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. 2016 Apr 1;22(7):1713-24.
doi: 10.1158/1078-0432.CCR-15-1275. Epub 2015 Nov 18.

Adrenergic Stimulation of DUSP1 Impairs Chemotherapy Response in Ovarian Cancer

Yu Kang  1 , Archana S Nagaraja  2 , Guillermo N Armaiz-Pena  2 , Piotr L Dorniak  2 , Wei Hu  2 , Rajesha Rupaimoole  2 , Tao Liu  2 , Kshipra M Gharpure  2 , Rebecca A Previs  2 , Jean M Hansen  2 , Cristian Rodriguez-Aguayo  3 , Cristina Ivan  4 , Prahlad Ram  5 , Vasudha Sehgal  5 , Gabriel Lopez-Berestein  3 , Susan K Lutgendorf  6 , Steven W Cole  7 , Anil K Sood  8
Affiliations

Adrenergic Stimulation of DUSP1 Impairs Chemotherapy Response in Ovarian Cancer

Yu Kang et al. Clin Cancer Res. .

Abstract

Purpose: Chronic adrenergic activation has been shown to associate with adverse clinical outcomes in cancer patients, but the underlying mechanisms are not well understood. The focus of the current study was to determine the functional and biologic effects of adrenergic pathways on response to chemotherapy in the context of ovarian cancer.

Experimental design: Increased DUSP1 production by sympathetic nervous system mediators (e.g., norepinephrine) was analyzed by real-time quantitative RT-PCR and by Western blotting. In vitro chemotherapy-induced cell apoptosis was examined by flow cytometry. For in vivo therapy, a well-characterized model of chronic stress was used.

Results: Catecholamines significantly inhibited paclitaxel- and cisplatin-induced apoptosis in ovarian cancer cells. Genomic analyses of cells treated with norepinephrine identified DUSP1 as a potential mediator. DUSP1 overexpression resulted in reduced paclitaxel-induced apoptosis in ovarian cancer cells compared with control; conversely, DUSP1 gene silencing resulted in increased apoptosis compared with control cells. DUSP1 gene silencing in vivo significantly enhanced response to paclitaxel and increased apoptosis. In vitro analyses indicated that norepinephrine-induced DUSP1 gene expression was mediated through ADRB2 activation of cAMP-PLC-PKC-CREB signaling, which inhibits JNK-mediated phosphorylation of c-Jun and protects ovarian cancer cells from apoptosis. Moreover, analysis of The Cancer Genome Atlas data showed that increased DUSP1 expression was associated with decreased overall (P= 0.049) and progression-free (P= 0.0005) survival.

