J Exp Clin Cancer Res. 2014 Sep 28;33(1):85. doi: 10.1186/s13046-014-0085-6.
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 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.
Expression 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.
Our 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.
The 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.
我们之前发现血小板激活因子受体 (PAFR) 在卵巢癌中过度表达，并发现其配体 PAF 通过磷酸化抗体微阵列诱导 EGFR 磷酸化。表皮生长因子受体 (EGFR) 在卵巢癌中也过度表达，并促进卵巢癌细胞的生长。在这里，我们研究了 PAFR 和 EGFR 信号在卵巢癌细胞中的串扰机制，以进一步确定 PAFR 和 EGFR 之间的相互作用是否协同促进卵巢癌的进展。
通过 Western blot、实时 PCR 和免疫荧光评估几种卵巢癌细胞系中 PAFR 的表达和定位。用 PAF 或 PAF 和一些实验中的药理学抑制剂刺激卵巢癌细胞。通过 Western blot 测量信号通路中蛋白质的磷酸化。通过酶联免疫吸附试验测量刺激的卵巢癌细胞上清液中 HB-EGF 的浓度。
我们的数据表明，PAF 通过 PAFR 在 SKOV-3 卵巢癌细胞中以时间和剂量依赖的方式增加 EGFR 磷酸化。这种转激活依赖于磷脂酶 C-β 和细胞内钙信号。该途径还依赖于Src 酪氨酸激酶和金属蛋白酶。PAF 通过金属蛋白酶依赖性方式增加肝素结合表皮生长因子样生长因子 (HB-EGF) 的释放来触发 EGFR 激活。涉及 G 蛋白偶联受体 (GPCR) 介导的 EGFR 转激活的几项研究表明，EGFR 依赖性增加 ERK1/2 磷酸化。然而，在 SKOV-3 细胞中，PAF 处理也以 EGFR 非依赖性方式增加 ERK1/2 磷酸化。
结果表明，在 SKOV-3 卵巢癌细胞中，PAF 转激活 EGFR 和下游 ERK 途径，从而使 GPCR 介导的信号多样化。PAFR 和 EGFR 之间的串扰表明炎症和生长因子信号在卵巢癌细胞之间存在潜在重要的信号联系。
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