Abstract

BackgroundWe previously identified platelet-activating factor receptor (PAFR) as being overexpressed in ovarian cancer and found that its ligand PAF evoked EGFR phosphorylation using the phospho-antibody microarray. Epidermal growth factor receptor (EGFR) are also overexpressed in ovarian cancer and contribute to the growth of ovarian cancer cells. Here, we investigated the mechanisms of crosstalk between PAFR and EGFR signaling in ovarian cancer cells to further determine whether the interaction between PAFR and EGFR synergistic contribute to the progression of ovarian cancer.MethodsExpression and localization of PAFR in several ovarian cancer cell lines were assessed by Western blot, realtime-PCR and immunofluorescence. The ovarian cancer cells were stimulated with PAF or PAF and in some experiments also pharmacological inhibitors. Phosphorylation of proteins in signaling pathways were measured by Western blot. HB-EGF concentrations of the supernatant from stimulated ovarian cancer cells were measured by enzyme-linked immunosorbent assay.ResultsOur data show that PAF increases EGFR phosphorylation through PAFR in a time- and dose- dependent manner in SKOV-3 ovarian cancer cells. This transactivation is dependent on phospholipase C-β and intracellular calcium signaling. This pathway is also Src tyrosine kinase- and metalloproteinase- dependent. PAF triggers EGFR activation through the increased heparin-binding EGF-like growth factor (HB-EGF) release in metalloprotease-dependent manner. Several studies involving EGFR transactivation through G-protein coupled receptor (GPCR) have demonstrated EGFR-dependent increase in ERK1/2 phosphorylation. Yet in SKOV-3 cells, PAF treatment also increases ERK1/2 phosphorylation in a EGFR-independent manner.ConclusionsThe results suggest that in SKOV-3 ovarian cancer cells, PAF transactivates EGFR and downstream ERK pathways, thus diversifying the GPCR-mediated signal. The crosstalk between PAFR and EGFR suggests a potentially important signaling linkage between inflammatory and growth factor signaling in ovarian cancer cells.

Highlights

  • We previously identified platelet-activating factor receptor (PAFR) as being overexpressed in ovarian cancer and found that its ligand Platelet-activating factor (PAF) evoked Epidermal growth factor receptor (EGFR) phosphorylation using the phospho-antibody microarray

  • PAF-induced EGFR transactivation is dependent on the PAF-receptor We investigated whether the PAF-receptor (PAFR) is involved in the transactivation of EGFR in ovarian cancer cells

  • (See figure on previous page.) Figure 4 Role of Ca2+ and PKC in responses to PAF in SKOV-3 cells. (A) SKOV-3 cells were pretreated for 1 h with the phospholipase C-β (PLCβ) inhibitor U73122 (20 μM) before stimulation with PAF (100 nM) or epidermal growth factor (EGF) (5 ng/ml) for 5 min. (B) SKOV-3 cells were pretreated for 1 h with the Ca2+ chelator BAPTAAM (20 μM) before stimulation with PAF (100 nM) or thapsigargin (1 μM) for 5 min. (C) SKOV-3 cells were loaded with the calcium probe Fura-2/ AM followed by stimulation with 100 nM PAF. (D) SKOV-3 cells were loaded with the calcium probe Fura-2/AM followed by stimulation with 100 nM

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Summary

Introduction

We previously identified platelet-activating factor receptor (PAFR) as being overexpressed in ovarian cancer and found that its ligand PAF evoked EGFR phosphorylation using the phospho-antibody microarray. Epidermal growth factor receptor (EGFR) are overexpressed in ovarian cancer and contribute to the growth of ovarian cancer cells. The epidermal growth factor (EGF) receptor (EGFR) is upregulated in ovarian cancer, and increased expression is associated with reduced survival rate [12,13,14,15]. The modest responses of EGFR blockade in response to the administration of monoclonal antibodies or tyrosine kinase inhibitors as single agents could be attributed to compensation through other signaling pathways

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