Conclusions: These findings provide a new understanding of the mechanisms by which adrenergic pathways can impair response to chemotherapy and have implications for cancer management.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
Catecholamines inhibit chemotherapy-induced apoptosis in ovarian cancer cells. HeyA8 (A and B) and SKOV3ip1 (C and D) cells were treated with the chemotherapeutic agents paclitaxel (A and C) or cisplatin (B and D), alone (IC50) or in combination with the catecholamines norepinephrine (NE; 10µM) or isoproterenol (ISO; 10µM). A beta-blocker (propranolol; 1µM) was administered 30 minutes prior to catecholamine exposure. Apoptosis assays were performed using annexin-V phycoerythrin/7AAD, followed by flow cytometry analysis. Results shown represent mean ± standard error of the mean, indicated by the error bar. *P < 0.05; **P < 0.01 compared with paclitaxel or cisplatin alone.
Figure 2
Figure 2
Catecholamines increase DUSP1 production through ADRB2. A, DUSP1 gene expression in HeyA8 and SKOV3ip1 ovarian cancer cells treated with norepinephrine (NE) compared with untreated cells (*P < 0.01) B, DUSP1 mRNA levels, determined by real-time reverse-transcription polymerase chain reaction in HeyA8 and SKOV3ip1 ovarian cancer cells after exposure to different concentrations (0, 0.1, 1, or 10µM) of NE or isoproterenol (ISO) for different time periods (1, 3, or 6 hours). The mean fold change in DUSP1 mRNA expression compared with control is shown. Error bars represent standard error of the mean. *P < 0.01 compared with vehicle-treated control condition. C, Western blots analysis of DUSP1 protein expression. HeyA8 cells were stimulated with 10µM NE for 3 hours, and protein was obtained from the cell lysate for Western blot analysis using a DUSP1 antibody. The quantification of band intensity relative to β-actin intensity is shown at the bottom. *P < 0.01 compared with the control. Adrenergic signaling plays a role in DUSP1 production. HeyA8 cells were pretreated with receptor-specific agonists or inhibitors and stimulated with norepinephrine (NE) for 3 hours; DUSP1 mRNA expression levels were then examined using real-time reverse-transcription polymerase chain reaction. Data are represented as a percentage of the control (medium only). The relative DUSP1 mRNA level is graphed as the mean fold change in DUSP1 production relative to control. Error bars represent standard error of the mean. D, ADRA1 antagonist prazosin and ADRA2 antagonist yohimbine. E, nonspecific β-adrenergic antagonist propranolol. *P < 0.01 compared with the NE-treated only. F, ADRB1 antagonist atenolol, ADRB2 antagonist ICI118,551, and ADRB3 antagonist SR59230A. *P < 0.01 compared with the NE-treated only. G, ADRB2 and ADRB3 siRNA. *P < 0.01 compared with control siRNA.
Figure 3
Figure 3
Norepinephrine (NE) plays a role in transcriptional control of DUSP1 promoter. A, DUSP1 promoter activity was determined by expression of a luciferase reporter gene in HeyA8 ovarian cancer cells after 3 hours of exposure to NE (1 or 10µM) or an equivalent volume of vehicle control solution. The role of specific β-adrenergic receptors in mediating NE effects on DUSP1 promoter activity was assessed by pretreating cells for 2 hours with 1µM concentrations of the α-adrenergic antagonist phentolamine, the ADRB1-selective antagonist metoprolol, the ADRB2-selective antagonist ICI118,551, or the ADRB3-selective antagonist SR59230A. To determine whether β-adrenergic signaling alone was sufficient to activate the DUSP1 promoter, cells were treated with 1µM concentrations of the nonselective β-agonist isoproterenol, the ADRB1-selective agonist dobutamine, the ADRB2-selective agonist terbutaline, or the ADRB3-selective agonist BRL37344. Values represent the mean ± standard error of 5 independent experiments. *P < 0.05 compared with vehicle-treated control condition. B-E, To identify the specific transcription factor and promoter response element mediating NE induction of the DUSP1 promoter, we conducted systematic mutagenesis of a luciferase reporter construct under control of the 914 bases upstream of the human DUSP1 transcription start site. In panels B, C, *P < 0.05 compared with vehicle-treated control condition; in panel E, *P < 0.05 compared with the full-length promoter construct in the magnitude of NE-induced luciferase activity. F, Transcription factors activated by NE. *P < 0.05 compared with vehicle-treated control condition.
Figure 4
Figure 4
