There are a number of dietary interventions capable of inhibiting mammary tumorigenesis; however, the effectiveness of dietary combinations is largely unexplored. Here, we combined 2 interventions previously shown individually to inhibit mammary tumor development. The first was the use of the omega-3 fatty acid, eicosapentaenoic acid (EPA), and the second was the implementation of calorie restriction. MMTV-Her2/neu mice were used as a model for human breast cancers, which overexpress Her2/neu. Six groups of mice were enrolled. Half were fed a control (Con) diet with 10.1% fat calories from soy oil, whereas the other half consumed a diet with 72% fat calories from EPA. Within each diet, mice were further divided into ad libitum (AL), chronic calorie-restricted (CCR), or intermittent calorie-restricted (ICR) groups. Mammary tumor incidence was lowest in ICR-EPA (15%) and highest in AL-Con mice (87%), whereas AL-EPA, CCR-Con, CCR-EPA, and ICR-Con groups had mammary tumor incidence rates of 63%, 47%, 40%, and 59%, respectively. Survival was effected similarly by the interventions. Consumption of EPA dramatically reduced serum leptin (P < 0.02) and increased serum adiponectin in the AL-EPA mice compared with AL-Con mice (P < 0.001). Both CCR and ICR decreased serum leptin and insulin-like growth factor I (IGF-I) compared with AL mice but not compared with each other. These results illustrate that mammary tumor inhibition is significantly increased when ICR and EPA are combined as compared with either intervention alone. This response may be related to alterations in the balance of serum growth factors and adipokines. Cancer Prev Res; 6(6); 540–7. ©2013 AACR.

A variety of nutritional interventions have been considered for breast cancer inhibition (1). This has included altering the type of fat included in the diet. The potential effects of fat are seen when comparing women who consume traditional Nordic diets with those who migrate to countries with western diets (2). The incidence of breast cancer in women who have immigrated increases and can triple in a single generation (3). The consumption of fish in the diets of the women after they have immigrated is significantly decreased and this may be one of the mitigating factors in the increase of breast cancer in these women as compared with women who have not immigrated (4, 5). Fish such as wild salmon, sardines, and mackerel are all high in omega 3 (ω-3) long-chain polyunsaturated fatty acids (LC-PUFA). Human case–controlled studies have reported an inverse relationship between the ratio of ω-3 and ω-6 LC-PUFAs and the incidence of breast cancer, and it has been proposed that the relative proportions of ω-3 and ω-6 LC-PUFAs in the diet are important for this effect (6).

A second area of nutrition under investigation for its role in breast cancer is calorie restriction. Chronic calorie restriction (CCR) has clearly been shown to reduce mammary tumor incidence in animal models. For example, a meta-analysis of 14 studies found that CCR in rodents resulted in 55% less tumors than controls regardless of what nutrient(s) were restricted (7). Another way to implement calorie restriction is in an intermittent fashion. In fact, intermittent caloric restriction (ICR) applied to rodents has been reported to protect in the development of mammary tumors to an even greater extent than CCR (8, 9).

Animal models of breast cancer also support a role for fish oil influencing the development of breast cancer. For example, MMTV-Her2/neu transgenic mice fed fish oil instead of corn oil as 25% of energy, had a mammary tumor incidence of 57% compared with 87% for mice fed corn oil (10). In addition, when mice with MDA-MB-435 human breast cancer cell line xenografts were fed a diet of 18% fish oil and 5% corn oil, they had significantly slower tumor growth and less metastases than mice fed 5% fish oil and 18% corn oil (11).

