Rapamycin Partially Mimics the Anticancer Effects of Calorie Restriction in a Murine Model of Pancreatic Cancer

Mice

The Institutional Animal Care and Use Committee of the University of Texas approved all mouse experiments. Male C57BL/6 mice were received from Jackson Laboratories (Bar Harbor, ME) between 4–6 weeks of age, singly housed in a semibarrier facility at the University of Texas at Austin Animal Resource Center, and fed a chow diet for a 2-week acclimation period before study initiation.

Interventions

Mice were randomized to receive one of two diets for 30 weeks: a) control diet (Research Diets, Inc., New Brunswick, NJ; #D12450B) consumed ad libitum, n=27; or b) CR diet (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 min. 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 biochemical 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% DMSO 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 rapamycin between 1–10 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 treatment period.

Tumor cell injection and tumor monitoring

Mouse pancreatic cancer cells (Panc02, generously provided by Drs. Ken Hance and Jefferey Schlom, NCI) (29) were cultured under an atmosphere of 5% CO₂ in a 37°C incubator with McCoy’s media (HyClone) supplemented with 10% fetal bovine serum (HyClone), penicillin/streptomycin, glutamine, non-essential 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 Molecular Cytogenetics Core Facility at The U.T. M.D. Anderson Cancer Center (Houston, TX).

At 22 weeks of study, mice were subcutaneously injected into the right flank with 1×10⁶ Panc02 cells. One mouse from the control and CR groups each and two mice from the control plus rapamycin group died before tumor development within two weeks of Panc02 injection, and were thus censored from subsequent analyses. Once palpable, tumors were measured with calipers weekly until approximately half of tumors from any group were ≥ 1.5 cm in diameter. Tumor volume was approximated using the formula for an ellipsoid (4/3π(r₁)²(r₂)). At study termination, all remaining mice 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. Blood samples were processed as detailed above. Pancreatic tumors were harvested and either snap-frozen in liquid nitrogen and stored at −80°C, or fixed with 10% neutral buffered formalin overnight and then switched to 70% ethanol, paraffin embedded, and used for histologic and immunohistochemical analyses as described below.

Glucose Tolerance Test (GTT)

A GTT was performed, as previously described (30), on randomly selected mice from the control and CR groups at 14 weeks of study (n=12 per group). At 22 weeks of study, following two weeks of rapamycin or vehicle treatment and before Panc02 cell injection, a second GTT was performed on a subset of 10 mice from each diet/drug group.

Energy balance-related serum hormones

Serum IGF-1 was measured using radioimmunoassay according to manufacturer’s instructions (DSL-2900 kit, DSL/Beckman Coulter Laboratories, Webster, TX). Serum insulin and leptin were measured using Lincoplex® bead-based assays (Millipore Corporation, Billerica, MA) on a BioRad Bioplex® analyzer (BioRad, Hercules, CA) according to manufacturer’s 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-µm thick sections and processed for either hematoxylin and eosin (H&E) or immunhistochemical staining at the Histology Core Laboratory at The U.T. M.D. Anderson Cancer Center, Science Park Research Division (Smithville, TX). Antibodies used for immunohistochemistry were optimized by core personnel using both positive and negative controls that were repeated with each analysis. Slides were deparaffinized and hydrated sequentially in ethanol and water. Antigen retrieval required microwaving slides for 10 min with 10mM citrate buffer. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min.

Nonspecific binding was blocked with Biocare blocking reagent (Biocare Medical, Concord, CA) for 30 min at RT, then sections were incubated with primary antibody diluted in blocking buffer. The following primary antibodies (source, dilution and incubation conditions presented parenthetically) were used: Ki-67 (Dako, Carpenteria, CA; 1:200, 4°C overnight); phospho-IGF1R T_yr1131 and IGF1R (Cell Signaling, Danvers, MA; 1:50, 4°C overnight); phospho-Akt_Ser473 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:50, 1 h RT); Akt (Cell Signaling; 1:100; 4°C overnight) phospho-GSK-3β_Ser9 (Santa Cruz Biotechnology; 1:50, 1 h RT); GSK-3β (Millipore; 1:200; 1 h RT); phospho-ERK_{Thr202/Tyr204} (Cell Signaling; 1:100; 1 h RT); ERK (Cell Signaling; 1:25; 1 h RT); phospho-Stat3_Tyr705 (Cell Signaling; 1:50; 4°C overnight); phospho-Stat3_Ser727 (Cell Signaling; 1:50, 4°C overnight); STAT3 (Cell Signaling; 1:100; 1 h RT); phospho-mTOR_Ser2448 (Cell Signaling; 1:50, 4°C overnight); mTOR (Cell Signaling; 1:100; 1 h RT); phospho-S6 ribosomal protein_(Ser235/236) (Cell Signaling; 1:50, 1 h RT); S6 (Cell Signaling; 1:100; 4°C overnight) and cyclin D1 (Santa Cruz Biotechnology; 1:500, 2h RT). Slides were washed twice in PBS, incubated with horseradish peroxidase (HRP)-labeled α-rabbit secondary antibody (Dako) for 30 minutes at RT at a concentration of 1:200 with the following exceptions: a) Ki-67 (rabbit anti-rat IgG; 1:200, 30 min RT; Vector, Burlingame, CA); b) phospho-STAT3 and phospho-mTOR (rabbit HRP-polymer; 30 min RT; Biocare, Concord, CA); and c) cyclin D1 (rabbit anti-mouse F(ab)’; 1:250, 15 min RT; Accurate Chemical, Westbury, NY). Slides were then washed five times with PBS. Diaminobenzidine was used to develop the antibody staining followed with a hematoxylin counterstain. Images were captured using a light microscope equipped with a digital camera (Leica Camera Inc, Allendale, NJ). 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 <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 50–75% of cells in field stained an equivalent intensity to control tumors), or 3=marked (majority of tumor section exhibited dark brown stain or >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 phospho-IGF1-R and phospho-ERK produced weak but consistent signal intensity; therefore, the percentage of stained cells in field was assessed. Sample size was determined based on quality of tissue fixation/staining. More tumors were used for analysis using the phosphorylated IGF1R, Akt, GSK-3β, ERK, STAT3, mTOR, and S6 ribosomal 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 40X magnification (with the exception of cyclin D1 which was captured at 20X) in 3–4 fields in a tumor section from each tumor analyzed.