Downstream signaling of ADRB2 is involved in norepinephrine(NE)-induced increases in DUSP1 expression. A, cAMP agonist forskolin. B, PLC inhibitor U73122 and PKC inhibitor staurosporine. C, PKC, CREB1 and SP1 siRNA. D, PI3K inhibitor LY294002 and AKT inhibitor AKT1/2. E, Epac inhibitor Brefeldin A and Epac agonist 8CPT-2Me-cAMP. F, PKA inhibitor KT5720 and H-89. G, MEK inhibitor U0126. H, p38 inhibitor SB203580. Error bars represent standard error of the mean. DUSP1 siRNA inhibits NE -induced dephosphorylation of JNK and c-Jun. I, Western blot analysis of p-JNK (p-JNK1 and p-JNK2) and p-c-Jun, evaluated using the appropriate phospho-specific antibodies. Total JNK and total c-Jun are shown for comparison. These results represent 3 independent experiments. J, Western blot analysis of JNK/c-Jun phosphorylation after 24 hours of treatment with NE and paclitaxel in DUSP1 siRNA-expressing cells.
Figure 5
Figure 5
Effects of chronic restraint stress on ovarian cancer chemosensitivity. One week after being intraperitoneally injected with HeyA8 A, or SKOV3ip1 B, cells, nude mice were subjected to 2 hours of daily restraint stress each morning until the end of the experiment. Mice were randomly assigned to 8 groups (10 mice each, 4 without stress and 4 with stress treated with control, paclitaxel alone, propranolol alone, or paclitaxel with propranolol). Treatment was initiated at 3–4 weeks after injection. Paclitaxel at a dose of 2 mg/kg (SKOV3ip1) or 2.5 mg/kg (HeyA8) was given intraperitoneally weekly; propranolol at a dose of 2 mg/kg was given intraperitoneally every day. At the end of the study, mice were killed and their tumors were harvested. Tumor weights (A and B, left) and tumor nodules (A and B, right) were quantified in the HeyA8 and SKOV3ip1 models. Immunohistochemical staining of tumor samples from the SKOV3ip1 model showing the effects of chronic restraint stress on cell apoptosis (C), proliferation (D), DUSP1 (E), and JNK phosphorylation (F). All photographs were taken at 200× magnification. The bars in the graphs correspond sequentially to the labeled columns of the images at left. Error bars represent standard error of the mean. *P < 0.05; **P < 0.01 compared with the control.
Figure 6
Figure 6
DUSP1 plays a role in mediating stress-induced chemoresistance. A, Analysis of apoptosis in SKOV3ip1 cells overexpressing DUSP1 in no-stress conditions. *P < 0.05 compared with the SKOV3ip1-EV group (cells transfected with pCMV6-entry vector). Error bars represent standard error of the mean. B, Analysis of apoptosis in HeyA8 cells expressing DUSP1 siRNA, treated with norepinephrine (NE) and paclitaxel. *P < 0.05 compared with the control siRNA, NE, and paclitaxel group. Error bars represent standard error of the mean. C and D, Effects of DUSP1 silencing on stress-mediated tumor growth. Nude mice were subjected to 2 hours of daily restraint stress each morning for 3–4 weeks using HeyA8 ovarian cancer models. Mice (n = 10 per group) were injected subcutaneously with HeyA8 cells and randomly assigned to 1 of 4 groups: (1) control siRNA twice weekly; (2) control siRNA twice weekly and intraperitoneal paclitaxel weekly; (3) DUSP1 siRNA twice weekly; or (4) DUSP1 siRNA twice weekly and intraperitoneal paclitaxel weekly. After 3–4 weeks of treatment, mice were killed and tumor weights and number of nodules were recorded. Mean tumor weight (C) and mean number of nodules (D) are shown in the graphs. *P < 0.05 compared with control siRNA and paclitaxel. Error bars represent standard error of the mean. E, Immunohistochemical analysis of cleaved caspase 3 staining in a sample of tumor tissue from each group. All photographs were taken at 200× magnification. **P < 0.01 compared with control siRNA and paclitaxel. Error bars represent standard error of the mean. Overall survival (F) and progression-free survival (G): Kaplan-Meier curves for ovarian cancer patients with high and low DUSP1 expression is shown. Median survival durations for each group are shown. Data compiled from The Cancer Genome Atlas Project. H, Working model of stress-induced chemoresistance. After being exposed to a stressor, catecholamines are released and bind to the β-adrenergic receptors on the tumor cell surface to initiate downstream signaling through cAMP-PLC-PKC and CREB, which initiates transcription of DUSP1 gene expression. DUSP1 protein is produced and JNK and c-Jun are dephosphorylated, protecting the cells from apoptosis.
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