The mechanisms of action of fish oil and calorie restriction are under active investigation. The ω-3 fatty acid eicosapentaenoic acid (EPA), which is thought to be one of the main active factors in fish oil, can reduce growth and increase apoptosis of some breast cancer cells in vitro (12, 13). The serum levels of a number of growth factors are altered by both EPA and calorie restriction (14, 15). Fish oil influences adipose tissue secretion of a specific group of growth factors known as adipokines (16). Adiponectin is one adipokine that circulates at high concentrations (2–20 μg/mL) in human serum and in contrast to most adipose-secreted proteins, is negatively correlated with body weight, body mass index, body fat, and serum leptin in humans (17). With respect to breast cancer, reduced serum adiponectin levels have been reported for postmenopausal women with breast cancer (18) and premenopausal women with a higher risk of additional neoplastic events (19). Calorie restriction decreases serum levels of the adipokine leptin (20), which has been associated with increased growth of breast cancer cells in vitro (21). Calorie restriction also decreases the serum levels of insulin-like growth factor 1 (IGF-I), which is involved in the growth of a number of different tumor types (22) and whose pathway is being targeted for antibreast cancer interventions in the clinic (23). It may be that identification of the optimal levels of adiponectin, leptin, and IGF-I is the key to breast cancer prevention (18, 24, 25).

In humans, Her2/neu is overexpressed in approximately 25% to 30% of patients with breast cancer and is associated with aggressive, hormone-independent breast cancer (26). The use of drug combinations has proven to be more effective than the use of single agents for reducing tumor recurrence and prolonging lifespan in women with Her2-neu–positive tumors (27). However, prevention is the ultimate goal and very little is known about whether combining potential interventions will be effective for this malignancy. The MMTV-Her2/neu mouse model overexpresses Her2/neu in mammary tissues and has close parallels to this type of human breast cancer (28). In the following study, we hypothesized that this model could be used to illustrate that combining 2 dietary interventions would lead to superior breast cancer inhibition compared with either intervention alone. Furthermore, this inhibition may be mediated by increased serum adiponectin and reduced serum leptin and IGF-I.

Mice and diets

Pairs of MMTV-Her2/neu homozygous transgenic mice were obtained from Jackson Laboratories and bred. Female pups were then enrolled into the study. The diet is based on the AIN-93M diet (29) that was originally designed for long-term maintenance of rodents in aging and tumorigenesis studies. The diets were obtained from Harlan Laboratories, Inc. Our base diet contains 10.1% fat calories derived from soy oil. No phytoestrogens have been found in the AIN-93 diet (30). All diets contained t-butylhydroquinone (TBHQ) for stabilization of the EPA or to control for effects of this ingredient on the study mice. These are the same levels of TBHQ per gram of fish oil as used for a prior study using the same MMTV-Her2/neu mice (10). All diets have 3.6 calories per gram. The intermittent-restricted diets are fed at 50% and designed so that the animals consume the same amounts in calories of protein, fat, vitamins, minerals, and TBHQ as the ad lib (AL) animals. The diets meet the recommended dietary linoleate requirements for mice of a minimum 0.68% of energy, and mice were monitored for signs of essential fatty acid deficiency such as hair loss, dermatitis, and scaly skin during the course of the experiment as well as fatty livers postmortem (31). Diets were handled under low-light conditions and stored at −20°C. The EPA diet contains 10.1% total fat calories with 7.25% fat calories from EPA and 2.85% fat calories from soy oil. The EPA was purchased as 90% Ethyl Ester from Sanmark LLC and incorporated into the appropriate diets by Harlan Laboratories. Table 1 shows the specific details of the diets. All procedures, protocols, and studies were approved by the University of Minnesota (Minneapolis), Institutional Animal Care and Use Committee, and the Hormel Institute Animal Facility (Austin, MN), which is Association for Assessment and Accreditation of Laboratory Animal Care accredited.

