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

描述（由申请方提供）：在动物模型和人类中，妊娠期间母体暴露于高脂肪（HF）饮食会增加雌性后代的雌激素受体（ER+）和ER-乳腺癌风险。这种影响可能不仅限于F1代女儿：我们发现，在怀孕期间暴露于含有炔雌醇（EE 2）的饮食也增加了孙女（F2代）和曾孙女（F3代）的乳腺癌风险。由于HF饮食会增加妊娠E2水平，因此我们建议在小鼠中研究母体暴露于HF饮食是否会增加F1-F4代后代发生ER+和/或ER-乳腺癌的风险。此外，我们将研究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确定了144个“命名”基因，与对照组相比，这些基因在EE 2暴露母鼠的F1-F3代后代乳腺中甲基化程度较低或较高。这些基因中有21%是多梳靶基因（PcGT），这反过来又包括一些肿瘤抑制基因（TSG），这表明怀孕期间的母亲饮食，包括HF饮食的消费，可能会诱导后代乳房中PcTG/TSG的甲基化。有趣的是，患乳腺癌风险高的女性表现出PcGT和TSG的甲基化。DNA甲基化的增加可能是由DNA甲基转移酶（DNMT 1，DNMT 3a和DNMT 3b）和polycomb（EZH 2，SUZ 12）的上调引起的，我们和其他人已经发现这发生在雌激素暴露的母鼠的后代中。此外，DNMT和polycomb的上调可能是由雌激素诱导的对靶向它们的非编码miRNA的抑制引发的，如在MCF-7人乳腺癌细胞和子宫内暴露于EE 2或HF饮食的大鼠乳腺中所见（我们的初步数据）。在这项研究中，我们测试了一个假设，即母亲暴露于 怀孕期间的HF饮食通过雌激素诱导的miRNA下调诱导PcTG/TSG的DNA甲基化而诱导F1-F4代后代中乳腺癌风险的跨代增加。将通过用组蛋白脱乙酰酶（HDAC）和DNMT抑制剂处理F1-F4代小鼠来研究涉及雌激素诱导的miRNA下调、DNMT和polycomb上调以及随后的PcGT/TSG甲基化的因果链，其导致F1-F4代后代中乳腺癌增加。我们的初步研究表明，在成年期暴露于HDAC+DNMT抑制剂完全逆转了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雌激素暴露母鼠F2-F4代中DNA甲基化增加和乳腺肿瘤发生增加，尚不清楚。最后，由于目前还没有办法知道谁可能已经暴露于子宫内高雌激素环境，我们将研究是否可以通过确定外周血中E2/ER调节的miRNA谱来识别这些个体。 公共卫生相关性：在这项研究中，我们将确定母亲在怀孕期间暴露于高脂肪饮食，这提高了母亲的雌二醇水平，增加乳腺癌的风险在F1-F4代小鼠后代通过诱导甲基化的PcGT/TSGs。我们还将确定甲基化的增加是否与靶向DNA甲基转移酶和polycombs的miRNA的抑制有关。最后，我们将确定外周RNA中miRNA的抑制是否作为子宫内暴露于升高的雌激素环境的标志。

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.