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. 2014 Apr;12(4):527-538.
doi: 10.1158/1541-7786.MCR-13-0567. Epub 2014 Feb 10.

The orphan nuclear receptor NR4A1 (Nur77) regulates oxidative and endoplasmic reticulum stress in pancreatic cancer cells

Syng-Ook Lee #  1   2 , Un-Ho Jin #  1 , Jeong Han Kang  3 , Sang Bae Kim  4 , Aaron S Guthrie  5 , Sandeep Sreevalsan  6 , Ju-Seog Lee  4 , Stephen Safe  1   6
Affiliations

The orphan nuclear receptor NR4A1 (Nur77) regulates oxidative and endoplasmic reticulum stress in pancreatic cancer cells

Syng-Ook Lee et al. Mol Cancer Res. 2014 Apr.

Abstract

NR4A1 (Nur77, TR3) is an orphan nuclear receptor that is overexpressed in pancreatic cancer and exhibits pro-oncogenic activity. RNA interference of NR4A1 expression in Panc-1 cells induced apoptosis and subsequent proteomic analysis revealed the induction of several markers of endoplasmic reticulum stress, including glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), and activating transcription factor-4 (ATF-4). Treatment of pancreatic cancer cells with the NR4A1 antagonist 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) gave similar results. Moreover, both NR4A1 knockdown and DIM-C-pPhOH induced reactive oxygen species (ROS), and induction of ROS and endoplasmic reticulum stress by these agents was attenuated after cotreatment with antioxidants. Manipulation of NR4A1 expression coupled with gene expression profiling identified a number of ROS metabolism transcripts regulated by NR4A1. Knockdown of one of these transcripts, thioredoxin domain containing 5 (TXNDC5), recapitulated the elevated ROS and endoplasmic reticulum stress; thus, demonstrating that NR4A1 regulates levels of endoplasmic reticulum stress and ROS in pancreatic cancer cells to facilitate cell proliferation and survival. Finally, inactivation of NR4A1 by knockdown or DIM-C-pPhOH decreased TXNDC5, resulting in activation of the ROS/endoplasmic reticulum stress and proapoptotic pathways.

Implications: The NR4A1 receptor is pro-oncogenic, regulates the ROS/endoplasmic reticulum stress pathways, and inactivation of the receptor represents a novel pathway for inducing cell death in pancreatic cancer.