Study design

Following weaning, all MMTV-Her2/neu transgenic mice were fed daily the AL-control diet from 6 to 8 weeks of age to adapt them to the daily feeding regimen with powdered food. At 8 weeks of age, one half of the mice were assigned to the EPA group and switched to the AL-EPA diet for 2 weeks. At 10 weeks of age, mice were divided into 6 groups. The mice assigned to the ICR groups were fed 50% of the average amount of food consumed by the corresponding AL group for 3 weeks and then 100% of the AL diet for 3 weeks for a total caloric restriction of 25%. The 6-week cycles of restriction and refeeding were maintained until the mice were 60 weeks of age or were euthanized because of mammary tumor burden. CCR groups were given 75% of the total calories that the AL age-matched groups consumed. To achieve these feeding regimens, it was necessary to stagger the experimental groups at the beginning of the study such that the AL mice were the first mice enrolled in the study and the other groups' food allotments were based on how much the AL mice ate. Mice were weighed weekly and carefully examined for mammary tumors. To facilitate accurate food intake measurements, mice were individually housed and food intakes were monitored daily. Food was placed in small glass cups placed inside larger cups to collect any spillage. This overall protocol is based on our earlier studies (9). When a mammary tumor on a mouse measured 7 × 7 mm, the age of the mouse was designated as the age of palpable mammary tumor detection. Some mammary tumors were detected when a mouse was euthanized because of the experimental end point. The age of the mouse at the time of euthanization was then designated as the age of mammary tumor detection. The termination of the study was 60 weeks of age. However, mice with a mammary tumor 20 mm in length were euthanized before 60 weeks. Criteria other than tumor size for early euthanasia included unexplained weight loss, not eating, open skin sores, or other signs of distress. In order that all mice were in a similar postprandial state before euthanasia, we always fed the mice in the afternoon between 14:00 and 17:00 hours. The ICR mice were sacrificed during a restricted feeding period.

Sample procurement and analysis

To obtain blood, mice were anesthetized at 7 weeks of age before the diets were implemented, in the middle of the study before palpable tumor formation at 25 to 28 weeks of age or at the end point when mice were 35 to 60 weeks of age. Approximately 100 μL of blood was obtained by orbital bleed. The blood was then allowed to clot, centrifuged, and serum stored at −70°C. Quantikine ELISA Kits from R&D Systems were used according to the manufacturer's instructions. All samples were analyzed in duplicate. At the time of euthanasia, mammary tumors were removed and weighed. A sample was placed in formalin and the remaining tissue was frozen at −70°C. In addition, livers, spleens, kidneys, ovaries, hearts, and lungs were removed, weighed, and examined for abnormalities. Samples of the lungs and any abnormalities were processed for histopathologic analysis. The remaining tissues were frozen at −70°C. Left and right parametrial and retroperitoneal fat deposits were removed, weighed, and frozen. Samples were then analyzed in a blinded fashion.

Histologic diagnosis

Part of the tumor tissue was fixed with formalin, embedded with paraffin, and stained with hematoxylin and eosin. Tumor histology was diagnosed by a trained pathologist and was graded on the basis of a combination of the Scarff–Bloom–Richardson (SBR) system and the Black method that emphasizes nuclear grading and excludes consideration of tubules as a criterion. All tumors were either well-differentiated (grade 1) or some-differentiated (grade 2) breast cancer.

Data analysis

Mammary tumor incidence, age of mammary tumor detection, and age of death were analyzed by ANOVA for general differences among the groups. When the ANOVA was significant, Neuman–Keul test was used to determine significance between specific groups.

AL control (Con) and AL-EPA groups ate similar amounts of food (Supplementary data) and as such the CCR-Con and CCR-EPA also ate similar amounts of food as did the ICR-Con and ICR-EPA groups. The AL-Con and AL-EPA mice gained weight at similar rates (Fig. 1). The final weights of the AL-Con and AL-EPA were somewhat different but this was not statistically significant for the overall experiment due to the very small numbers of AL-Con mice that survived to the 60-week end point. The body weights of the CCR-Con and CCR-EPA were also similar to each other. The ICR-EPA mice were slightly heavier (P < 0.05) than the ICR-Con mice despite consuming similar quantities of food.

