Prepubertal exposure to soy or its biologically active component genistein reduces later breast cancer risk in both animal models and human populations. We investigated whether that might be due to reported estrogenic properties of genistein. Our study indicated that daily prepubertal exposures between postnatal days 7 and 20 to 10 microg 17beta-estradiol (E2) reduced later risk of developing 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors. Assessment of mammary gland morphology revealed that both prepubertal E2 and genistein (50 microg daily) exposures reduced the size of mammary epithelial area and number of terminal end buds (TEBs) and increased the density of lobulo-alveolar structures, suggesting that these exposures induced elimination of targets for malignant transformation by differentiation. Next, the mechanisms mediating the protective effects of E2 and genistein were investigated. E2 is shown to up-regulate BRCA1, a tumor suppressor gene that participates in DNA damage repair processes and cell differentiation and that down-regulates the activity of estrogen receptor (ER)-alpha. The expression of BRCA1 mRNA was up-regulated in the mammary glands of rats exposed to E2 or genistein during prepuberty, when determined at the ages of 3, 8 and 16 weeks. Prepubertal E2 exposure reduced ER-alpha levels in the mammary gland, while prepubertal genistein exposure had an opposite effect. Our results suggest that prepubertal estrogenic exposures may reduce later breast cancer risk by inducing a persistent up-regulation of BRCA1 in the mammary gland.

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Epidemiological studies have investigated whether a high birth weight is associated with increased breast cancer risk, but the results remain inconclusive. This study was designed to determine whether high birth weight increases later susceptibility to carcinogen-induced mammary tumorigenesis in an animal model and to determine mechanisms mediating this association. Pregnant female Sprague Dawley rats were fed either a control or a high-fat diet during the extent of gestation. Maternal exposure to the highfat diet increased pregnancy leptin levels and offspring's birth weight, but had no effect on pregnancy estradiol or insulin-like growth factor 1 levels. Changes in the offspring's mammary gland morphology and protein expression were assessed. The mammary epithelial tree of the high-birth-weight offspring was denser, contained more terminal end buds and exhibited higher number of proliferating cells. Further, their mammary glands expressed lower levels of ER-a, but higher levels of activated MAPK. No alterations in apoptosis were noted. High-birth-weight rats developed 7,12-dimethylbenz[a]anthracene-induced mammary tumors significantly earlier, and tumors grew larger than in the controls. The tumors in this group expressed higher levels of leptin receptor and activated Akt, and contained fewer apoptotic cells than those in the controls. Our results indicate that high birth weight is related to shortened latency to develop mammary tumors-perhaps reflecting an increase in activated MAPK levels and increased tumor growth-perhaps caused by a lower apoptotic response due to higher leptin receptor and activated Akt levels in the tumors. ' 2006 Wiley-Liss, Inc.Key words: birth weight; breast cancer; animal model; leptinIn 1990, Dr. Trichopoulos proposed that some breast cancers originate already in utero as a consequence of high estrogenic environment.1 Since then, several human studies have addressed this hypothesis using different proxy measures of in utero estrogenic environment, including birth weight. Most epidemiological studies have reported that high birth weight increases a woman's breast cancer risk, 2-7 albeit some have not. 8The hypothesis that the in utero hormonal environment affects breast cancer risk can be tested more directly in animal models than in humans. Studies in rats have shown that an exposure to estradiol during pregnancy increases the female offspring's mammary tumorigenesis.9 How might this happen? One possibility is that high prenatal hormone levels alter the normal developmental programming of the mammary gland, causing changes in gland morphology and associated gene expression, most likely through epigenetic changes. In rats exposed to elevated levels of estradiol in utero, these morphological alterations are manifested as an increase in the number of terminal end buds (TEBs) 9,10 -structures that give rise to malignant mammary tumors 11 -and changes in the expression of genes that lead to increased cell proliferation and inhibition of apoptosis (Shajahan et al., unpublished data). The...

Findings in humans and animal models suggest that in utero hormonal and dietary exposures increase later breast cancer risk. Since alcohol intake by adult women consistently increases their breast cancer risk, we wondered whether maternal alcohol consumption during pregnancy increases female offspring's mammary tumorigenesis. In our study, pregnant female rats were pair-fed isocaloric diets containing either 0 (control), 16 or 25 g alcohol kg À1 feed between days 7 and 19 of gestation. These alcohol exposures generate blood alcohol levels that correspond to low and moderate alcohol consumption and are lower than those that induce foetal alcohol syndrome. Serum oestradiol levels were elevated in pregnant rats exposed to alcohol (Po0.003). When adult, female offspring of alcohol-exposed dams developed significantly more 7,12-dimethylbenz[a]anthracene -induced mammary tumours, compared to the controls (tumour multiplicity; mean7s.e.m., controls: 2.070.3, 16 g alcohol: 2.770.4 and 25 g alcohol: 3.770.4; Po0.006). In addition, the mammary epithelial tree of the alcohol-exposed offspring was denser (Po0.004) and contained more structures that are susceptible for the initiation of breast cancer (Po0.001). Immunohistochemical assessment indicated that the mammary glands of 22-week-old in utero alcohol-exposed rats contained elevated levels of oestrogen receptor-a (Po0.04) that is consistent with the changes in mammary gland morphology. In summary, maternal alcohol intake during pregnancy increases female offspring's mammary tumorigenesis, perhaps by programming the foetal mammary gland to exhibit persistent alterations in morphology and gene expression. It remains to be determined whether an increase in pregnancy oestradiol levels mediated alcohol's effects on offspring's mammary tumorigenesis.

