Examining the cell cycle-dependence of progesterone receptor activity

描述（由申请人提供）：最近的研究表明孕激素在乳腺癌的病因学中起作用;然而，孕激素促进肿瘤形成/进展的机制尚未确定。孕激素的作用是由孕激素受体（PR）介导的，它已被证明介导孕激素在乳腺癌细胞系中的生长刺激和生长抑制作用。这些观察结果强调了乳腺癌中PR信号的复杂性，以及进一步研究PR功能调节的必要性。乳腺癌细胞中PR靶基因表达的研究通常是在血清条件下生长的细胞中进行的，这些细胞在G1期将其捕获，我们以前的研究表明，PR活性可能在细胞周期的各个阶段具有独特的活性，可能是通过差异共调节因子的募集。我们以前的研究也表明，PR的核转位在细胞周期中是不同的，与G2/M相比，S期的转位量更大。这一观察结果表明，不需要PR与DNA结合的快速信号传导可能在G1或G2/M中最高，其中在细胞质中发现残留的PR。此外，我们的研究表明，细胞周期蛋白A/Cdk 2/Cdk 1活性在S期基因表达中起作用。我将通过以下方法确定PR活性是否随细胞周期而不同：1）使用微阵列鉴定在细胞周期中差异表达的基因，2）评估PR诱导的快速信号传导是否是细胞周期依赖性的，3）使用siRNA或使用Cdk 1/2抑制剂耗尽细胞周期蛋白A2，以询问S期基因是否优先对细胞周期蛋白A/Cdk活性敏感。我还将利用新的，高分辨率的显微镜分析，1）进行免疫荧光筛选，以确定新的PR辅助调节，2）确定这些辅助调节的招聘是否不同的细胞周期的功能，和3）检查细胞信号转导和Cdk依赖性途径的影响，对辅助调节招聘使用MAPK和Cdk抑制剂。总的来说，这些研究将有助于我们了解PR活动的调节。了解PR在乳腺癌中的调节方式很重要，因为PR通过调节靶基因表达直接介导乳腺癌细胞增殖，并通过与其他促有丝分裂细胞信号通路的相互作用间接介导乳腺癌细胞增殖。对PR活性所必需的因素的研究可能最终揭示乳腺癌的潜在治疗靶点。

DESCRIPTION (provided by applicant): Recent studies have suggested that progestins play a role in the etiology of breast cancer; however, the mechanisms by which progestins promote tumor formation/progression have not been defined. Progestin action is mediated by the progesterone receptor (PR), which has been shown to mediate both growth stimulatory and growth inhibitory effects of progestins in breast cancer cell lines. These observations highlight the complexity of PR signaling in breast cancer and the need for further study of the regulation of PR function. Studies of PR target gene expression in breast cancer cells are typically performed with cells grown under serum conditions that arrest them in G1, and our previous studies suggest that PR activity is likely to have unique activities in the various phases of the cell cycle, possibly through differential co-regulator recruitment. Our previous studies have also shown that nuclear translocation of PR is different across the cell cycle, with a greater amount of translocation in S-phase, compared to G2/M. This observation suggests that rapid signaling, which does not require PR binding to DNA, might be highest in G1 or G2/M where residual PR is found in the cytoplasm. Furthermore, our studies suggest that cyclin A/Cdk2/Cdk1 activity plays a role in S-phase gene expression. I will determine whether PR activity differs as a function of cell cycle by 1) using microarrays to identify genes that are differentially expressed across the cell cycle, 2) assessing whether PR-induced rapid signaling is cell cycle-dependent and 3) depleting cyclin A2 using siRNA or by using Cdk1/2 inhibitors to ask whether S-phase genes are preferentially sensitive to cyclin A/Cdk activity. I will also utilize novel, high througput microscopy analyses to 1) perform an immunofluorescence screen to identify novel PR co-regulators, 2) determine whether recruitment of these co-regulators differs as a function of cell cycle, and 3) examine the effects of cell signaling and Cdk-dependent pathways on co-regulator recruitment using MAPK and Cdk inhibitors. Collectively, these studies will contribute to our knowledge of the regulation of PR activity. Understanding how PR is regulated in breast cancer is important because PR mediates breast cancer cell proliferation directly by regulating target gene expression and indirectly through cross-talk with other mitogenic cell signaling pathways. Investigation of the factors necessary for PR activity may eventually unveil potential therapeutic targets for breast cancer.