CCL18 from tumor-cells promotes epithelial ovarian cancer metastasis via mTOR signaling pathway

Abstract

CCL18 is a chemotactic cytokine involved in the pathogenesis and progression of various disorders, including cancer. Previously, our results showed high levels of CCL18 in the serum of epithelial ovarian carcinoma patients suggesting its potential as a circulating biomarker. In this study, we determined that CCL18 expression was up-regulated in ovarian carcinoma compared with adjacent tissue and was expressed in carcinoma cells in the tumor and not in normal ovarian epithelial cells by laser capture microdissection coupled with real-time RT-PCR. Moreover, correlation analysis showed that the CCL18 level was positively correlated with the metastasis of patients with ovarian cancer. Survival analysis also revealed that an increased level of CCL18 was associated with worse survival time in ovarian cancer patients. Over-expression of CCL18 led to enhanced migration and invasion of the Skov3 ovarian cancer cell line in vitro and in vivo. Finally, proteomics analysis demonstrated that CCL18-mediated ovarian cancer invasiveness was strongly correlated with the mTORC2 pathway. These findings suggest that the CCL18 chemokine has an important role in chemokine-mediated tumor metastasis, and may serve as a potential predictor for poor survival outcomes for ovarian cancer. © 2015 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

Keywords: chemokine (C-C motif) ligand 18; epithelial ovarian carcinoma; metastasis; signaling pathway.

Figure 1

CCL18 Expression in ovarian cancer tissues and serum. (A) Expression of CCL18 mRNA from the LCM of ovarian carcinomas samples, **P < 0.001; (B) Results of overall survival (OS) analysis in patients stratified according to the CCL18 mRNA level (Kaplan–Meier test), P < 0.001; Delete: death cases. (C) CCL18 level of serum of patients with ovarian cancer and benign ovarian tissue, **P < 0.001; (D) Expression of CCL18 protein was observed by immunohistochemistry. Hemalum colors nuclei of cells were blue. CCL18 immunoreactivity in tumor cells was confined to the intercellular with brown, no staining in the nuclei of cells. Non‐epithelial ovarian carcinomas, absence of CCL18 immunostaining.

Figure 2

Effects of CCL18 overexpression on the proliferation of Skov3 cells. (A) Expression of CCL18 mRNA in different ovarian cancer cell lines. (B) Transfections of CCL18 was detected by real time fluorescence quantitative PCR. (C) Transfections of CCL18 was detected by western blot. (D) Effects of over‐expression CCL18 on subcutaneous tumor formation, five repeats. (E) Comparison of tumor weight in over‐expression CCL18 after xenograft growth 30 d, five repeats. (F) The effect of CCL18 on growth of Skov3 cell line in vitro by using MTT assay. *P < 0.05; **P < 0.001.

Figure 3

Influence of CCL18 on ovarian cancers invasion and metastasis in vitro and in vivo by orthotopic transplantation model of human ovarian cancer in nude mice. (A) The effects of CCL18 on the migration of Skov3 cells line in vitro. (B) The effects of CCL18 on the invasion of Skov3 cells line in vitro. (C) The effects of CCL18 on metastasis of ovarian tumors in vivo using a fluorescence stereomicroscope. (D–H) Hematoxylin and eosin staining (H&E) in metastasic organs, 200×. Hemalum colors nuclei of cells was blue, cytoplasm was eosin dyed red. Malignant tumor cells contained multiple or irregular nuclei. Over‐expressed CCL18 led to massive metastasis in the gastrointestinal tract, livers, and kidneys of the mice bearing CCL18_Skov3 xenografts (Figure 3D–F), Skov3_GFP cells as compared with Skov3_GFP cells (Figure 3G,H).

Figure 4

Proteomics analysis of xenograftion with CCL18_Skov3 and Skov3 cell lines using iTRAQ approach. (A) Plotting the iTRAQ ratios of the final 136 altered proteins from both biological replicates identified the correlation coefficient (R ₂) at 0.904, which interpreted acceptable variation of iTRAQ ratios of the significantly regulated proteins in both biological replicates. (B) Western blot studies to validate differential expression pattern of 8 candidate proteins. Up‐regulation of EIF2S2, EIF5a, EIF5b, EIF4A1, Rac2, Kras. (C) Down‐regulation of BAX and CDKN2A in xenografts were observed by Western blot studies. (D) The schema of CCL18‐stimulated mTOR signaling. (E) Western blot analysis of mTOR signaling on mTOR biomarkers. (F) Immunohistochemical images of p‐S6 (S235/236) staining in tumor xenografts, 200×.