Research Grants Awarded
Calorie Restriction And Eicosapentaenoic Acid For The Inhibition Of Breast Cancer
Career Catalyst Research
Background: This proposal focuses on combining two dietary interventions that have been shown to separately impact breast cancer (brca) prevention. First, a number of human epidemiological studies have shown inverse associations between per capita consumption of marine fatty acids with incidence and mortality rates of brca. Several animal models have also suggested that fish oil can influence the development of brca. In particular, in a study using MMTV-HER-2/neu transgenic mice those fed fish oil had a mammary tumor (MT) incidence of 57% compared to 87% for corn oil fed mice. The major active ingredients of fish oil are the long-chain omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which in addition to their anticancer roles have the ability to modify lipid profiles, reduce inflammation and positively regulate neurological disorders. Human and rodent studies have shown that EPA consumption increases serum levels of adiponectin (adipocyte complement-related protein of 30 kDa (Acrp30)). Interestingly elevated Acrp30 levels are associated with reduced incidence of brca.
A second protective dietary intervention for mammary tumorigenesis is caloric restriction. Chronic caloric restriction (CCR) has clearly been shown to reduce MT incidence in a number of animal models. A meta-analysis of 14 studies found that CCR rodents developed 55% fewer tumors than controls regardless of what nutrient(s) were restricted. We have recently reported the results of three separate studies investigating intermittent caloric restriction (ICR) in two different transgenic mouse models. In the two studies utilizing the MMTV-TGF-?-mice we found that multiple periods of ICR resulted in 96% and 85% reductions of oncogene-induced MT incidence while a similar degree of CCR resulted in only 40% and 64% decreases. In a third study utilizing a second strain of transgenic mice, MMTV-HER-2/neu, ICR reduced MT incidence by 40% while there was no effect of CCR on MT development.
Objective: The mechanisms of the protective actions for EPA and ICR are unknown. However, the interventions influence the secretion Acrp30 as well as leptin. Higher levels of serum Acrp30 and lower levels of serum leptin are associated with brca prevention. Acrp30 is increased by consumption of EPA in humans and mice while serum leptin levels are lowered with decreasing body fat such as occurs during ICR. We propose the hypothesis that the two interventions, EPA consumption and ICR, can be combined to achieve even greater inhibition of brca formation than either alone and that the ratio of Acrp30 and leptin is linked to the inhibition of brca.
Specific Aims: Specific Aim 1. To determine the effects of EPA consumption on serum Acrp30/leptin ratios and mammary tumorigenesis during intermittent caloric restriction.
a. Characterize serum Acrp30 and leptin levels and identify correlations to mammary tumorigenesis during intermittent caloric restriction in the presence and absence of EPA.
b. Determine if EPA combined with intermittent caloric restriction provides protection from mammary tumor development as reflected by tumor incidence, latency and burden.
Specific Aim 2. To determine the effects of intermittent caloric restriction in the presence or absence of EPA on mammary tissue and mammary tumor apoptosis, proliferation, inflammation, oxidative stress and expression of leptin/Acrp30 receptors.
Study Design: Six groups of MMTV-HER-2/neu transgenic mice will be utilized. From 10 weeks of age half of the mice will be fed a diet with fat derived from corn oil and the other half will consume a diet with significant proportion of fat calories from EPA. Mice will be further divided into ad libitum (AL), intermittent caloric restricted (ICR) and chronic caloric restricted (CCR) groups. AL groups (corn oil and EPA) will receive unrestricted access to their diets. The ICR groups will be fed calories equal to 100% of the AL age-matched groups for three weeks followed by three weeks of calories equal to 50% of the AL age-matched groups. CCR mice will be pair-fed and receive a mixture of the two diets in the same proportions and total amounts as the ICR mice average during each 6-week period. The 6 week cycle of restriction and refeeding will be maintained until the mice are 82 weeks or are euthanized due to MTs. Blood samples will be obtained in cycles 0, 3, 6, 9 and at the end of both final restriction and refeeding periods. Serum will be analyzed for Acrp30, leptin, C-reactive protein, a marker for inflammation and F(2)-isoprostanes, a biomarker of in-vivo lipid peroxidative damage.