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Figures

Figure 1
Figure 1. 2D-PAGE gels showing differentially expressed proteins in Panc-1 cells transfected with siNR4A1 and functional classification of the proteins
Cells were transfected with either siScr or siNR4A1 for 60 hr, and whole cell lysates (400 μg of protein) were prepared, and the pH gradient in the first dimension was from 3 to 10. The second dimension was a 12% acrylamide SDS-PAGE. Gels were stained by colloidal staining with Coomassie blue G250 (A). Arrows point to the proteins of interest, and the numbers assigned to the spots correspond to the numbers listed in Supplemental Table 2. Functional distribution (B) and canonical pathway (C) of the 38 identified proteins. Assignments were made based on information from the NCBI, the Swiss-Prot/TrEMBL Protein Knowledge Base, and the Ingenuity Pathways Knowledge Base. Enlarged images of protein spots for which image analysis software reported ≥2-fold differences in accumulation in siNR4A1-transfected cells are shown (D, left panel). Panc-1 cells were transfected with either siScr or siNR4A1 for 60 hr, and whole cell lysates were analyzed by western blot analysis (D, right panel).
Figure 2
Figure 2. Knockdown of NR4A1 induces ER stress and apoptosis in multiple cancer cell lines
(A) Panc-1 cells were transfected with either siScr or siNR4A1, and TEM analysis was performed at 60 hr after transfection. The structure of the ER (arrows) in cells transfected with siNR4A1 showed morphological disorders. (B, C, and D) Panc-1, L3.6pL, MCF-7/RKO cells, respectively, were transfected with either siScr or siNR4A1 for 72 hr, and whole cell lysates were analyzed by western blot analysis. β-Actin was used as a loading control.
Figure 3
Figure 3. Knockdown of NR4A1 induces ER stress-mediated apoptosis by increasing ROS production in pancreatic cancer cells
(A) Panc-1 cells were transfected with each indicated siRNA for 72 hr, and whole cell lysates were analyzed by western blot analysis. (B-D) Cells were transfected with siScr or siNR4A1 for 6 hr. At 60 hr after transfection, ROS production was measured by the oxidation of non-fluorescent DCFH-DA to fluorescent DCF using a fluorescence plate reader (B). #P<0.001 vs. siScr without reduced glutathione (GSH). At 24 hr after transfection, the cells were treated with GSH for an additional 48 hr and whole cell lysates were analyzed by western blot analysis (C and D). β-Actin was used as a loading control. The multiple lanes represent lysates from different experiments
Figure 4
Figure 4. DIM-C-pPhOH, the NR4A1 inactivator, induces ER stress-mediated apoptosis by increasing ROS production in Panc-1 cells
(A) Cells were treated with either DMSO or DIM-C-pPhOH for 24 hr. (B) Cells were transfected with either Ad-Null or Ad-NR4A1 for 12 hr, and treated with DIM-C-pPhOH (25 μM) for another 24 hr. Whole cell lysates were analyzed by western blot analysis. (NR4A1 protein levels were induced 4-fold by Ad-NR4A1). (C) Cells were treated with DMSO or DIM-C-pPhOH for 18 hr, and ROS production was measured by the oxidation of non-fluorescent DCFH-DA to fluorescent DCF using either flow cytometric analysis (left panel) or a fluorescence plate reader (right panel). (D) Cells were treated with DMSO or DIM-C-pPhOH in the presence or absence of GSH for 24 hr and whole cell lysates were analyzed by western blot analysis. β-Actin was used as a loading control.
Figure 5
Figure 5. TXNDC5 is a novel NR4A1-regulated gene involved in ER stress-mediated apoptosis
(A) Heat map of genes including ROS metabolism genes regulated by siNR4A1 in Panc-1 cells (left panel). Each cell in the matrix represents the expression level of a gene feature. Red and green reflect relatively high and low expression levels of genes, respectively, as indicated in the scale bar (a log2-transformed scale). (A, right panel and B) Panc-1 cells were transfected with each indicated siRNA for 48 hr and mRNA levels were determined by real-time PCR, as described in the Materials and Methods. TATA-binding protein (TBP) was used as an internal control and mRNA levels are presented as means with s.d. of three experiments. *P<0.05 and #P<0.001 vs. siScr. (C) Panc-1 cells were transfected with each indicated siRNA for 72 hr (left panel) for western blot analysis or 60 hr (right panel) for measurement of ROS. (D) Panc-1 cells were transfected with siScr or siTXNDC5 for 6 hr. At 60 hr after transfection, ROS production was measured by the oxidation of non-fluorescent DCFH-DA to fluorescent DCF using a fluorescence plate reader (left panel). #P<0.001 vs. siScr without GSH. At 24 hr after transfection, the cells were treated with GSH for an additional 48 hr and whole cell lysates were analyzed by western blot analysis (right panel). β-Actin was used as a loading control and the two lanes for each siRNA represent different experiments.
Figure 6
Figure 6. Regulation of TXNDC5 transcriptional activity by NR4A1
(A) Panc-1 cell swere transfected with siNR4A1 for 72 hr (left panel) or treated with DIM-C-pPhOH for 24 hr (middle and right panel), and TXNDC5 protein and mRNA levels were determined by western blot analysis and real-time PCR, respectively. (B, left panel) A putative NBRE in the TXNDC5 promoter. (B, middle panel) Cells were cotransfected with each siRNA and pTXVDC5-Luc (−1444/+25), and luciferase activity (relative to β-galactosidase activity) was determined. (B, right panel) Panc-1 cells were transfected with pTXVDC5-Luc (−1444/+25) for 4 hr and treated with DIM-C-pPhOH for another 18 hr. Luciferase activity (relative to β-galactosidase activity) was determined, and the corresponding empty vector was used as a control. (C, left panel) ChIP assay. Panc-1 cells were treated with DIM-C-pPhOH for 6 hr, and the ChIP assay was performed as described in the Materials and Methods. (C, right panel) DNA binding assay. Nuclear extracts of Panc-1 cells were tested for NR4A1-DNA binding activity as described in Materials and Methods. (D) Schematic diagram illustrating induction of apoptosis by inactivation of NR4A1.
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