At present, we do not know what causes sporadic breast cancer. Environmental factors,particularly diet, appear to explain at least 70% of newly diagnosed breast cancers, but it is not clear what these factors are. We propose that the lack of progress in this area is due to a lack of considering the effect of timing of environmental and dietary exposures on the breast. The evidence provided above suggests that an in utero exposure to an estrogenic environment-including that caused by diet [high (n-6) PUFA or genistein]-increases breast cancer risk. This increase may be mediated by an increased presence of TEB in the mammary epithelial tree and increased ER-alpha levels, reduced ER-beta levels or both. Prepubertal estrogenic exposure, in contrast, reduces later risk of developing breast cancer. The protective effect of estrogens may be mediated by early epithelial differentiation, reduced presence of ER-alpha and increased levels of ER-beta in the mammary gland. The challenge we are now facing is to determine whether the data obtained mainly through the use of animal models is relevant to women and if so, how we might be able to modulate pregnancy and childhood estrogenic exposure by appropriate dietary modifications to reduce breast cancer risk in women.

Dietary exposures during childhood may influence later breast cancer risk. We tested in an animal model the hypothesis that prepubertal intake of (n-3) PUFAs, present mainly in fish, reduces susceptibility to breast cancer. Between postnatal days 5 to 25, rat pups were fed (n-3) PUFA-containing diets at a 2:1 ratio of (n-6):(n-3) PUFAs (typical of prehistoric societies) or a control (n-6) PUFA diet at a 17:1 ratio of (n-6):(n-3) PUFAs (comparable with current Western societies). These fatty acids were given in a low- or high-fat context (16 or 39% energy from fat). The low-(n-3) PUFA diet reduced while the high-(n-3) PUFA diet increased carcinogen-induced mammary tumorigenesis. The low-(n-3) PUFA diet reduced mammary cell proliferation and increased apoptosis, particularly in the terminal end buds (the mammary source of malignant breast tumors). The high-(n-3) PUFA diet had opposite effects on these 2 key biomarkers and increased phospho-Akt levels, a survival factor. Microarray analyses identified genes that were permanently upregulated in the low-(n-3) PUFA-exposed glands and function in oxidative damage repair. Serum levels of 8-hydroxy-2'deoxyguanosine, a marker of DNA damage, were significantly reduced in these low-(n-3) PUFA-fed rats, and increased in the high-(n-3) PUFA-exposed group. The latter group exhibited reduced expression of BRCA1, a DNA repair gene. Our results indicate that the opposing susceptibilities to mammary tumorigenesis between the low- versus high-fat (n-3) PUFA-exposed groups were associated with altered DNA damage repair and gene expression linked to proliferation, survival, and differentiation.

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson's disease and beta-amyloid and tau in Alzheimer's disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases. Video LinkThe video component of this article can be found at

Findings in BRCA1 mutation carriers suggest that physical activity, particularly during childhood, may be linked to a reduced risk of developing breast cancer. We investigated whether physical activity at puberty alters the expression of BRCA1 and two other tumor suppressor genes-p53 and estrogen receptor (ER)-β-in rats. In addition, the effects on ER-α expression, mammary proliferation and functional epithelial differentiation were investigated as markers of altered mammary cancer risk in rats exposed to regular physical activity at puberty. Female Sprague Dawley rat pups were randomized to voluntary exercise, sham-exercise control and nonmanipulated control groups. Treadmill training (20-25 m/min, 15% grade, 30 min/day, 5 days/ week) started on postnatal day 14 and continued through day 32. Third thoracic mammary glands © Springer Science+Business Media, LLC. 2008 Correspondence to: L Hilakivi-Clarke, clarkel@georgetown.edu. Authors' contributions The work described in the manuscript was designed to test a hypothesis proposed by Dr. Leena HilakiviClarke who provided overall direction for the study. Drs. Kim Westerlind and Robert Strange performed the animal study and provided tissues for the analysis, Dr. Mingyue Wang did most of the gene and protein expression experiments, together with Drs. Bin Yu, Galam Khan and Dipti Patil. Graduate student Kelly Boeneman processed the mammary glands and performed morphological assessment. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript (n = 5 per group and age) were obtained at days 32, 48 and 100 and assessed for changes in morphology through wholemounts, and at 100 days cell proliferation by using Ki67 staining, protein levels of ER-α and ER-β by immunohistochemistry, and mRNA expression levels of BRCA1, p53, ER-α and ER-β by real-time PCR. Mammary glands of rats exposed to exercise during puberty contained fewer terminal end buds (TEBs) and a higher number of differentiated alveolar buds and lobules than the sham controls. However, cell proliferation was not significantly altered among the groups. ER-α protein levels were significantly reduced, while ER-βlevels were increased in the mammary ducts and lobular epithelial structures of 100-day old rays which were voluntarily exercised at puberty, compared to sham controls. ER-β BRCA1 and p53 mRNA levels were significantly higher in the mammary glands of 100-day-old exercised versus sham control rats. Pubertal physical activity reduced mammary epithelial targets for neoplastic transformation through epithelial differentiation and it also up-regulated tumor suppressor genes BRCA1, p53 and ER-β and reduced ER-α/ER-β ratio in the mammary gland. It remains to be determined whether the up-regulation of BRCA1, and perhaps p53, explains the protective effect of childhood physical activity against breast cancer in women who carry a germline mutation in one of the BRCA1 alleles.