Mammary tumor incidence was the lowest in the ICR-EPA group (15.4%) and highest in the AL-Con group (86.7%), whereas the AL-EPA, CCR-Con, CCR-EPA, and ICR-Con had mammary tumor incidence rats of 55.6%, 47.2%, 40%, and 58.6%, respectively (Fig. 2A). The reduction in mammary tumor incidence was accompanied by a delay in the identification of the first tumors in the groups although this was not significant. The main criteria for identification of a tumor was palpation of a tumor that was 7 × 7 mm or larger. However, a few mice were only identified as tumor positive at the termination of the study, and the age of mammary tumor palpation was considered to be 60 weeks for these mice. The number of mice with mammary tumors identified at euthanasia for each group was: AL-Con = 5, AL-EPA = 3, CCR-Con = 3, CCR-EPA = 2, ICR-Soy = 1, and ICR-EPA = 0. Figure 2B shows a similar trend for the survival curves. The AL-Con group had the lowest percentage of mice surviving to 60 weeks of age with only 40.0%, whereas the ICR-EPA had the highest percentage of mice surviving to 60 weeks of age with 88.5% (P < 0.01). The AL-EPA, CCR-Con, CCR-EPA, and ICR-Con survival percentages were 48.1%, 72.2%, 73.3%, and 62.1%, respectively (Fig. 2B). Mice with tumors were euthanized because of tumor burden (AL-Con = 18, AL-EPA = 11, CCR-Con = 13, CCR-EPA = 8, ICR-Soy = 11, and ICR-EPA = 1), tumor necrosis (AL-Con = 0, AL-EPA = 1, CCR-Con = 0, CCR-EPA = 1, ICR-Soy = 0, and ICR-EPA = 0), or end point of the study (AL-Con = 8, AL-EPA = 3, CCR-Con = 4, CCR-EPA = 3, ICR-Soy = 6, and ICR-EPA = 1). When examining the survival curves for effects of calorie restriction, we found that the AL-Con was significantly different from the CCR-Con (P < 0.003) and that the AL-EPA was significantly different from the CCR-EPA and the ICR-EPA (P < 0.04 and P < 0.001, respectively). When examining the survival curves for the effects of EPA, we found that the ICR-Con was significantly different from the ICR-EPA (P < 0.02).

An overview of the experimental results is provided in Table 2. We examined the amounts of food that the mice consumed over the course of the experiment. When the control mice from each dietary restriction regimen were compared with the EPA-fed mice there were no significant differences. The percentage of tumor-free mice was significantly lower in the AL-Con group when compared with all other groups, and the percentage of tumor-free mice was significantly higher in the ICR-EPA group as compared with all other groups over the course of the 60-week study. The ICR-EPA group had 22 of the 26 mice remaining tumor free during the 60-week study, whereas only 4 of the 30 mice from the AL-Con groups remained tumor free. The average age of the mice in weeks when a tumor was palpated was earliest in the AL-fed groups. The fact that the experiment ended when the mice were 60 weeks of age influenced the average age of survival. However, the average survival until terminal end point was the longest in the ICR-EPA group at 59.2 weeks and shortest in the AL-Con group at 52.6 (P < 0.0002). The average number of tumors per mouse for mice that developed at least one tumor and the total tumor burden per animal was not significantly different between any of the groups despite the fact that the average tumor burden was 0.98 g for ICR-EPA mice and 1.78 g for AL-Con mice. This was due to the very small number of mice with mammary tumors in the ICR-EPA group. All tumors were breast cancer and not benign tumors although they were low grades that were well differentiated (grade 1) or with some differentiation (grade 2). No significant differences in tumor grade between the groups were found. There was a single metastasis in the AL-Con group.

To better understand the effects of the interventions, we examined serum for alterations in growth factors and adipokines. Figure 3A shows that serum IGF-I was significantly reduced by calorie restriction with the ICR-Con being significantly different than the AL-Con (P < 0.01) and the AL-EPA (P < 0.001). The presence of EPA in the diet did not significantly change the levels of IGF-I. We found that serum leptin was significantly higher in the AL-Con mice compared with the corresponding calorie-restricted mice (AL-Con > CCR-Con > ICR-Con). The presence of EPA in the diet significantly reduced the levels of leptin in the AL-EPA mice compared with the AL-Con mice (P < 0.02) but combining EPA with either CCR or ICR did not result in any significant changes versus the CCR-Con or ICR-Con groups. This was true regardless of whether the sera were obtained during the middle of the study at 25 to 28 weeks of age (Fig. 3B) before the appearance of palpable tumors or at the terminal end points of the study when the mice were 35 to 60 weeks of age (Fig. 3C). The levels of adiponectin in the AL-Con, CCR-Con, and ICR-Con mice compared with the mice fed EPA were significantly lower (overall ANOVA P < 0.0001) but dietary restriction did not significantly change the adiponectin levels at the study midpoint (Fig. 3D). The levels of serum adiponectin at the end point (Fig. 3E) were more similar between the groups although there were significant differences between some groups (overall ANOVA P < 0.0036).

