Deregulation of angiopoietin-like 4 slows ovarian cancer progression through vascular endothelial growth factor receptor 2 phosphorylation

Abstract

Background As a tissue-specific proangiogenic or antiangiogenic agent, angiopoietin-like 4 (ANGPTL4) has recently gained attention in many diseases, such as metabolic syndrome, cardiovascular disease and cancer. However, the roles of ANGPTL4 in angiogenesis and tumor growth in epithelial ovarian cancer, the most lethal gynecologic malignancy, remain unclear. Objective To identify a novel mechanism of ANGPTL4 inhibition in epithelial ovarian cancer. Methods Western blot, quantitative reverse transcription PCR, and immunofluorescence analyses were applied to evaluate ANGPTL4 expression in ovarian cancer cell lines. Cell proliferation, migration, and invasion were investigated through 5-ethynyl-2′-deoxyuridine (EdU) incorporation, CCK-8 and Transwell assays. The expression of epithelial-mesenchymal transition (EMT)-related proteins in ovarian cancer cells and tumor-bearing mice was evaluated. CD31 staining was used to identify tumor angiogenesis. Immunoprecipitation was performed to examine the regulatory relationship between ANGPTL4 and the vascular endothelial growth factor receptor 2 (VEGFR2)/vascular endothelial (VE)-cadherin/Src complex. VEGFR2 phosphorylation at Y949 and VE-cadherin expression were assessed by western blotting. Inactivation of VEGFR2 Y949 phosphorylation was achieved in a MISIIR-TAg VEGFR2 Y949F/Y949F mouse model. Results Here, we demonstrated that ANGPTL4 was overexpressed in A2780 and CAOV3 ovarian cancer cells. In vitro assays indicated that inhibition of ANGPTL4 by lentiviral small interfering RNA does not alter ovarian cancer cell proliferation, migration, invasion, and EMT, while ANGPTL4 silencing exhibited significant inhibitory effects on tumor angiogenesis, growth, and metastasis in vivo. Immunoprecipitation analysis showed that suppression of ANGPTL4 was accompanied by dissociation of the VEGFR2/VE-cadherin/Src complex and phosphorylation of VEGFR2 Y949 in A2780 and CAOV3 ovarian tumors. Inactivation of VEGFR2 Y949 phosphorylation in MISIIR-TAg VEGFR2 Y949F/Y949F mice abolished all tumor-suppressive effects of ANGPTL4 inhibition in spontaneous ovarian carcinoma. Conclusions Overall, our results indicate that ANGPLT4 silencing delays tumor progression in specific types of ovarian cancer and may be a potential target for individualized treatment of ovarian cancer.