Stress effects on FosB- and interleukin-8 (IL8)-driven ovarian cancer growth and metastasis

Abstract

A growing number of studies indicate that chronic stress can accelerate tumor growth due to sustained sympathetic nervous system activation. Our recent findings suggest that chronic stress is associated with increased IL8 levels. Here, we examined the molecular and biological significance of IL8 in stress-induced tumor growth. Norepinephrine (NE) treatment of ovarian cancer cells resulted in a 250-300% increase in IL8 protein and 240-320% increase in its mRNA levels. Epinephrine treatment resulted in similar increases. Moreover, NE treatment resulted in a 3.5-4-fold increase in IL8 promoter activity. These effects were blocked by propranolol. Promoter deletion analyses suggested that AP1 transcription factors might mediate catecholamine-stimulated up-regulation of IL8. siRNA inhibition studies identified FosB as the pivotal component responsible for IL8 regulation by NE. In vivo chronic stress resulted in increased tumor growth (by 221 and 235%; p < 0.01) in orthotopic xenograft models involving SKOV3ip1 and HeyA8 ovarian carcinoma cells. This enhanced tumor growth was completely blocked by IL8 or FosB gene silencing using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoliposomes. IL8 and FosB silencing reduced microvessel density (based on CD31 staining) by 2.5- and 3.5-fold, respectively (p < 0.001). Our findings indicate that neurobehavioral stress leads to FosB-driven increases in IL8, which is associated with increased tumor growth and metastases. These findings may have implications for ovarian cancer management.

FIGURE 1.

Effects of NE or Epi on IL8 protein levels. IL8 levels were assessed by ELISA in SKOV3ip1 and HeyA8 ovarian carcinoma cells after 1, 3, and 6 h of exposure to NE (A) or Epi (B). Average percent increase in IL8 levels compared with controls is reported. Error bars represent S.E.

FIGURE 2.

Effect of NE on IL8 mRNA levels. IL8 mRNA levels were determined by real time RT-PCR in SKOV3ip1 ovarian cancer cells after exposure to NE (10 μm) at different time points. Average fold change in IL8 mRNA compared with control is reported. Error bars represent S.E.

FIGURE 3.

NE transcriptional control of the IL8 promoter. IL8 promoter activity was assayed by expression of a luciferase reporter gene in HeyA8 ovarian cancer cells after 3 h of exposure to NE (10 μm). The role of β-adrenergic signaling was evaluated by pretreatment of NE-exposed cells with the β-blocker propranolol. The role of PKA activity was assessed by pharmacological activation by Bt₂cAMP (db-cAMP) (100 μm), and the role of PKC activity was assessed by pharmacological activation by phorbol 12-myristate 13-acetate (PMA) (10 ng/ml). Similar results were observed for SKOV3ip1 ovarian carcinoma cells (data not shown). Error bars represent S.E.

FIGURE 4.

Role of adrenergic signaling in IL8 expression. IL8 expression was assessed in SKOV3ip1 ovarian cancer cells at protein (A) and mRNA levels (B) by ELISA and real time RT-PCR, respectively, after 6 h of exposure to NE (10 μm). The role of β-adrenergic signaling was evaluated by pretreatment of NE-exposed cell with the β-blocker propranolol (10 μm). The role of PKA activity was assessed by pharmacological activation by Bt₂cAMP (db-cAMP) (100 μm) and forskolin (10 μm). The importance of PKA in mediating NE-induced cellular responses was assessed by pretreatment of NE-exposed cells with a PKA-specific blocker KT5720. Similar results were observed for HeyA8 ovarian cancer cells (data not shown). Error bars represent S.E.

FIGURE 5.

Role of AP1 in NE-mediated IL8 induction. A, analysis of full-length and truncated IL8 promoter sequences identified NE-responsive transcription elements within 133 bp upstream of the transcription start site. Site-directed mutagenesis of a predicted AP1 response element and a predicted CCAAT/enhancer-binding protein (C/EBP) (NFIL6) site each partially abrogated NE-mediated induction of the IL8 promoter, whereas mutagenesis of a predicted NF-κB response element enhanced IL8 promoter response to NE. B, electrophoretic mobility shift assay of HeyA8 nuclear extracts using a fluorescence-tagged consensus oligonucleotide target to assess activation of AP1 or Sp1 15 min after exposure to NE. C, role of AP1 complex components in mediating NE-induced IL8 expression was evaluated using real time RT-PCR in ovarian cancer cells after silencing Fos, FosB, Jun, and JunB. Average fold change in IL8 mRNA is presented. Error bars represent S.E.

FIGURE 6.

Effect of IL8 or FosB silencing on stress-mediated tumor growth. Nude mice were subjected to 2-h daily restraint stress each morning for 3–4 weeks using HeyA8 and SKOV31ip1 ovarian cancer models. A and B, IL8 and FosB silencing was achieved with specific siRNAs incorporated into neutral nanoliposomal DOPC, injected twice weekly. C, ADRB activity was blocked with propranolol (β-blocker). At the end of each study, mice were sacrificed, and tumors were harvested. Average tumor weight (bar graph) is shown. Error bars, represent S.E.

FIGURE 7.

Effect of daily stress on patterns of metastasis. SKOV3ip1 cells were injected directly into the mouse ovary through a surgical skin incision. One week later, nude mice were subjected to a 2-h daily restraint stress each morning for 4 weeks. A, IL8 silencing was achieved with specific siRNA incorporated into neutral nanoliposomal DOPC, injected twice weekly. At the end of the study, mice were sacrificed, and tumors were harvested. Average number tumor of nodules (bar graph) is shown. Error bars represent S.E. B, representative images of extent of metastatic spread in stress versus nonstress mice. Metastatic areas are outlined with white dots. C, bar graph represents percent of animals in each study arm with metastasis to intraperitoneal and distant organ sites. Para-aortic lymph nodes are depicted as para-aortic LN.

FIGURE 8.

Effect of IL8 silencing on tumor microenvironment. A, immunohistochemical staining analysis of IL8, MMP-2, and MMP-9 expression in SKOV3ip1 tumors harvested from the chronic stress ovarian cancer model described in Fig. 6. Representative images (original magnification ×200) are shown as follows: nuclei (blue) and IL8, MMP-2, and MMP-9 (brown). Tumor cell proliferation (B) and angiogenesis (C) was assessed by immunohistochemical staining for Ki 67 and CD31 antigens in tumors harvested at the end of in vivo experiments from all treatment groups. Representative photomicrographs representing Ki 67 (proliferative nuclei are brown; nonproliferative tumor cell nuclei are blue) and CD31 (mouse endothelial cells are blue; tumor cell nuclei are blue) staining (original magnification ×100) are shown. The graph on the right shows proliferative index and mean number of MVD from each treatment group. Average MVD was calculated by averaging MVD counts from five random fields per slide, and at least three slides were examined for each treatment group. Error bars represent S.E.

FIGURE 9.

Role of IL8 and FosB in mediating the effects of chronic stress response on tumor growth. Chronic stress activates ADRB2 signaling, resulting in increased FosB and IL8 levels, which in turn promote tumor angiogenesis, growth, and metastasis. AC, adenylate cyclase.