The goals of this project were to determine whether 2 nutritional interventions that individually protect against mammary tumor development would provide even greater protection when combined and to identify potential mechanisms of action. The first nutritional intervention was alteration of the ratio of ω-3 to ω-6 fatty acids in the diet by addition to the ω-3 fatty acid EPA. The second nutritional intervention was calorie restriction. Highlights of our findings include that ICR in combination with EPA, resulted in significantly increased survival and tumor-free periods compared with all other treatments (Fig. 2). This may be related to the alteration of serum growth regulatory molecules. The first dietary intervention, addition of the ω-3 fatty acid EPA, was able to alter serum levels of adiponectin (Fig. 3D and E) and the second intervention, calorie restriction, was able to alter levels of serum IGF-I and leptin (Fig. 3A–C).

When we evaluated each intervention alone, we found that the addition of EPA to the diets of the AL-fed animals resulted in a trend toward increased times to survival and longevity as well as a significant difference in the absolute percentage of animals that developed tumors as compared with the AL-Con–fed animals (Table 2). EPA may be acting in part through both leptin and adiponectin. The addition of EPA to the diet resulted in a significant decrease in serum leptin in all groups as compared with the AL-Con mice regardless of whether the serum was obtained before or after palpable tumors had arisen (Fig. 3B and C). Leptin is positively correlated with total body fat and is present in the serum of almost all humans. Leptin has been implicated as a growth promoter in breast cancer (32). One dramatic example is that mice deficient in leptin, LepobLepob, or with nonfunctioning leptin receptors, LeprdbLeprdb, did not develop transgene-induced mammary tumors (33, 34). Some human studies indicate that the presence of the leptin receptor, Ob-Rb, in breast cancer (35) and high serum leptin are associated with poor prognosis (36). Calorie restriction is well documented to result in a reduction in serum leptin in humans (37) and a decrease in tumorigenesis in animal studies (7, 38). Therefore it is interesting that the addition of EPA to the diet was able to mimic the effects of calorie restriction on serum leptin (Fig. 3B and C).

Adiponectin was significantly increased by EPA at the middle time point (Fig. 3D) and to a lesser degree at the end point of the study (Fig. 3E). Increased serum adiponectin has been associated with decreased risk of breast cancer, particularly in postmenopausal women (39). Adiponectin has been shown to be negatively correlated with body fat (17) in humans, and it was originally assumed that the amount of body fat was the factor regulating how much adiponectin was in the serum. However, this study as well as others (14) indicate that regulation of serum adiponectin may actually be related to dietary components such as EPA.

When we evaluated the effects of calorie restriction in the absence of EPA, both by chronically or intermittently restricting calories, we found significant differences in tumorigenesis between the restricted groups and the AL-fed groups. The AL-Con mice had significantly reduced tumor free and survival times compared with the CCR-Con mice (P < 0.01) as well as significantly more tumor-bearing mice than either the CCR-Con (P < 0.05) or the ICR-Con (P < 0.05) groups. We did not find any significant differences between the CCR-Con and the ICR-Con groups. Previously, in a different transgenic mouse model, we found that the ICR diet regimen inhibited mammary tumorigenesis to a greater extent than the CCR diet regimen (8, 9). It may be that Her2/neu-overexpressing mammary tumors in mice are less responsive to the ICR diet regimen than the tumors in mice that overexpress TGF-α. It has become evident that breast cancer is composed of many different types of cancer that respond differently to treatment (40). Evaluation of the clinical meaning of the differences between the Her2/neu-overexpressing tumors compared with the TGF-α–overexpressing tumors may help elucidate the possible role of different diet regimens as they pertain to specific types of breast cancer.

