Role of DGAT1 in Mammary Gland Development and Cancer

描述(申请人提供)：我们建议利用缺乏DGAT1的小鼠来分析三酰甘油(TG)代谢在乳腺上皮发育和乳腺癌中的作用。 (Dgat1-/-)，一种从脂肪酸和二酰甘油底物合成TGS的主要酶。在正常的乳腺发育过程中，TGS在分泌乳汁的上皮细胞中合成，并储存在脂肪细胞中，脂肪细胞是乳腺基质中含量最丰富的细胞类型。以脂肪组织中过多的甘油三酯储存为特征的肥胖，正在作为乳腺癌的风险因素进行研究。在动物研究以及人类的一些营养和流行病学研究中，总脂肪的高摄入量与乳腺癌风险的增加有关。然而，这些影响的具体机制在很大程度上是未知的。在Dgat1-/-小鼠中，怀孕期间乳腺上皮细胞的生长和分化受到损害。近年来，在正常乳腺发育和癌症中，乳腺间质在上皮生长中的作用已被证实。乳腺移植实验表明，Dgat1-/-小鼠上皮生长的减少是由间质效应局部介导的。根据初步实验，与野生型相比，将原始乳腺癌细胞注射到Dgat1-/-小鼠的乳房脂肪垫中也可以减少乳腺肿瘤的发生。该方案中的实验将探索DGAT1-/-乳腺间质中脂代谢变化与正常和恶性上皮生长之间的联系机制。我们推测，在没有DGAT1的情况下，乳腺中的脂质代谢产物或其他因素会发生变化，从而损害乳腺上皮细胞发育的信号。我们将使用生化技术来测量Dgat1-/-乳腺的脂质含量和组成。我们还将进行微阵列实验，以分析Dgat1/-乳腺脂肪细胞与野生型相比的基因表达变化。作为识别改变因素的补充方法，将开发一种上皮和脂肪细胞共培养的体外系统。为了探索DGAT1缺陷在乳腺癌中的作用，我们将使用Dgat1-/-小鼠的乳腺癌细胞原位移植，以及Dgat1-/-小鼠与两个乳腺癌发病率增加的转基因小鼠模型之间的杂交。我们将测量肿瘤发生率、肿瘤负荷和形态，并确定DGAT1缺乏的保护作用是否对这些肿瘤模型中的一个具有特异性。最后，基于显示Dgat1/-腹股沟淋巴结中白细胞分布改变的初步数据，我们将检验免疫系统在小鼠肿瘤排斥反应中发挥作用的假设。

DESCRIPTION (provided by applicant): We propose to analyze the role of triacylglycerol (TG) metabolism in mammary epithelial development and breast cancer using mice lacking DGAT1 (Dgat1-/-), a major enzyme that synthesizes TGs from fatty acids and diacylglycerol substrates. In normal mammary gland development, TGs are synthesized in the epithelium for milk secretion and stored in adipocytes, the most abundant cell type in the mammary stroma. Obesity, characterized by excess TG storage in adipose tissue, is under study as a risk factor for breast cancer. High intake of total fat has been associated with increased breast cancer risk in animal studies and in some nutrition and epidemiological studies in humans. However, specific mechanisms for these effects are largely unknown. In Dgat1-/- mice, mammary epithelial growth and differentiation are impaired during pregnancy. In recent years, the role of the mammary stroma in epithelial growth has been established in both normal breast development and cancer. Mammary transplantation experiments showed that the reduction in epithelial growth in Dgat1-/- mice is mediated locally by stromal effects. Based on preliminary experiments, breast tumor development is also reduced when primary breast cancer cells are injected into the mammary fat pad of Dgat1-/- mice compared to wild-types. Experiments in this proposal will explore mechanisms linking alteration of lipid metabolism in Dgat1-/- mammary stroma with both normal and malignant epithelial growth. We hypothesize that lipid metabolites or other factors are altered in mammary gland in the absence of DGAT1 and impair signaling for mammary epithelial development. We will use biochemical techniques to measure the lipid content and composition of Dgat1-/- mammary glands. We will also perform microarray experiments to analyze gene expression changes in Dgat1-/- mammary adipocytes compared to wild-types. As a complementary approach to identify altered factors, an in vitro system of co-culture of epithelium and adipocytes will be developed. To explore effects of DGAT1 deficiency in breast cancer, we will use orthotopic grafts of breast cancer cells in Dgat1-/- mice and crosses between Dgat1-/- mice and two transgenic mouse models with increased incidence of breast tumors. We will measure tumor incidence, tumor load, and morphology, and determine whether the protective effect of DGAT1 deficiency is specific for one of these tumor models. Finally, based on preliminary data showing altered distribution of leukocytes in Dgat1-/- inguinal lymph nodes, we will test the hypothesis that the immune system plays a role in tumor rejection by mice.