Role of eotaxin-1 signaling in ovarian cancer

Abstract

Purpose: Tumor cell growth and migration can be directly regulated by chemokines. In the present study, the association of CCL11 with ovarian cancer has been investigated.

Experimental design and results: Circulating levels of CCL11 in sera of patients with ovarian cancer were significantly lower than those in healthy women or women with breast, lung, liver, pancreatic, or colon cancer. Cultured ovarian carcinoma cells absorbed soluble CCL11, indicating that absorption by tumor cells could be responsible for the observed reduction of serum level of CCL11 in ovarian cancer. Postoperative CCL11 levels in women with ovarian cancer negatively correlated with relapse-free survival. Ovarian tumors overexpressed three known cognate receptors of CCL11, CC chemokine receptors (CCR) 2, 3, and 5. Strong positive correlation was observed between expression of individual receptors and tumor grade. CCL11 potently stimulated proliferation and migration/invasion of ovarian carcinoma cell lines, and these effects were inhibited by neutralizing antibodies against CCR2, CCR3, and CCR5. The growth-stimulatory effects of CCL11 were likely associated with activation of extracellular signal-regulated kinase 1/2, MEK1, and STAT3 phosphoproteins and with increased production of multiple cytokines, growth factors, and angiogenic factors. Inhibition of CCL11 signaling by the combination of neutralizing antibodies against the ligand and its receptors significantly increased sensitivity to cisplatin in ovarian carcinoma cells.

Conclusion: We conclude that CCL11 signaling plays an important role in proliferation and invasion of ovarian carcinoma cells and CCL11 pathway could be targeted for therapy in ovarian cancer. Furthermore, CCL11 could be used as a biomarker and a prognostic factor of relapse-free survival in ovarian cancer.

Figure 1. Serum CCL11 in ovarian cancer

Serum CCL11 concentrations were analyzed in healthy controls and patients with early (I-II) and late (III-IV) stages of ovarian cancer (A) and in age-matched women with ovarian (all stages), endometrial, breast, lung, pancreatic, and colon cancers (B) using bead-based immunoassay technology. Reactions were performed according to manufacturer’s protocol (Invitrogen). C, Postoperative levels of CCL11 in sera of ovarian cancer patients. CCL11 was measured in postoperatively collected sera using Luminex bead-based assay. Patient group was divided into two subgroups with RFS < 3 yrs and RFS > 3 yrs. D, Absorption of CCL11 by cultured ovarian carcinoma cells. Cells (10⁷) were trypsinized and resuspended in 3 ml of cell culture medium and CCL11 was added to a final concentration of 1 ng/ml. Cells were incubated with shaking for 1 hr, supernatant was collected, and CCL11 concentration was determined by bead-based immunoassay. In this and following Figures, * denotes statistical significance of differences between cancer and healthy at p < 0.05; ** - p < 0.01; *** - p < 0.001.

Figure 2. Expression of CCL11 (A-C) and CCR2,3,5 receptors in ovarian carcinoma

A, CCL11 protein expression in ovarian carcinoma cell lines; B, CCL11 protein expression in primary ovarian tumor cells. Primary ovarian carcinoma cells were obtained from ascites fluid. Adherent cells were cultured for 7 days in 96-well plates. These primary and established OVCAR-3 and SKOV-3 cells were pre-incubated with monensin for 48 h, fixed and incubated with primary Abs against CCL11 and secondary Alexa 488 conjugated Abs, counterstained with antibody against cytokeratin 19 (CK) and with Hoechst 33342 and analyzed by Cellomics Array scan (40x objective). C, CCL11 RNA expression in normal ovarian epithelium pooled from 5 healthy women (1), in individual ovarian tumors (2-3), and in cultured ovarian carcinoma cells, OVCAR3 (4) and SKOV3 (5). 6 - negative control; 7 -positive control (Actin). D, CCL11 receptors expression in established cell lines. CCR2,3, and 5 were stained in cultured cells using specific monoclonal FITC-conjugated antibody, and analyzed by flow cytometry. Non-specific antibody binding observed after staining the cells with isotype-matched FITC-conjugated IgG control, was subtracted. E, Analysis of CCL11 receptors in ovarian tumors. CCR2,3,and 5 were analyzed in ovarian carcinoma Tissue Array using primary polyclonal Abs optimized to work in FFPR tissues as described in Methods. Tumor sections from 75 different patients and normal ovary sections from 30 healthy women were examined for each receptor with a representative section presented (x200). (1) Negative control on ovarian cancer; (2) Negative control on normal ovary; (3) CCR2 in ovarian cancer (arrow); (4) CCR2 in healthy ovarian epithelium; (5) CCR3 in ovarian cancer (arrow); (6) CCR3 in normal ovary; (7) CCR5 in ovarian cancer; (8) CCR5 in normal ovary.

