type II diabetics by
62% (32), and also prevents development of carcinogen-
induced pancreatic lesions enhanced by a high-fat diet in
hamsters (33), suggesting improved glucose metabolism
and/or mTOR inhibition may contribute to decreased
pancreatic cancer development.
In addition to improved responsiveness to insulin in the
CR group, there was a significant decrease in serum levels of
IGF-1. This is important given the connection between
pancreatic cancer risk and serum levels of IGF-1 in human
subjects (11, 12), and the implication that reduced IGF-1
underlies many of the anticancer effects of CR (8, 10). The
protective effects of reduced IGF-1 signaling in a skin carci-
nogenesis model have been associated with an abrogation
in mTOR signaling (9). The current study recapitulates this
connection between CR, reduced circulating IGF-1, and
subsequently diminished Akt/mTOR signaling. It also
shows that CR dampened other components of prosurvival
signaling such as GSK-3b, ERK, and STAT3. Although this is
not a comprehensive assessment of proliferation and survi-
val signaling pathways, these findings suggest that the
ability to suppress multiple pathways underlies the potent
and broad-acting anticancer effects of CR.

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 Published OnlineFirst May 18, 2011; DOI: 10.1158/1940-6207.CAPR-11-0023
Our studies also aimed to establish and understand
the pancreatic cancer therapeutic potential of rapamy-
cin. Recently, several reports have offered evidence that
mTOR inhibitors might have cancer preventive activity,
at least in part by mimicking some effects of CR (2427).
For example, low-dose rapamycin treatment of HER-2/
neu mice significantly diminished tumor formation,
with one third of mice having no detectable tumors at
sacrifice (26). In a mouse prostate model expressing
human AKT1, the rapamycin analog, RAD001, reversed
prostatic intraepithelial neoplasia by enhancing apop-
tosis and abrogating expression of HIF-1a genes (25).
Another rapamycin analog, CCI-779, abrogates the pro-
gression of lung adenomas that are induced by activating
K-RAS mutations (2527, 34). Sensitivity to mTOR
inhibition is enhanced in tumors that rely on PI3K/
Akt signaling such as those with a deficiency in the
phosphatase and tensin homolog tumor suppressor
(3537). Excessive activation of the PI3K/AKT pathway
may be largely responsible for the detrimental effects of
obesity (38). Thus, it seems plausible that rapamycin
treatment could overcome the growth-promoting effects
of excessive energy intake.
Our findings show that treatment with rapamycin cir-
cumvents some of the protumorigenic effects of the calorie-
dense control diet. This effect is seen in our rapamycin-
treated mice despite insulin resistance, elevated serum
levels of IGF-1 and leptin, and other metabolic parameters
typically associated with enhanced tumor growth. Rapa-
mycin treatment actually resulted in a worsened sensitivity
to a glucose bolus than the control group, despite having
no effect on caloric intake and body weights. The alteration
in glucose homeostasis in response to rapamycin is con-
sistent with findings in the Psammomys obesus diabetic rat
model in which insulin resistance is enhanced by rapamy-
cin because of increased b cell apoptosis, decreased b cell
mass, and reduced glucose-induced insulin biosynthesis
and secretion from islet cells (39). Moreover, chronic
rapamycin treatment stimulates hepatic gluconeogenesis
(40), which could further disrupt glucose homeostasis.
These metabolic outcomes of mTOR inhibition potentially
enhance glucose bioavailability to cancer cells, possibly
offsetting some of the growth-inhibitory effects of rapamy-
cin. Despite these metabolic alterations, including high
levels of circulating mitogens, mTOR signaling was sub-
stantially inhibited by rapamycin (even to a greater extent
than by CR), resulting in pancreatic tumor growth suppres-
sion. This further supports the mTOR pathway as an
important target for pancreatic cancer prevention and
control.
