Structure-dependent activation of NR4A2 (Nurr1) by 1,1-bis(3′-indolyl)-1-(aromatic)methane analogs in pancreatic cancer cells

Abstract

NR4A2 (Nurr1) is an orphan nuclear receptor with no known endogenous ligands and is highly expressed in many cancer cell lines including Panc1 and Panc28 pancreatic cancer cells. Structure-dependent activation of NR4A2 by a series of 1,1-bis(3′-indolyl)-1-(aromatic)methane (C-DIM) analogs was determined in pancreatic cancer cells transfected with yeast GAL4-Nurr1 chimeras and a UASx5-luc reporter gene or constructs containing response elements that bind NR4A2. Among 23 different structural analogs, phenyl groups containing p-substituted trifluoromethyl, t-butyl, cyano, bromo, iodo and trifluoromethoxy groups were the most active compounds in transactivation assay. The p-bromophenyl analog (DIM-C-pPhBr) was used as a model for structure–activity studies among a series of ortho-, meta- and para-bromophenyl isomers and the corresponding indole 2- and N-methyl analogs. Results show that NR4A2 activation was maximal with the p-bromophenyl analog and methylation of the indole NH group abrogated activity. Moreover, using GAL4-Nurr1 (full length) or GAL-Nurr1-A/B and GAL4-Nurr1-(C-F) chimeras expressing N- and C-terminal domains of Nurr1, respectively, DIM-C-pPhBr activated all three constructs and these responses were differentially affected by kinase inhibitors. DIM-C-pPhBr also modulated expression of several Nurr1-regulated genes in pancreatic cancer cells including vasoactive intestinal peptide (VIP), and the immunohistochemical and western blot analyses indicated that DIM-C-pPhBr activates nuclear NR4A2.

Introduction

The nuclear receptor (NR) superfamily of transcription factors are characterized by their structural homology which includes N- and C-terminal domains (A/B and E/F, respectively), a DNA binding domain (C), and an adjacent hinge region (D) [1], [2], [3]. Both N- and C-terminal regions may contain activation functions (AFs), and the ligand binding domain (LBD) resides in the C-terminus of NRs [1], [2], [3], [4]. The 48 members of the NR superfamily can be subdivided into three broad categories, namely, the endocrine nuclear receptors, adopted orphan receptors, and orphan receptors such as NR4A for which cognate ligands have not yet been identified [1], [2], [3], [4]. The NR4A orphan receptor subfamily includes NR4A1 (Nur77, NGFI-B, TR3), NR4A2 (Nurr1, NOT), and NR4A3 (Nor-1, MINOR) [5], [6], [7], [8], and it has been suggested that the failure to identify an endogenous ligand may be due to the lack of a typical NR ligand binding pocket in the LBD domain of NR4A receptors [9].

NR4A receptors are immediate-early genes that are induced by diverse stimuli in multiple tissues, and there is increasing evidence that these receptors play important roles in maintaining tissue homeostasis and in pathophysiological processes including cancer [5], [6], [7], [8]. NR4A receptors have specific functions in the brain, T-cells/thymocytes, adipose tissue, steroidgenesis, muscle, blood vessels, macrophages, and cardiovascular system; however, the role of NR4A receptors in carcinogenesis is less well understood [7], [8]. Knockdown of NR4A1 by RNA interference in cancer cell lines induced apoptosis or inhibited growth in multiple cell lines (reviewed in [8]) and NR4A2 knockdown also induced apoptosis and decreased metastasis in cancer cells lines [10], [11], [12].

The effects and mechanisms of action of drug-induced activation or deactivation of NR4A receptors are complex and dependent on both cell context and structure. Studies with phorbol esters, retinoids and other apoptosis-inducing agents have unraveled a novel NR4A1-dependent proapoptotic pathway that involves nuclear export of the receptor which in some cell lines form a mitochondrial proapoptotic bcl-2–NR4A1 complex [13], [14]. Studies in this laboratory have demonstrated that among 1,1-bis(3′-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs), the p-methoxy (DIM-C-pPhOCH₃) and p-hydroxy (DIM-C-pPhOH) derivatives induce nuclear NR4A1-dependent apoptosis and growth inhibition of colon, pancreatic and bladder cancer cells through activation or deactivation of the receptor [15], [16], [17], [18]. Cytosporone B and related compounds bind directly the receptor and appear to activate nuclear NR4A1-mediated transcription; these receptor agonists also induce nuclear NR4A1 export [19], [20].

6-Mercaptopurine (6-MP) activates NR4A2 in CV1 and HEK293 cells through the N-terminal A/B domain and this pathway involves metabolic activation of 6-MP [21]. Several benzimidazoles also induce NR4A2-dependent transactivation, and a p-chloro-substituted C-DIM analog (DIM-C-pPhCI) also activates Nurr1 in bladder cancer cells [22], [23]. In this study, we have investigated the structure-dependent activation of NR4A2 in pancreatic cancer cells by twenty-three C-DIM analogs containing various p-substituted phenyl and heteroaromatic substituents and their activity was compared to 6-MP. Among the most active compounds were the p-trifluoromethyl (DIM-C-pPhCF₃), p-bromo (DIM-C-pPhBr), p-t-butyl (DIM-C-pPhtBu), p-cyano (DIM-C-pPhCN), p-iodo (DIM-C-pPhI), and p-trifluoromethoxy (DIM-C-phOCF₃) analogs. Using one or more NR4A2-active C-DIMs as models, these compounds also induced transactivation in cells transfected with constructs containing three copies of an NGFI-B response element (NBRE₃-luc) and three copies of a Nur response element (NuRE_x3-luc). Nurr1-active C-DIMs also activated a wild-type GAL4-Nurr1 variant and GAL4-Nurr1-(A/B) and GAL4-Nurr1-(C-F) chimeras containing N- and C-terminal regions of NR4A2, respectively, and the prototypical model NR4A2 activator DIM-C-pPhBr induced expression of several NR4A2-dependent genes in Panc1 and Panc28 that were confirmed by RNA interference. These studies demonstrate that the C-DIM structure is an excellent scaffold for developing NR4A2-active compounds.