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-间充质转化（EMT）的特征在于上皮细胞中细胞-细胞粘附和细胞极性的丧失以及获得运动和侵袭特性。虽然对于发育是必不可少的，但EMT是肿瘤可以获得进行组织侵袭和转移的能力的一种机制。因此，重要的是要确定新的疗法，可以抑制EMT，但很少有EMT抑制剂的高通量筛选（HTS）的分析已经开发。成纤维细胞生长因子受体2（FGFR 2）剪接的变化发生在EMT期间，并使用创新的基于内切酶的剪接报告基因测定，我们先前进行了全基因组高通量cDNA表达筛选，用于该剪接开关的调节剂。该筛选鉴定了上皮细胞类型特异性剪接调节剂ESRP 1和ESRP 2，证明了在高通量、基于阵列的筛选中基于细胞的剪接测定的可行性。在EMT过程中，大量的ESRP调节的外显子切换剪接，表明在此过程中发生了选择性剪接的全局变化。因此，这种剪接网络的变化是EMT的动态特征，并且ESRP调节的靶标的剪接变化可以用作EMT的生物标志物。在本申请中，我们将使用ESRP调节的外显子开发更稳健的下一代剪接报告基因测定，所述外显子在剪接中经历深刻的“开关样”变化，并使用分子文库生产中心网络（MLPCN）将其配置用于HTS测定。在目标1中，我们将适应现有的小基因报告含有ESRP调节外显子和侧翼内含子调控序列的HTS在我们建立的基于内切酶的报告小基因的背景下。报告基因将包括其包含被激活的外显子以及在EMT期间经历跳跃的外显子。还将开发更多的报告器，用于计数器屏幕，以优先考虑HTS命中。在目标2中，这些筛选将被配置用于以384孔格式筛选，并且将使用几种小化合物文库以及几种先前描述的化合物进行中试筛选，所述化合物已显示出作为剪接的一般调节剂起作用。将在间充质细胞中筛选这些化合物的剪接变化，其指示间充质向上皮转化（MET）的逆转过程，并在上皮细胞中筛选这些化合物的诱导型EMT的抑制或逆转。在第一年成功完成这一资助机制（PAR-10-182）的试验阶段，将使我们能够使用MLPCN化合物库提交这些测定用于更大规模的筛选阶段。这种筛选很有希望产生新的剪接小分子调节剂，包括广泛促进上皮特异性剪接途径以抑制或逆转EMT并阻断癌症转移的子集。此类化合物将潜在地包括影响信号传导途径或其他上游事件的那些化合物，这些上游事件可能有效地激活抑制EMT的广泛的转录和转录后基因表达程序。 公共卫生关系：上皮间质转化（EMT）是癌细胞从原发部位逃逸并转移到远处的过程，因此是新型癌症治疗的目标。我们已经确定了控制上皮剪接网络的选择性剪接的调节器，该网络在EMT期间丢失，这表明间充质剪接程序可以促进EMT，并且这些剪接变化可以作为该过程的生物标志物。目前的申请将使用创新的剪接测定来筛选抑制这种剪接转变的新型化合物，从而鉴定用于预防肿瘤转移的药物的先导化合物。