How CCR and ICR are able to inhibit mammary tumorigenesis as compared with AL feeding and each other is probably multifaceted. We found that ICR was able to decrease serum IGF-I compared with AL-fed mice but that there was not a significant difference between CCR and ICR (Fig. 3A) groups. We also found that both CCR and ICR interventions were able to reduce levels of serum leptin as compared with AL feeding but not as compared with each other (Fig. 3B and C). These results parallel with the mammary tumor inhibition that we found and illustrate the correlation in this model between levels of serum IGF-I and leptin with mammary tumor inhibition.

Calorie restriction and weight loss either through conventional dietary restriction or bariatric surgery have been reported to be linked to breast cancer inhibition in humans (41, 42). The effects of ICR versus CCR in humans have not been well investigated. However, Harvie and colleagues found that serum leptin levels were reduced by approximately 40% in women who underwent either CCR or ICR for 6 months to attain an overall 25% reduction in caloric intake, whereas adiponectin increased by 10% in the intermittent group versus 1% in the chronic group although this was not a significant difference (43). There was no significant effect on the adiponectin:leptin ratio. A study of the effects of anorexia nervosa on the incidence of breast cancer has been published (44). It was found that in the anorexia nervosa group, there was a 53% reduction in breast cancer incidence with a 23% reduction in nulliparous women and a 76% reduction in incidence in parous women compared with the general population. These results suggest that both CCR and ICR may be beneficial in reducing breast cancer risk in humans.

Finally, we evaluated the combined effects of both CCR and ICR with EPA. The addition of EPA to the CCR-fed mice did not result in a significant difference in mammary tumor development as compared with the CCR control mice. However, addition of EPA to the ICR protocol resulted in significant differences in the percentage of tumor-free mice, the average tumor-free time, and survival compared with the ICR-Con mice with soy oil as the fat source (Table 2). These results suggest an additive or possibly even a synergistic effect when ICR was combined with EPA for the inhibition of breast cancer. EPA alone was able to significantly decrease serum leptin and increase serum adiponectin levels as compared with AL mice.

ICR was able to decrease serum IGF-I and serum leptin as compared with the AL-fed mice. It seems likely that alteration of these growth regulators is part of the mechanism by which the ICR-EPA mice were able to inhibit mammary tumorigenesis. However, there was also significant inhibition of mammary tumorigenesis in the ICR-EPA mice as compared with the CCR-EPA mice but without any differences in the serum levels of IGF-I, leptin, or adiponectin. This suggests that additional mechanisms of action are most likely influencing the ICR-EPA to CCR-EPA differences in mammary tumor inhibition.

In summary, this project was conducted to characterize a novel combination of dietary interventions for inhibition of breast cancer and to examine the role of growth regulators in breast cancer inhibition. The work provides research to help evaluate their use of fatty acids and calorie restriction for breast cancer prevention on which to base future investigations. The best inhibition was achieved by the combination of ICR with EPA. On a practical note, it may be easier for some humans to tolerate short periods of caloric restriction and substitutions of EPA for a portion of their fat intake than a lifetime of CCR and a low-fat diet.

No potential conflicts of interest were disclosed.

Conception and design: M.E. Grossmann

Development of methodology: N.K. Mizuno, M.E. Grossmann

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N.K. Mizuno, O.P. Rogozina, C.M. Seppanen, J.D. Liao, M.E. Grossmann

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N.K. Mizuno, C.M. Seppanen, J.D. Liao, M.P. Cleary, M.E. Grossmann

Writing, review, and/or revision of the manuscript: M.P. Cleary, M.E. Grossmann

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): O.P. Rogozina

Study supervision: M.P. Cleary, M.E. Grossmann

This work was financially supported by Susan G. Komen for the Cure grant KG081178 and The Hormel Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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