Estrogen/GPER/PKA/FGF2 signal axis in cancer-associated fibroblasts enhances the invasion of breast cancer cells

Objective To investigate the effect of activated cytoplasmic G protein-coupled estrogen receptor (GPER) from co-cultured cancer-associated fibroblasts (CAFs) in the regulation of fibroblast growth factor 2 (FGF2) and the invasion of breast cancer cells. Methods The intracellular GPER localization in CAFs cultured alone or co-cultured with ER- positive (MCF-7) and ER- negative (MDA-MB-468) breast cancer cells was examined using immunofluorescence assays. Real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were utilized to detect the expression of FGF2 mRNA in the CAFs and FGF2 secretion in the supernatant, respectively. The activation of GPER downstream ERK, AKT and PKA signal pathways was determined by Western blotting. The changes in the invasiveness of breast cancer cells were examined by Transwell assays. Results Co-culture with the 2 breast cancer cells both induced translocation of GPER from the cell nucleus to the cytoplasm in the CAFs. In the cell co-cultures, 17-β estradiol (E2) significantly increased FGF2 levels in the CAFs and the supernatant, and this effect was obviously blocked by mutation of the nucleus export signal (NES) in GPER amino acid sequence (P < 0.05); these results could not be observed in the CAFs cultured alone. E2 treatment of the co-cultured CAFs resulted in obvious activation of GPER/AKT and GPER/PKA signal pathways (P < 0.05) and increased the protein expression of p-AKT and p-PKA; Mutation of the NES in GPER sequence significantly inhibited PKA signaling and up-regulated the expression of p-ERK (P < 0.05). The E2-induced increase of FGF2 expression in CAFs was significantly blocked by the application of the PKA inhibitor H-89 but not by the AKT inhibitor WM (P < 0.05). More importantly, the secreted FGF2 from E2-treated CAFs significantly promoted the invasion of the breast cancer cells, which was abolished by the neutralizing antibody of FGF2. Conclusion Estrogen enhances the activation of the downstream PKA signaling in a cytoplasmic GPER-dependent manner in the CAFs, and the enhanced FGF2 paracrine promotes the invasion of breast cancer cells. These signal pathways may play important roles in the progression of breast cancer