Figure 3. CCL11 induces migration/invasion and proliferation, and neutralizing antibodies against CCL11 and its receptors inhibit proliferation and induce apoptosis in ovarian carcinoma cell lines

A, B, Migration/Invasion. Cells were incubated with 0-10 ng/ml CCL11 for 24 hrs. Migration/invasion was measured using BD BioCoat Matrigel Invasion Chambers. A, Dose-curve of CCL11; B, Effects of neutralizing anti-CCR2,3,5 antibodies and their combination on migration/invasion of ovarian carcinoma cells. OVCAR-3 and SKOV-3 cells were pre-treated with 100 ng/ml neutralizing antibodies against CCR2,3, 5 or IgG isotype control (at 100 ng/ml) for 24 h and cell migration was assessed. C,D, Proliferation. Cell proliferation was evaluated by cell counts using imaging cytometry. C, Dose-curve of CCL11; D, Effects of neutralizing anti-CCL11 and anti-CCR2,3,5 antibodies and their combination on proliferation of untreated and CCL11-treated ovarian carcinoma cells. OVCAR-3 cells were incubated with neutralizing antibodies or IgG isotype control as above for 72 h and cell proliferation was assessed by imaging cytometry. E, Effects of neutralizing anti-CCL11 and anti-CCR2,3,5 antibodies and their combinations on apoptosis of cisplatin treated ovarian carcinoma cells. OVCAR-3 and SKOV-3 cells were incubated with neutralizing antibodies as above for 48 h, cisplatin was added at final concentration of 5 μM and incubation continued for additional 24 h. Results for OVCAR-3 cells are presented. Similar results for SKOV-3 cells are not shown. *** denotes statistical significance of differences at P < 0.001. In B and C *** over CCL11+IgG columns indicates statistical significance of differences between CCL11 treated vs. untreated control cells; *** over horizontal lines indicate significance of differences between neutralizing Ab treated cells vs. CCL11 treated cells; *** over anti-CCR2,3,5 combination treated cells indicates significance of differences between cells treated with combination of Abs vs. cells treated with each single neutralizing Ab.

Figure 4. CCL11 induced cytokine production and signal transduction in ovarian carcinoma cell lines

A,B, Induction of cytokines. Media were harvested and cytokine concentrations were measured using xMAP technology with multiplexed cytokine Bio-Rad kit. Data are presented on a log-scale. Vertical lines indicate statistical errors, SE. All differences between control and CCL11 treated cells are highly statistically significant (P < 0.001). A, SKOV-3 cells; B, OVCAR-3 cells. C. CCL11 induced phosphorylation of ERK1/2, STAT3, and MEK-1 in OVCAR-3 cells. Tumor cells were treated with 5 ng/ml of CCL11 for 0-30 min. Cell lysates were prepared and analyzed using 8-Plex phosphoprotein kit as described in Methods. B, Transcription factors expression in CCL11 treated tumor cells. OVCAR-3 and SKOV-3 cells were treated with 5 ng/ml of CCL11 for 0, 5, and 30 min. Nuclear extracts were prepared and transcription factor analysis was performed using 40-plex kit. Representative TFs that demonstrated the most robust response to CCL11 treatment are presented.