NUR77 Agonists: New Targets for Pancreatic Cancer Chemotherapy

描述(申请人提供)：神经生长因子I-B(NGFI-B)孤儿受体家族包括NGFI-B？(Nur77，TR3)，NGFI-B？(Nurr1)和NGFI-B？(Nor1)，NGFI-B蛋白的配体尚未见报道。凋亡/分化诱导剂通过核(反式激活)途径或通过核-线粒体或核-胞质易位激活Nur77依赖的细胞凋亡。我们现在首次在反式激活实验中鉴定出至少三个化合物能激活野生型Nur77，而这种反应需要Nur77的配体结合域(LBD)。NUR77活性1，1-bis(3‘-indolyl)-1-(p-substitutedphenyl)methanes含有CF3(dim-C-pPhCF3)、H(dim-C-Ph)和OCH3(dim-C-pPhOCH3)取代基。这些C-取代的二吲哚甲烷(DIMS)通过核途径诱导胰腺癌细胞的凋亡，我们假设C-取代的DIMS代表一类独特的配体，激活Nur77并诱导胰腺癌细胞的凋亡。在目标1中，将在六个表达Nur77蛋白的胰腺癌细胞系中确定一系列C-取代DIMS的环和苯基取代的衍生物对Nur77的结构依赖性激活。最具活性的化合物(至少3个)将被用作典型的Nur77激动剂，用于在凝胶迁移率改变分析中研究配体依赖的Nur77-DNA结合，以及配体诱导的Nur77-辅活化子相互作用和增强的反式激活。目的2将重点介绍Nur77激动剂诱导细胞凋亡的途径，并将这些反应与部分胰腺癌细胞系中观察到的诱导凋亡的药物(TPA、丁酸盐和类维A酸类CD437)的反应进行比较。这种方法将解决以前报告中的明显差异，并区分配体激活的Nur77(核)和核外诱导细胞凋亡的途径。此外，还将探讨Nur77激动剂诱导促凋亡基因表达的分子机制。在目标3中，将测定Nur77激动剂的体内抗肿瘤活性。这项拟议的研究将确定通过选定的C-取代DIMS激活Nur77的作用机制，这些DIMS是这种孤儿受体的第一个已知配体。这些研究将有助于强调选择性Nur77调节剂作为一类治疗胰腺癌的新型化疗药物的潜在作用。

DESCRIPTION (provided by applicant): The nerve growth factor I-B (NGFI-B) family of orphan receptors include NGFI-B? (Nur77, TR3), NGFI-B? (Nurr1), and NGFI-B? (Nor1), and ligands for NGFI-B proteins have not been reported. Apoptosis/differentiation inducing agents activate Nur77-dependent apoptosis via nuclear (transactivation) pathways or by nuclear-mitochondrial or nuclear-cytosolic translocation of Nur77. We have now identified for the first time at least three compounds that activate wild-type Nur77 in transactivation assays, and this response requires the ligand binding domain (LBD) of Nur77. The Nur77-active 1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes contain CF3 (DIM-C-pPhCF3), H (DIM-C-Ph), and OCH3 (DIM-C-pPhOCH3) substituents. These C-substituted diindolylmethanes (DIMs) induce apoptosis in pancreatic cancer cells through a nuclear pathway, and we hypothesize that C-substituted DIMs represent a unique class of ligands that activate Nur77 and induce apoptosis in pancreatic cancer cells. In Aim 1, the structure-dependent activation of Nur77 by a series of ring- and phenyl-substituted derivatives of C-substituted DIMs will be determined in six pancreatic cancer cell lines that express Nur77 protein. The most active compounds (at least 3) will be used as prototypical Nur77 agonists for investigating ligand-dependent Nur77-DNA binding in electrophoretic mobility shift assay and ligand-induced Nur77-coactivator interactions and enhanced transactivation. Aim 2 will focus on induction of apoptotic pathways by Nur77 agonists and compare these responses to those observed for apoptotic inducing agents (TPA, butyrate and the retinoid CD437) in selected pancreatic cancer cell lines. This approach will resolve apparent differences in previous reports and distinguish between ligand-activated Nur77 (nuclear) vs. extranuclear pathways for induction of apoptosis. Moreover, the molecular mechanisms of Nur77 agonist-induced expression of pro-apoptotic genes will also be investigated. In Aim 3, the in vivo antitumorigenic activity of Nur77 agonists will be determined. The proposed studies will define the mechanism of action of Nur77 activation by selected C-substituted DIMs that are the first known ligands for this orphan receptor. These studies will serve to highlight the potential role of selective Nur77 modulators as a new class of chemotherapeutic drugs for treatment of pancreatic cancer.