Transgenerational effects of maternal high fat diet during pregnancy on breast ca

描述(由申请人提供)：在动物模型和人类中，母亲在怀孕期间暴露于高脂肪(HF)饮食会增加雌性后代中雌激素受体(ER+)和ER-乳腺癌的风险。这种影响可能并不局限于F1代的女儿：我们发现，在怀孕期间接触含有乙炔雌二醇(EE2)的饮食会增加孙女(F2代)和曾孙女(F3代)患乳腺癌的风险。由于HF饮食增加了孕期E2水平，我们建议在小鼠中调查母亲摄入HF饮食是否会增加F1-F4代后代患ER+和/或ER-乳腺癌的风险。此外，我们还将调查这些跨代效应是否涉及DNA甲基化的变化。我们使用大规模平行测序进行的初步分析发现，与对照相比，在暴露于EE2的母鼠的F1-F3代后代的乳腺中，有144个“命名”基因处于低甲基化或高甲基化状态。这些基因中有21%是多梳靶基因(PcGts)，其中包括一些肿瘤抑制基因(TSGs)，这表明孕期母亲的饮食，包括食用HF饮食，可能会诱导后代乳房中PcTGs/TSGs的甲基化。有趣的是，患乳腺癌的高危女性表现出PcGTs和TSGs的甲基化。DNA甲基化的增加可能是由于DNA甲基转移酶(DNMT1，DNMT3a和Dnmt3b)和多聚体(EZH2，SUZ12)的上调所致，我们和其他人已经发现，这些基因发生在暴露于雌激素的母鼠的后代中。此外，DNMT和Polycomb的上调可能是由雌激素诱导的针对它们的非编码miRNAs的抑制启动的，就像在宫内暴露于EE2或HF饮食的MCF-7人乳腺癌细胞和大鼠乳腺中看到的那样(我们的初步数据)。在这项研究中，我们检验了一种假设，即母亲暴露于 怀孕期间的HF饮食通过诱导PcTGs/TSGs的DNA甲基化，通过雌激素诱导miRNAs的下调，在F1-F4代后代中诱导乳腺癌风险的跨代增加。通过用组蛋白脱乙酰酶(HDAC)和DNMT抑制剂治疗F1-F4代小鼠，将研究涉及雌激素诱导的miRNA下调、DNMT和Polycomb上调以及随后的PcGts/TSG甲基化导致F1-F4代小鼠乳腺癌增加的因果链条。我们的初步研究表明，成年期间暴露于HDAC+DNMT抑制剂可完全逆转子宫内雌激素暴露小鼠乳腺癌风险的增加，但这些暴露是否逆转了F2-F4代雌激素暴露母鼠增加的DNA甲基化和增加的乳腺肿瘤发生尚不清楚。最后，由于目前还没有办法知道谁可能在子宫雌激素样环境中暴露于高水平，我们将研究是否可以通过检测外周血中E2/ER调节的miRNA图谱来识别这些人。 公共卫生相关性：在这项研究中，我们将确定孕妇在怀孕期间暴露于高脂肪饮食是否会增加F1-F4代小鼠患乳腺癌的风险，这是通过诱导PcGTs/TSG的甲基化来实现的。我们还将确定甲基化增加是否与靶向DNA甲基转移酶和多聚体的miRNAs抑制有关。最后，我们将确定外周RNA中miRNAs的抑制是否可以作为子宫内暴露于高雌激素环境的标志。

DESCRIPTION (provided by applicant): Maternal exposure to a high fat (HF) diet during pregnancy increases estrogen receptor (ER+) and ER- mammary cancer risk among female offspring in animal models and in humans. The effect may not be limited to F1 generation daughters: we found that an exposure during pregnancy to a diet containing ethinyl estradiol (EE2) increased mammary cancer risk also in granddaughters (F2 generation) and great granddaughters (F3 generation). Since HF diet increases pregnancy E2 levels, we are proposing to investigate in mice whether maternal exposure to HF diet increases the risk of developing ER+ and/or ER- mammary cancer in F1-F4 generation offspring. In addition, we will investigate whether these transgenerational effects involve changes in DNA methylation. Our preliminary analysis performed using massively parallel sequencing identified 144 "named" genes which were either hypo- or hypermethylated in the mammary glands of F1-F3 generation offspring of EE2 exposed dams, compared to controls. 21% of these genes were polycomb target genes (PcGTs), which in turn included some tumor suppressor genes (TSGs), suggesting that maternal diet during pregnancy, including consumption of a HF diet, may induce methylation of PcTGs/TSGs in the offspring's breast. Interestingly, women at high risk of developing breast cancer exhibit methylation of PcGTs and TSGs. The increase in DNA methylation may be caused by up-regulation of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) and polycombs (EZH2, SUZ12), which we and others have found to occur in the offspring of estrogen exposed dams. Further, up-regulation of DNMTs and polycombs may have been initiated by estrogen-induced suppression of non-coding miRNAs which target them, as seen in MCF-7 human breast cancer cells and mammary glands of rats exposed to EE2 or HF diet in utero (our preliminary data). In this study, we test a hypothesis that maternal exposure to a HF diet during pregnancy induces a transgenerational increase in mammary cancer risk in the F1-F4 generation offspring by inducing DNA methylation of PcTGs/TSGs, via estrogen-induced down-regulation of miRNAs. A causal chain involving estrogen-induced down-regulation of miRNA, up-regulation of DNMTs and polycombs and subsequent methylation of PcGTs/TSGs, leading to increased mammary cancer in F1-F4 generation offspring, will be investigated by treating F1-F4 generation mice with histone deacetylase (HDAC) and DNMT inhibitors. Our preliminary study indicates that an exposure to HDAC+DNMT inhibitors during adult life completely reverses the increase in mammary cancer risk in in utero estrogen exposed mice, but whether these exposures reverse increased DNA methylation and increased mammary tumorigenesis in F2-F4 generations of estrogen exposed dams, is not known. Finally, as there is currently no way of knowing who might have been exposed to high in utero estrogenic environment, we will study whether these individuals can be identified by determining E2/ER regulated miRNA profile in the peripheral blood. PUBLIC HEALTH RELEVANCE: In this study, we will determine whether maternal exposure to a high fat diet during pregnancy, which elevates maternal estradiol levels, increases mammary cancer risk in F1-F4 generation mouse offspring by inducing methylation of PcGTs/TSGs. We also will determine whether increased methylation is associated with suppression of miRNAs which target DNA methyltransferases and polycombs. Finally, we will determine whether suppression of miRNAs in the peripheral RNA serves as a marker of having been exposed to elevated estrogenic environment in utero.