Non-genomic actions of estrogen in breast cancer

说明(申请人提供)：雌激素会增加罹患乳腺癌的风险。雌激素的非基因组效应来自于膜上的信号转导，部分原因是ER和EGF受体之间的串扰。基于细胞的系统将证实，通过ERK和PL3激酶阻断E2信号3天实质上阻止了培养的MCF-7和ZR-75-1(ER+)细胞的增殖。G1期细胞周期靶点和GI/S进展与ERK和Pl3K有关。有研究认为，E2与BRCA1相互作用促进乳腺癌的发生。在肝癌-1937细胞(表达突变的BRCA1)中，野生型BRCA1的表达下调了E2激活ERK的能力，而突变的BRCA1的表达没有下调。该模型还将确定wtBRCA1是否能在培养3天的细胞中阻断E2的增殖和ERK诱导作用。我们将通过表达一个仅取消内源性膜ER功能的显性负ER来牵涉膜受体，并将E结构域靶向膜。据推测，E2结合膜上的ER，激活离散的G蛋白，从而激活Src。SRC激活特定的基质金属蛋白酶，释放HB-EGF，反式激活EGFR或ErbB2。这会导致乳腺癌细胞中ERK和PI3K的激活。这将通过使用显性负结构siRNA和缺乏EGFR或ErbB2的细胞来显示。我们认为受体也必须二聚化才能从膜上发出信号，我们将使用二聚体突变体的表达来说明这一点。在体内，针对膜或核的E结构域在ER-乳腺癌细胞中稳定表达。这些细胞被注射到裸鼠体内，并在雌激素重复的条件下进行生长测定。我们的目标是定义雌激素的非基因组作用，这些作用可能在细胞膜上被拮抗，但又能在细胞核内保留E2所希望的效果。

DESCRIPTION (provided by applicant): Estrogen enhances the risk of developing breast cancer. Non-genomic effects of estrogen result from signal transduction originating at the membrane, in part from cross talk between ER and the EGF receptor. Cell based systems will establish that blocking E2-signaling through ERK and Pl3 kinase for 3 days substantially prevents the proliferation of cultured MCF-7 and ZR-75-1 (ER+) cells. G1 cell cycle targets and GI/S progression are ERK and Pl3K entrained. It is proposed that E2 interacts with BRCA1 to promote breast cancer. The ability of E2 to activate ERK will be shown to be down regulated by expression of wild type but not mutant BRCA1 in HCC-1937 cells (express a mutant BRCA1), co-transfected to express ER. This model will also determine whether wtBRCA1 can block the proliferative and ERK-inducing effects of E2, in cells cultured for 3 days. We will implicate the membrane receptor by expressing a dominant negative ER that only abrogates endogenous membrane ER function, and by targeting the E domain to the membrane. It is proposed that E2 binds the membrane ER, activates discrete G proteins that activate Src. Src activates specific matrix metalloproteinases that liberate HB-EGF and transactivates the EGFR or ErbB2. This leads to ERK and PI3K activation in breast cancer cells. This will be shown using dominant negative constructs SiRNA, and cells deficient for EGFR or ErbB2. We propose that the receptor must also dimerize to signal from the membrane, and we will use expression of a dimer mutant to show this. In-vivo, E domain targeted to membrane or nucleus are stably expressed in ER - breast cancer cells. These cells are injected into nude mice, and growth under estrogen-repleted conditions is determined. The goal is to define non-genomic actions of estrogen that could potentially be antagonized at the membrane, yet preserve the desirable effects of E2 in the nucleus.