Western blotting

Pancreatic tumors were homogenized and lysed in RIPA buffer (Sigma, St. Louis, MO) with protease inhibitor tablet (Roche Applied Sciences, Indianapolis, IN) and phosphatase inhibitor cocktails I and II (Sigma). Protein lysates (50–80µg) were resolved by SDS-PAGE using 6, 10, or 12% gels, transferred to PVDF membranes (Bio-Rad, Hercules, CA), and blocked using LI-COR Blocking Buffer (LI-COR Biotechnologies, Lincoln, NE). Membranes were incubated overnight at 4°C with primary antibody (from Cell Signaling unless otherwise stated) diluted in blocking buffer and specific for: phospho-Akt_SER473 (1:500); Akt (1:1000); actin (1:1000); phospho-GSK-3β_Ser9(Santa Cruz, 1:1000); GSK-3β (Millipore, 1:1000); phospho-ERK_{Thr202/Tyr204}(1:1000); ERK (1:1000); phospho-mTOR_Ser2448(1:500); mTOR (1:1000); phospho-p70/S6K_Thr389 (1:500); p70/S6K (1:1000); and myosin IIa (1:1000). 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 min each) in 0.1% Tween-20/PBS (PBS-T), membranes were incubated for 1 h at RT in species-specific secondary antibody (LI-COR Biotechnologies) diluted in LI-COR blocking buffer (1:5000). Following three washes in PBS-T, membranes were scanned using the Odyssey infrared fluorescent imaging system. Densitometry was performed using LI-COR software (LI-COR Biotechnologies). Relative expression of phosphorylated proteins was calculated using 3 tumors per group.

Statistical analyses

Data are presented as mean ± standard deviation, except serum hormone levels and Ki-67 which are presented as mean ± standard error of the mean. Statistical analyses were conducted using SPSS (Apache Software Foundation, Wilmington, DE). Temporal differences between groups with respect to body weight and caloric intake were assessed using repeated measures analysis; final measurements were compared using independent t-tests. Pretumor serum hormone levels between the control and CR groups were compared using independent t-tests. Pair-wise comparisons 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 1) Ki-67 were determined by one-way ANOVA followed by Tukey’s post-hoc test of significance and 2) 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.

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 significantly reduced body weights beginning as early as 3 weeks on study (p<0.01) (Fig. 1A–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 bolus injection in CR mice peaked at 15 minutes and averaged 332±78 mg/dL, while 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 the control diet plus rapamycin group, n=9) or vehicle (control, 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 control plus rapamycin group (Fig. 1A). However, this decrease did not approach statistical significance, nor did it significantly affect body weight (Fig. 1B). This modest differential between the two groups subsided by week 25. Between weeks 20–27 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 circulating energy balance-related hormones relative to the control 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).

Effects of CR and rapamycin on Panc02 tumor growth and histology

To compare the anticancer effects of CR and rapamycin interventions, we injected mice from each diet/drug group with Panc02 cells at week 22 and monitored tumor growth during the next 8 weeks. Tumors in rapamycin-treated mice (p=0.009) and CR mice (p<0.0001) grew significantly slower than in control mice receiving vehicle. CR exerted a more dramatic inhibitory effect than rapamycin as evidenced by a significant difference between their tumor growth (p=0.005; Fig. 2A). Final tumor volumes from rapamycin-treated mice (374±206 mm³; p=0.04) and CR mice (221±107 mm³; p<0.0001) were significantly smaller on average than controls (550±147 mm³). The 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 compared 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 immunohistochemical 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 proliferation (based on Ki-67 immunopositivity) relative to controls (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 was 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 displayed reduced expression of phosphorylated Akt (p=0.03), ERK (p=0.05), STAT-3_Tyr705 (p=0.02), STAT-3_Ser727 (p=0.01), mTOR (p=0.02), S6 ribosomal protein (p=0.004), GSK-3β (p=0.06), IGF1-R (p=0.11), and cyclin D1 (p=0.11) (Fig. 3A–B). Rapamycin produced a more selective signaling profile than CR in which only pSTAT-3_Ser727 (p=0.02) and mTOR pathway components were inhibited relative to control, including phospho-mTOR (p=0.02), phospho-S6 ribosomal protein (p=0.006) (Fig. 3A–B), but not phospho-IGF-1R (p=1), phospho-Akt (p=0.3), phospho-GSK-3β (p=1), phospho-ERK (p=0.54), or phospho-STAT3_TYR705 (p=0.24). No appreciable changes to total protein expression were noted (data not shown).

Immunoblotting of various signaling intermediates confirmed the dampening of other survival signals in CR tumors (Fig. 4A). CR (n=3) significantly reduced phosphorylation of Akt (p=0.006), GSK-3β (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 phosphorylated proteins when averaged over three tumors (Fig. 4B). Rapamycin potently and consistently reduced phosphorylation of mTOR (p=0.005) and p70/S6K (p=0.02) when compared to 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-3β 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, GSK3b, or ERK (Fig. 4A).