PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

Prostaglandin E2 (PGE2) interacts with tyrosine kinases receptor signaling in both tumor and stromal cells supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, A549 and GLC82, PGE2 promotes nuclear translocation of epidermal growth factor receptor (nEGFR), affects gene expression and induces cell growth. Indeed, cyclin D1, COX-2, iNOS and c-Myc mRNA levels are upregulated following PGE2 treatment. The nuclear localization sequence (NLS) of EGFR as well as its tyrosine kinase activity are required for the effect of PGE2 on nEGFR and downstream signaling activities. PGE2 binds its bona fide receptor EP3 which by activating SRC family kinases, induces ADAMs activation which, in turn, releases EGFR-ligands from the cell membrane and promotes nEGFR. Amphiregulin (AREG) and Epiregulin (EREG) appear to be involved in nEGFR promoted by the PGE2/EP3-SRC axis. Pharmacological inhibition or silencing of the PGE2/EP3/SRC-ADAMs signaling axis or EGFR ligands i.e. AREG and EREG expression abolishes nEGFR induced by PGE2. In conclusion, PGE2 induces NSCLC cell proliferation by EP3 receptor, SRC-ADAMs activation, EGFR ligands shedding and finally, phosphorylation and nEGFR. Since nuclear EGFR is a hallmark of cancer aggressiveness, our findings reveal a novel mechanism for the contribution of PGE2 to tumor progression.

were seeded and incubated for 24 h. Next, cells were harvested and seeded for MTT, clonogenic assay and RNA isolation. (A) Cell growth was assessed by MTT assay after 48 h treatment with 25 ng/ml EGF or 1μM PGE2. Data are presented as mean ± SEM of triplicate cultures, expressed as % of control. **p < 0.01 vs Ctrl; ## p < 0.01 vs Vector; § § p < 0.01 vs EGFR WT. (B) Clonal outgrowth was assessed by counting number of clones (> 50 cells) 12 days after treatment with 25 ng/ml EGF or 1 μM PGE 2 . Data are presented as mean ± SEM of triplicates, expressed as % of control. **p < 0.01, ***p < 0.001 vs Ctrl; # p < 0.05, ## p < 0.01 vs Vector; § p < 0.05, § § p < 0.01 vs EGFR WT. (C) RNA was isolated after 2, 4, 8, 12 h treatment with 1μM PGE2 and analyzed by qRT-PCR for regulated nuclear EGFR target genes. The data are presented as fold change ± SEM of three independent experiments, relative to non-treated cells (Control), which were assigned to 1. *p < 0.05 vs Ctrl.

Supplementary Figure 5: Three-dimensional reconstruction of A549 cells showing EGFR nuclear translocation upon EP3 agonist treatment. 3D reconstruction of confocal stacks showing EGFR localization in untreated A549 cells (left
panels) or exposed to the EP3 agonist Sulprostone at 1 μM for 60 min (right panels). Confocal images were reconstructed in XY, XZ and YZ axis. EGFR is visualized in green and the nuclei in blue.

Supplementary Figure 6: EP3 receptor mediates PGE 2 -induced EGFR nuclear translocation in GLC82. (A)
Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in GLC82 cells exposed for 60 min to 1 µM EP3 agonist. (B) Confocal analysis of EGFR localization in GLC82 treated with EP3 agonist for 60 min. EGFR was stained in green, DAPI (blue) was used to counterstain the nuclei. Confocal pictures were acquired in the middle section of nuclei at 63× magnification. Scale bars, 20 μm. Boxed areas are shown in detail in the inset.

Supplementary Figure 7: Three-dimensional reconstruction of confocal imaging stacks of EP3-silenced A549 cells. 3D reconstruction of confocal stacks showing EGFR localization in A549 cells transfected with siRNA control or siRNAs
against EP3 receptor. 48 h post transfection, cells were treated or not with 1 µM PGE 2 for 60 min as indicated in the panels. Images were reconstructed in XY, XZ and YZ axis. EGFR is visualized in green and the nuclei in blue.

Supplementary Figure 8: EP3 knockdown does not alter EGF-induced EGFR nuclear translocation. A549 cells
were transfected with siRNA control or siRNAs against EP3 receptor for 24 h. After, cells were starved overnight and treated with 25 ng/ml EGF for 10 min. Cells were stained for EGFR (green) and DAPI (blue). Confocal pictures were acquired in the middle section of nuclei at 63× magnification. Scale bars, 20 μm. Boxed areas are shown in detail in the inset.
Immunoblotting analysis of EGFR localization in A549 exposed for 10 min to 25ng/ml EGF with or without 10 µM PP1 or SU6656. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction, respectively. (C) A549 cells were transfected with Vector control or a plasmid bearing constitutive-active SRC (pcSRC-Y527F) for 24 h and 48 h. Immunoblotting analysis of SRC phosphorylated on Tyr 416 in A549 transfected for 24 h or 48 h was performed as control for plasmid expression and SRC activation. Actin was used as loading control (left panel). EGFR expression at 24 h or 48 h in cytosolic and nuclear fraction in A549 transfected with pcSRC-Y527F (right panel).