Abstract 3638: Hypermethylation of BRCA1, and hypomethylation and overexpression of unfolded protein response (UPR) regulators EIF2AK3, IKBKB and NFkB in breast cancer may originate from elevated in utero estrogenic environment.
Authors: Hilakivi-Clarke Leena A. , Zhang Xiao , Luoto Riitta , Nguyen Nguyen , Zhang Xiyuan , Xuan Jason , Lu Jin , Zwart Alan , Clarke Robert 
Published: August 2014

Abstract
High in utero estrogenic environment increases later breast cancer risk in women and preclinical models. This exposure also causes permanent epigenetic alterations in the mammary glands in animals that are inherited not only by the daughters, but the granddaughters and great granddaughters as well. Further, we have found that both de novo tamoxifen resistance and recurrence of breast cancers are significantly increased in rats exposed to excessive levels of estradiol (E2) in utero. Treatment with histone deacetylase and DNA methyltransferase inhibitors prevents the resistance to antiestrogen therapy in the E2 exposed rats. In this study, we investigated whether DNA methylation patterns are altered in daughters who are naturally exposed to high in utero E2 levels. Mothers’ E2 levels were determined on gestation weeks 8-12 and 26-28, and buccal swab DNA was obtained from 22 of their one-year-old daughters. Analysis using Illumina Infinium Methylation 450 arrays was performed and the results were explored by comparing methylation patterns among daughters whose mothers had the highest versus lowest quintile (3-fold lower) of E2 levels during pregnancy. A total of 223 genes exhibited significantly higher, and 666 genes significantly lower, DNA methylation patterns in the top E2 quintile daughters, compared with the bottom E2 quintile daughters. Of the hypermethylated genes in the buccal swab DNA, 23% were also more methylated in tumors compared with normal mammary tissues in data from women in the Cancer Genome Atlas (TCGA). Of the hypomethylated genes in buccal swab DNA, 25% were hypomethylated in the tumors in TCGA. Further, 21% of the methylated genes were confirmed to be down-regulated, and 31% of the hypomethylated genes were over-expressed in TCGA breast tumors. The most strongly hypermethylated gene in the high E2 exposed daughters was BRCA1 (3.7-fold, p&lt;0.006), and this gene was also methylated in TCGA tumors. However, BRCA1 mRNA expression was not reduced in breast tumors in TCGA, and was only marginally reduced in the mammary glands of rats or mice exposed to excess E2 in utero. Among the hypomethylated genes were several members of the unfolded protein response (UPR) pathway, including EIF2AK3 (p&lt;0.007), IKBKB (p&lt;0.01) and NFkB (p&lt;0.05); these UPR genes were also significantly up-regulated in the breast tumors in TCGA. Our findings thus suggest that epigenetically-induced alterations in the expression of UPR genes originate from in utero estrogenic exposures. Since abnormalities in UPR are linked to breast cancer risk and antiestrogen resistance, hypomethylation of UPR genes may partially explain high breast cancer risk and increased de novo and acquired tamoxifen resistance in in utero estrogen exposed females.
Citation Format: Leena A. Hilakivi-Clarke, Xiao Zhang, Riitta Luoto, Nguyen Nguyen, Xiyuan Zhang, Jason Xuan, Jin Lu, Alan Zwart, Robert Clarke. Hypermethylation of BRCA1, and hypomethylation and overexpression of unfolded protein response (UPR) regulators EIF2AK3, IKBKB and NFkB in breast cancer may originate from elevated in utero estrogenic environment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3638. doi:10.1158/1538-7445.AM2013-3638