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Impact of soy isoflavones on DNA methylation in rat mammary glands - Dose-response aspects, critical time windows, and influence on carcinogenesis

Pudenz, Maria

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Epidemiological studies indicate a reduced risk and a lower incidence of hormone-dependent cancer types in populations following a traditional Asian diet rich in phytoestrogens such as isoflavones (IF). Lifestyle and timing of exposure to IF seems to be a critical factor. In rodent models, especially multi-generational or prepubertal exposure was shown to modulate mammary gland (MG) morphology, resulting in anti-tumorigenic activity. Many processes during early development are regulated by epigenetic mechanisms, therefore IF might alter the epigenetic reprogramming of the MG affecting normal cell growth and susceptibility to breast cancer. Several investigations provide evidence for the impact of IF on DNA methylation but most of the work focused on selected candidate genes. Investigations of genome-wide changes of DNA methylation in the MG upon IF treatment are scarce. The present thesis was aimed to analyzed DNA methylation profiles in MGs of healthy rats and during estrogen-induced mammary carcinogenesis on a genome-wide scale. Our focus was to clarify the impact of different IF doses and identify critical developmental windows of exposure in Wistar rats, and to investigate the chemopreventive efficacy of IF during estrogen-induced MG tumor development in ACI rats. Genome-wide methylation analyses were initially performed with Wistar rats bred on a diet with medium IF levels that were switched to either IF-free or high dose IF diet for two weeks after ovariectomy (OVX). We observed only few IF-related DNA methylation changes (measured by MCIp-Seq technology). This weak epigenetic effect was attributed to the fact that all rats had been exposed to the phytoestrogens during all critical developmental phases until adult life. Most methylation changes were observed outside of classical regulatory regions (promoters, CpG islands or 5’ UTRs) but interestingly were enriched for binding sites for ETS-domain and basic leucine zipper-domain containing transcription factors. For validation of the genome-wide results, selected candidate genes were analyzed quantitatively by EpiTYPER MassARRAY technology. Two groups of rats with lifelong exposure (from conception to post OVX) to low and high IF doses were included to consider dose effects. We observed a U-shaped dose-response pattern, with the low IF concentration reducing methylation levels, whereas medium and high doses increased methylation compared to an IF-unexposed control group. The relevance of these changes needs to be further investigated. However, lifelong exposure to high IF levels significantly reduced estrogenic and proliferative response of the MG, whereas lower IF concentrations were not sufficient to induce the beneficial health effects and even provoked opposing effects, i.e., significantly induced PCNA and PR protein expression. Different from the lifelong exposure to high IF levels, IF intervention exclusively during puberty exhibited only minor effects on DNA methylation and mRNA expression levels of candidate genes. Also, expression of proliferation markers were not affected. An exception was an increase in Vdr and Gata3 mRNA expression which might sensitize the MG towards enhanced differentiation. In contrast, short term IF exposure only after OVX affected DNA methylation patterns of candidate regions mostly in an opposed direction when compared to the lifelong IF exposure. Post OVX exposure to IF also induced pro-proliferative as well as pro-estrogenic properties and sensitized the animals towards the estrogen treatment. These results contradict the use of high IF concentrations, e.g., for hormone replacement therapy after menopause. In order to investigate lifelong IF-mediated effects on estrogen-induced rat mammary carcinogenesis, ACI were exposed lifelong to the highest IF dose. IF intervention reduced incidence and multiplicity (56 %, p=0.018) of MG tumors but shortened tumor latency by 5 weeks (p<0.0001) and enhanced tumor growth (>2 fold), if tumors escaped the preventive effect. IF intake increased the estrogenic and proliferative response of the MG during puberty when carcinogenesis was induced by exogenous estrogen exposure, and epigenetic modifying enzymes such as DNMT3a and 3b were significantly downregulated. Genome-wide methylation profiling analyzed by quantitative reduced representation bisulfite sequencing (RRBS) again indicated hypo- and hypermethylation mainly outside of classical regulatory regions. Genes with differential promoter methylation induced during carcinogenesis which could be modulated by IF exposure were enriched for biological processes and signaling pathways involved in reproductive tissue homeostasis and endocrine system. After applying stringent selection criteria, we were able to validate selected methylation changes from the RRBS analysis by MassARRAY. In all regions investigated, the carcinogenic process significantly modified methylation levels up to 30 %, inducing both, hypo- and hypermethylation. Interestingly, lifelong intervention with the highest IF dose prevented this carcinogenesis-mediated loss and/or gain in methylation. For half of the selected candidate genes methylation changes significantly correlated with mRNA expression. Interestingly, lifelong high dose IF exposure significantly reduced mRNA levels of DNMT1 in healthy MGs and prevented the estrogen-induced upregulation of DNMT1 during tumor formation. In conclusion, we identified genome wide DNA methylation changes induced by dietary IF. The impact of IF on DNA methylation is highly dependent on exposure time and IF dose. Lifelong exposure to IF reduced estrogen-induced MG tumor incidence but shorted tumor latency. This phenomenon might be partly explained by the downregulation of epigenetic modifiers such as DNMTs during early MG development as well as during breast carcinogenesis. Additional investigations are required to gain a comprehensive insight on IF-induced epigenetic regulation unraveling the functional mechanisms of biological effects exerted by phytoestrogenic soy IF.

Item Type: Dissertation
Supervisor: Popanda, PD Dr. Odilia
Place of Publication: Germany
Date of thesis defense: 23 March 2016
Date Deposited: 01 Jun 2016 07:12
Date: 2016
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
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