Synergistic effects of combined platelet-activating factor receptor and epidermal growth factor receptor targeting in ovarian cancer cells

BACKGROUND Genetic alterations, including the overexpression of epidermal growth factor receptor (EGFR), play a crucial role in ovarian carcinogenesis. To date, EGFR targeting has shown limited antitumor effects in ovarian cancer when administered as monotherapy. We previously identified platelet-activating factor receptor (PAFR) as being overexpressed in ovarian cancer and found that its ligand PAF evoked EGFR phosphorylation. To determine whether PAFR targeting can enhance the antitumor efficacy of EGFR inhibition, we investigated the effects of a PAFR antagonist (WEB2086) in conjunction with an EGFR inhibitor (AG1478). METHODS The expression of EGFR and PAFR in CAOV-3 and SKOV-3 ovarian cancer cell lines was measured by Western blot and immunocytochemistry. Synergy was determined using isobologram analysis. The effects of combined PAFR and EGFR targeting on both cells were assessed by using CCK-8, transwell, flow cytometry, western blot analysis. In vivo studies were conducted using CAOV-3 cells xenografted in nu/nu mice. RESULTS Treatment with combination WEB2086 and AG1478 resulted in significantly greater inhibition of proliferation and invasion compared to either drug alone. When examining equipotent combinations of WEB2086 and AG1478 to determine potential synergy, a combination index (CI) of 0.49 was identified for CAOV-3 cells and a CI of 0.58 for SKOV-3 cells indicating synergy. This co-inhibition induced significantly more apoptosis and arrested the cells at G0/G1 phase in both cell lines. The activation of PAFR and/or EGFR induced phosphorylation of the mTOR, AKT, and MAPK pathways. Combined PAFR and EGFR targeting synergistically diminished the expression of PAFR and EGFR phosphorylation and downstream signaling. In vivo studies further verified the antitumor effects of combined PAFR and EGFR targeting in a CAOV-3 xenograft model. CONCLUSIONS These results suggest that WEB2086 and AG1478 are synergistic in ovarian cancer cells with high expression of both PAFR and EGFR. The presented approach may have important therapeutic implications in the treatment of ovarian cancer patients.