Mice will be weighed weekly and examined for MTs which will then be measured. At the time of euthanasia, MTs will be removed, location recorded and tumors weighed. Protein expression levels for Acrp30, leptin and their respective receptors will be determined in mammary tissue and MTs. MT incidence, age of MT detection and age of death will be analyzed as well as the relationships of Acrp30:body weight, Acrp30:fat pad weight, Acrp30:leptin ratios, etc using appropriate statistical analysis. Effects on metastasis will also be assessed.
This study will combine two proven nutritional interventions for inhibition of brca in a novel manner. The primary endpoint of brca inhibition should be achieved. The serum and tissue samples collected will allow us to meet the secondary endpoint of confirmation of our hypothesis concerning the mechanisms of action of the interventions. Finally, the study is such that it will provide the ability to define significant and immediate recommendations for clinical trials.
We are interested in two areas of diet that may play a role is breast cancer (brca). The first area is consumption of the fatty acid EPA which is found in fish oil. A number of human epidemiological studies have shown inverse associations between per capita consumption of marine fatty acids with incidence and mortality rates of brca. The second is periods of intermittent caloric restriction (ICR). Although there is some controversy about the consequences of weight cycling on health in general, particularly in the popular press, very few negative outcomes have been reported in the scientific literature. Also, women who report weight cycling do not have increased incidence of breast cancer and ICR has been suggested to provide protection from various diseases as well as being associated with delayed aging thus providing potential benefits even if they are utilized in individuals who are not at risk to develop cancer. Furthermore there is increasing acceptance that this is probably the way that humans have existed up until recently. Our own work using ICR in transgenic models has shown dramatic reductions in tumor incidence in several studies using mammary cancer models. For this study we will utilize the HER-2/neu mouse model which may have close parallels to the approximately 20% of human brca in which Her2/neu is over expressed. HER2/neu is an oncogene and there are no chemopreventive agents available for HER2/neu over expressing brca. In this model both consumption of EPA containing fish oil and intermittent calorie restriction lower the incidence of mammary tumors when the interventions are independently implemented. Our proposed research will combine these two preventive strategies and determine the outcome on tumor incidence, latency and burden. This is an innovative and novel strategy for mammary tumor prevention.
The mechanisms of action of EPA and ICR are unknown. However, both influence the secretion of proteins called adipokines from fat tissue in the body and these appear to play a role in brca inhibition and growth. Brca inhibition has been linked to increased levels of the adipokine, adiponectin, which has been shown to be increased by consumption of fish oil in humans as well as mice. Brca inhibition has also been linked to decreased levels of leptin, and we and others have found that leptin levels are decreased with decreasing body fat such as occurs during ICR. We propose that the two methods, EPA consumption and ICR, can be utilized together to achieve even greater inhibition of brca formation than either method alone and that the ratio of the adipokines Acrp30 and leptin are linked to the inhibition of brca. In the proposed study we will obtain serum samples over the course of the study to prospectively determine the role of these two adipokines and their ratio in association with mammary tumor development. This will clarify the mechanisms of action for these protective effects. Understanding how these interventions work at the cellular level may provide the opportunity in the future to develop compounds that could be used as chemopreventive agents to elicit similar effects to those obtained from dietary interventions.
This study will serve as a stepping stone to future work to determine levels of EPA required for protection. Additionally, in combination with intermittent calorie restriction the presence of EPA may allow for a more modest reduction in calories and increase the acceptability of this intervention. The results of this investigation will provide significant immediate improvement in the ability to define nutritional trials and recommendations. Furthermore, it would clearly be easier to implement defined periods of caloric restriction rather than having to contemplate a lifetime of chronic restriction for a prevention strategy. Likewise the substitution of many presently consumed dietary fats with EPA is far superior from a practical standpoint than a lifetime of low fat diets. These nutritional interventions are possible in the real world at the present time. Thus novel combinations of nutritional approaches may provide excellent opportunities for real world brca interventional strategies.