C-FAR Functional Foods SRI
Year 1 Final Report

Objective: Breast Cancer & Functional Foods

Effects of Soybean Constituents on Breast Cancer

William J. Banz,^1,2, Stuart Adler ^3,4, Todd A. Winters,^1,2

Department of Animal Science, Food and Nutrition,¹ and Department of Physiology,²
Southern Illinois University,
Carbondale, IL 62901

Department of Obstetrics and Gynecology,³ and Department of Cell Biology and Physiology,⁴
Washington University School of Medicine,
St. Louis, MO 63110

Brief Project Review

Breast cancer remains a major health problem. Although there are increased efforts directed towards early detection and improved treatments, efforts are also necessary to try to prevent the development of breast cancer. Life style changes, such as dietary modification, have been successful for prevention of other life threatening conditions, notably heart disease. While not all studies agree, epidemiologic evidence indicates that populations that consume Asian diets high in vegetables and soy products have a lower incidence of breast cancer. Chemical compounds from soy and other plants have been identified that have the potential to act as mimics of the natural female steroid hormone, estradiol, and these "phytoestrogens" have received attention as one approach for preventing breast cancer by interfering with the body's own natural hormonal regulation of breast tissue development that may lead to cancer. This proposal is based on the idea that the body has ways to adapt and to compensate for the presence of these natural food components in the diet, so that regulation by estrogen receptors can distinguish between the natural signals and these dietary mimics. We further propose that some beneficial effects of these phytoestrogens are a result of these adaptations that make breast tissue less susceptible to abnormal stimulation that promotes the development of breast cancer. We believe that soy phytoestrogens will beneficially alter the mechanisms/pathways that facilitate the development of breast cancer. Since breast cancer affects 1 of every 11 women, such beneficial effects will greatly increase the consumer demand for soy foods containing these functional components.

Hypothesis/Purpose

Our goal is to test the hypothesis that phytoestrogen action is modulated by the cellular ratio of co-activators and co-repressors. Furthermore, we hypothesize that the ratio of co-repressors and co-activators is itself altered by exposure to phytoestrogens, and that this change in the levels or ratios of co-regulators contributes to the protective effects of these compounds on breast cancer.

Specific Aims

SA #1: To fully characterize the activities of the phytoestrogens in gene regulation by ER and ERß. Established cell culture systems for assaying transcriptional activation and repression by both ER and ERß will be used and reporter gene activity determined for genistein, and daidzein. Activity as antagonists will also be determined.

SA #2: To determine whether phytoestrogen activity is modulated by the cellular ratios or levels of co-activators and co-repressors. Cotransfection of various ratios of co-regulators will be used, and reporter gene activity determined for the phytoestrogens using hydroxytamoxifen and estradiol as controls. This will determine if phytoestrogens exhibit an agonist to antagonist transition with changes in co-regulator expression.

SA #3: To determine whether phytoestrogens alter the level of expression of co-regulators in cell lines and in vivo. The levels of co-regulators will be determined in cultured cells and in rat organs before and after treatment with estradiol, hydroxytamoxifen, genistein and daidzein.

Key Accomplishments in this Time Period

1. We have established a quantitative assay using RNAse protection to determine the mRNA expression levels of co-regulators in particular cell types. RNAse protection assays are, by their intrinsic specificity for exact nucleotide basepairing, species-specific. This first assay is specific for expression of the SRC-1 co-activator, and is designed for specific application to rat tissues and rat cell lines. We are in the process of validating a similar assay for SMRT, the corepressor molecule, also designed for use in applications using rat tissue or rat cell lines. Minor modifications to these assay can be made later for specific detection in human tissues or human cell lines.

2. Using the assay for SRC-1 mRNA we have confirmed that estradiol does indeed modulate the expression of SRC-1 in the rat GC cell line we have been using for our transfection experiments. This was expected and is similar to results published previously by others using the related rat GH cell line.

3. For the first time estrogenic compounds other than estradiol have been evaluate. These compounds include the phytoestrogens coumestrol, genistein, and daidzein and the estrogen receptor antagonist, ICI 164384. All have been tested in our preliminary RNAse protection assays.

4. These phytoestrogens, as estrogenic compounds, would also be expected to regulate expression of SRC-1 mRNA. In fact, that is the case, as the estrogenic compounds also appear to regulate expression of SRC-1 mRNA in the GC cell line. Interestingly, and in confirmation of our transfection experiments evaluating the effect of phytoestrogens on gene expression, there appears to be two classes of agonistic compounds based on their effect on gene expression and coregulator modulation: estradiol and coumestrol appear to act similarly, while the effects of genestein and daidzein act similarly to each other, but differently than estradiol and coumestrol.

5. Use of agonists and antagonist in these assays of coregulator modulation indicates that previous classifications as antagonist or agonist is imprecise for description of modulation of co-regulator expression. Whether the effects observed with a particular compound will be similar to or different from effects observed with estradiol are, at this point, unpredictable. For example, the previously described "pure antagonist" ICI 164384 has effects similar to those of estradiol, and the phytoestrogens coumestrol and genistein have very different effects, while both would have previously been classified as estrogen receptor agonists of similar potency.

6. Another major goal of this project was to determine the effects of changes in co-regulator expression on gene expression of estrogen regulated genes. Reporter gene assays have been performed using luciferase reporter genes and expression plasmid vectors for introduction of estrogen receptor alpha or estrogen receptor beta. We have extended these transfection assays now by including expression vectors for SRC-1 and for SMRT, the relevant co-activator and co-repressor. By introducing various amounts of the different plasmids, we can artificially increase the level of expression of the co-regulator proteins and/or in the relative ratios of co-activator to co-repressor and thus determine whether changes like those observed in mRNA in the RNAse protection assays would have affects on the expression of ER-regulated genes.

7. Using co-transfection assays in GC cells and in Hela cells, it appears that the level or ratio of co-regulator expression does differentially modulate the activities of genistein, estradiol, and ICI 164384, and that, the observed effects are unpredictable based only on the previous classifications of compounds as agonists or antagonists.

8. These preliminary data describe above are extremely encouraging. We anticipate follow-up in vitro experiments will give use novel insight into the mechanisms that make the soy phytoestrogens unique (i.e., exhibit numerous beneficial effects, while displaying minimal undesirable effects often associated with other estrogenic compounds) as compared to classical estrogenic compounds.

9. The initial animal studies will begin early this summer. They will help elucidate whether soy phytoestrogens alter the level of expression of co-regulators in vivo. Initially, the levels of co-regulators will be determined in rats randomly assigned to one of three treatment groups: High-Phytoestrogen Soy Protein; Low-Phytoestrogen Soy Protein; or Non-Soy (Casein) Protein. Future animal studies will focus on defining and optimal dosage of soybean constituents to maximize their beneficial effects in vivo.

Indeed, these preliminary cellular studies support the initial hypothesis that phytoestrogen action is modulated by the cellular ratio of co-activators and co-repressors. Moreover, that the ratio of co-repressors and co-activators is itself altered by exposure to phytoestrogens, and that this change in the levels or ratios of co-regulators contributes to the protective effects of these compounds on breast cancer. Since breast cancer affects 1 of every 11 women, such beneficial effects will greatly increase the consumer demand for soy foods containing these functional components.