High throughput assays for modulators of splicing switches during the EMT

EMT 期间拼接开关调制器的高通量测定

• 批准号: 8181147
• 负责人: RUSS Paul CARSTENS
• 金额: $ 16万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8181147
• 关键词: Affect; Alternative Splicing; Benchmarking; Biological Assay; Biological Markers; Cell Line; Cell Polarity; Cell-Cell Adhesion; Cells; Chemicals; Development; Distant; Drug Compounding; Ensure; Epithelial; Epithelial Cells; Event; Exons; Fibroblast Growth Factor Receptor 2; Funding Mechanisms; Gene Expression; Global Change; Lead; Libraries; Luciferases; Mesenchymal; Molecular Bank; Neoplasm Metastasis; Pathway interactions; Phase; Play; Process; Production; Property; RNA Splicing; Reporter; Role; Screening procedure; Signal Pathway; Site; Testing; Therapeutic; Tissues; Work; base; cDNA Expression; cancer cell; cancer therapy; cell behavior; cell type; epithelial to mesenchymal transition; genome-wide; high throughput screening; inhibitor/antagonist; innovation; meetings; next generation; novel; prevent; programs; small molecule; tumor

DESCRIPTION (provided by applicant): The epithelial-mesenchymal transition (EMT) is characterized by the loss of cell-cell adhesion and cell polarity in epithelial cells and the acquisition of motile and invasive properties. While essential for development, the EMT is one mechanism by which tumors can acquire the capability to undergo tissue invasion and metastasis. It is therefore important to identify novel therapies that can inhibit the EMT, but few assays for EMT inhibitors in high throughput screens (HTS) have developed. A change in fibroblast growth factor receptor 2 (FGFR2) splicing occurs during the EMT and using an innovative luciferase-based splicing reporter assay we previously carried out a genome-wide high throughput cDNA expression screen for regulators of this splicing switch. This screen identified the epithelial cell type specific splicing regulators ESRP1 and ESRP2 demonstrating the feasibility of cell-based splicing assays in high throughput, array-based screens. An extensive set of ESRP-regulated exons switch splicing during the EMT, indicating that global changes in alternative splicing occur during this process. A change in this splicing network is a thus a dynamic feature of the EMT and changes in splicing of ESRP-regulated targets can be used as a biomarker for the EMT. In this application we will develop more robust next generation splicing reporter assays using ESRP- regulated exons that undergo profound "switch-like" changes in splicing and configure them for HTS assays using the Molecular Libraries Production Centers Network (MLPCN). In Aim 1, we will adapt existing minigene reporters containing ESRP regulated exons and flanking intronic regulatory sequences for HTS in the context of our established luciferase-based reporter minigenes. The reporters will include exons whose inclusion is activated as well as those that undergo skipping during the EMT. Additional reporters will also be developed for use in counter-screens to prioritize HTS hits. In Aim 2, these screens will be configured for screening in 384 well format and pilot screens will be carried out using several small compound libraries as well as several previously described compounds that have been shown to function as general modulators of splicing. These compounds will be screened in mesenchymal cells for splicing changes indicative of the reverse process of mesenchymal to epithelial transition (MET) and in epithelial cells for inhibition or reversal of an inducible EMT. Successful completion of this pilot phase of this funding mechanism (PAR-10-182) in year one will enable us to submit these assays for the larger scale screening phase using the MLPCN library of compounds. Such screens hold great promise to yield novel small molecule regulators of splicing, including a subset that broadly promote epithelial-specific splicing pathways to inhibit or reverse the EMT and block cancer metastasis. Such compounds will potentially include those that affect signaling pathways or other upstream events that might potently activate broad transcriptional and post-transcriptional gene expression programs that inhibit the EMT. PUBLIC HEALTH RELEVANCE: The epithelial to mesenchymal transition (EMT) is the process by which cancer cells can escape from the primary site and metastasize to distant sites and is therefore a target for novel cancer therapies. We have identified regulators of alternative splicing that control an epithelial splicing network that is lost during the EMT, suggesting that a mesenchymal splicing program can promote the EMT and that these splicing changes serve as biomarkers for this process. The current application will use innovative splicing assays to carry out screens for novel compounds that inhibit this splicing transition and thereby identify lead compounds for drugs to prevent tumor metastasis.

描述（由申请人提供）：上皮 - 间质转变（EMT）的特征是上皮细胞中细胞 - 细胞粘附和细胞极性的丧失以及动力和入侵性质的获取。虽然对发育至关重要，但EMT是一种机制，肿瘤可以通过该机制获得组织入侵和转移的能力。因此，重要的是要鉴定可以抑制EMT的新型疗法，但是在高吞吐量筛选（HTS）中，EMT抑制剂的测定很少。成纤维细胞生长因子受体2（FGFR2）的变化发生在EMT期间，并使用基于创新的荧光素酶的剪接报告报告基准测定器，我们以前对此跨基因组进行了跨基因组高吞吐量cDNA表达筛选。该屏幕确定了上皮细胞类型特异性剪接调节器ESRP1和ESRP2，证明了基于高吞吐量的基于阵列的屏幕的基于细胞的剪接测定的可行性。在EMT期间，一组广泛的ESRP调节的外显子开关剪接，表明在此过程中发生了替代剪接的全局变化。因此，该剪接网络的变化是EMT的动态特征，ESRP调节目标的剪接变化可以用作EMT的生物标志物。在此应用程序中，我们将使用ESRP调节的外显子开发更健壮的下一代剪接报告基因测定，这些外显子使用分子库生产中心网络（MLPCN）进行剪接的深刻“开关样”变化，并将其配置为HTS分析。在AIM 1中，我们将在我们既定的基于荧光素酶的记者小型烯的背景下，适应含有ESRP调控外显子和内含子调节序列的现有微型记者。记者将包括激活包含的外显子，以及在EMT期间进行跳过的外显子。还将开发其他记者，以在反屏幕中使用，以优先考虑HTS命中率。在AIM 2中，这些屏幕将被配置为以384井格式进行筛选，并将使用几个小型化合物库以及几种先前描述的化合物进行筛选，这些化合物已被证明是拼接的一般调节剂。这些化合物将在间充质细胞中筛选，以进行剪接变化，以表明间质向上皮转变（MET）和上皮细胞的反向过程，以抑制或反转诱导EMT。在第一年，该资金机制（PAR-10-182）的这个试点阶段的成功完成将使我们能够使用MLPCN化合物库提交大规模筛查阶段的这些测定法。这样的筛选巨大的希望可以产生新型的小分子调节剂，其中包括广泛促进上皮特异性剪接途径的子集，以抑制或逆转EMT并阻止癌症转移。这样的化合物可能包括影响信号通路或其他上游事件的化合物，这些事件可能会有效激活抑制EMT的广泛转录和转录后基因表达程序。 公共卫生相关性：间充质转变（EMT）的上皮是癌细胞从主要部位逃脱并转移到远处的过程，因此是新型癌症治疗的靶标。我们已经确定了控制EMT期间丢失的上皮剪接网络的替代剪接的调节剂，这表明间充质剪接程序可以促进EMT，并且这些剪接变化是该过程的生物标志物。当前的应用将使用创新的剪接测定法对抑制这种剪接过渡的新型化合物进行筛选，从而鉴定药物的铅化合物以防止肿瘤转移。