In addition to disparate responses to insulin and glu-
cose between the CR and rapamycin interventions, sig-
naling patterns were also divergent. Although both
interventions diminished mTOR signaling, rapamycin
did not inhibit other survival signals such as ERK, GSK-
3b, and STAT3TYR705. Akt phosphorylation in the tumors
from the rapamycin-treated group was generally higher
than in tumors from the control group. This is consistent
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for Cancer Research.
Rapamycin and Calorie Restriction Inhibit Pancreatic Cancer
with a known diminution of an S6K-mediated feedback
inhibition that can result in Akt activation in response to
mTOR inhibitors (4144). However, rapamycin reduced
phosphorylation of an mTOR-sensitive residue (serine
727) on p-STAT3 that is necessary for full transcriptional
activation of STAT3 (45). It is plausible that the transcrip-
tional activity of STAT3 (which we did not assess in our
system) is dampened by rapamycin when compared with
control tumors, but not CR tumors, especially consider-
ing CR resulted in reduced phosphorylation at both the
TYR705 and SER727 sites.
A histological difference between tumors from CR and
rapamycin-treated mice was also noted. Adipocytes were
consistently present in the tumors from mice receiving
the control diet, regardless of whether they received
rapamycin or vehicle. In contrast, CR clearly reduced
adipocyte infiltration into tumors. Zhang and colleagues
(46) showed that stromal and endothelial cells from
adipose tissue migrate to tumors and promote tumor
growth, and endogenous white adipose tissue
(enhanced in an overweight or obese state) was the
source of the adipose progenitors infiltrating the tumors
(46). Furthermore, studies show that coadministration
of tumor cells with mouse adipose cells promote tumor
take and growth (47, 48). Further contributing to poten-
tial growth-promoting effects of local adipocytes is the
release of soluble adipokines/cytokines (49), one of
which (leptin) was elevated in the serum of mice in
our study receiving the high-calorie control diet, irre-
spective of treatment with rapamycin or vehicle. As
recently suggested by Subbaramaiah and colleagues,
multiple energy balancerelated signals contribute to
inflammatory cross talk between macrophages, adipo-
cytes, and epithelial tumor cells (50, 51).
Taken together, our findings show that CR had a
stronger inhibitory effect on Panc02 tumor growth than
did rapamycin. CR and rapamycin decreased phosphor-
ylation of mTOR pathway components, but only CR
reduced circulating IGF-1 and leptin levels and phosphor-
ylation of Akt, GSK-3b, ERK/mitogen-activated protein
kinase, and STAT3bTYR705. Thus, treatment with rapamy-
cin, which specifically inhibited mTOR and significantly
decreased Panc02 tumor growth relative to control diet,
only partially mimics (at the dose used) the more potent
effects of CR on multiple progrowth signals and pancrea-
tic tumor growth inhibition. This observation that
CR, relative to rapamycin, is a more potent suppressor
of pancreatic tumor growth and is more promiscuous in
terms of targeting multiple signaling pathways suggests
that combination approaches (such as an mTOR inhibi-
tor plus a lifestyle or pharmacologic intervention that
targets other key pathways) will be most effective for the
prevention and control of pancreatic cancer. Future stu-
dies are being designed to verify the hypothesis that a
synergistic effect is exerted on tumor inhibition when the
broader acting effects of CR are combined with low-to-
intermediate doses of pharmacologic mTOR inhibitors
such as rapamycin.
Cancer Prev Res; 4(7) July 2011 1049
 Published OnlineFirst May 18, 2011; DOI: 10.1158/1940-6207.CAPR-11-0023
Lashinger et al.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Dr. J. DiGiovanni and Dr. P. Dennis for assistance with
rapamycin dose selection, Dr. K. Hance and Dr. J. Schlom for generously
providing the Panc02 cells, and Dr. D. Kusewitt for assistance with quanti-
fication of immunohistochemical staining patterns.
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Grant Support
R01 CA135386 (S. Hursting and S. Fischer); R25T CA57730; and T32
CA135386 (L. Lashinger).
The costs of publication of this article were defrayed in part by the
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Cancer Prev Res 2011;4:1041-1051. Published OnlineFirst May 18, 2011.

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