Fig 2: VIP–VIPR2 signaling regulates the phosphorylation of AKT in MDA-MB-231 cells. (A) MDA-MB-231 cells were starved for 3 h and then stimulated with the indicated doses of VIP for 10 min. Treated cells were lysed and evaluated by western blotting using specific antibodies for the indicated proteins. A set of representative images is shown. The graph shows the density of pAKT (Thr308) normalized against the corresponding density of the respective pan AKT. The data are presented as means ± SD (n = 3); ***p < 0.001 (versus 0 nM VIP; Kruskal–Wallis test followed by Dunn’s multiple comparison test). (B) MDA-MB-231 cells transfected with the indicated siRNAs were stimulated with 100 nM VIP for 10 min. Cell lysates were analyzed by western blotting to detect the indicated proteins. The graphs show the density of pAKT (Thr308 or Ser473) normalized against the corresponding density of the respective pan-antibody bands. The data are presented as means ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups (Kruskal–Wallis test followed by Dunn’s multiple comparison test). n.s. indicates not statistically significant. (C) MDA-MB-231 cells stably expressing EGFP (Empty) or VIPR2-EGFP were stimulated with VIP for 10 min. Cell lysates were analyzed by western blotting using the indicated specific antibodies. The graphs show the density of pAKT (Thr308 or Ser473) normalized against the corresponding density of the respective pan AKT. The data are presented as means ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups (Kruskal–Wallis test followed by Dunn’s multiple comparison test) ns, not significant..

Fig 3: Schematic representation of the role of VIPR2 in breast cancer cell migration. Increased VIPR2 mRNA expression and/or VIPR2 gene copy number has been found in invasive breast carcinoma. VIPR2 couples with Gαs, Gαi and Gαq proteins. Gαi-coupled VIPR2 and its Gβγ subunits regulate PI3Ks activity. PI3Ks convert phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] into phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3]. The synthesized PI(3,4,5)P3 promotes the translocation of WASP family verprolin homologous protein 2 (WAVE2) to the plasma membrane. WAVE2 drives lamellipodium formation by enhancing actin nucleation via the actin-related protein 2 and 3 (ARP2/3) complex, resulting in promotion of breast cancer cell migration.

Fig 4: Cell motility in VIPR2-silenced and VIPR2-expressing MDA-MB-231 cells. (A and B) Migration of EGFP-expressing MDA-MB-231 cells transfected with either control siRNA or VIPR2 siRNA1 (A) and that of MDA-MB-231 cells stably transfected with either VIPR2-EGFP or EGFP plasmid (B) was measured using a transwell migration assay. VIP (200 nM) was added to the lower chamber of the vessel. Cells were added to the upper chamber and incubated for 48 h (A) or 30 h (B) at 37°C. Quantification of migrating cells is shown in graphs. The data are presented as means ± SD [n = 9 (3 independent experiments; 3 areas were randomly selected in each group at each experiment)]. *p < 0.05, ***p < 0.001 between the indicated groups (Kruskal–Wallis test followed by Dunn’s multiple comparison test). (C) Wound healing in vitro in the scratch assay using confluent monolayer of VIPR2-silenced or VIPR2-expressing MDA-MB-231 cells was monitored for 18 h after 100 nM VIP stimulation. The dotted line indicates top position of the cell population. The distance migrated during 6 h was measured at 10 randomly selected points (see schematic diagram), and the average speed is shown in graphs. The data are presented as means ± SD [n = 90 (3 independent experiments; 10 points in 3 areas randomly selected in each group at each experiment)]. *p < 0.05, ***p < 0.001 between the indicated groups (Kruskal–Wallis test followed by Dunn’s multiple comparison test). (D) Migration of MDA-MB-231 cells stably transfected with VIPR2-EGFP was measured using a transwell migration assay. VIP (200 nM) was added to the lower chamber of the vessel. The indicated doses of KS-133 were added to both chambers. Cells were added to the upper chamber and incubated for 48 h at 37°C. Quantification of migrating cells is shown in graph. The data are presented as means ± SD [n = 9 (3 independent experiments; 3 areas were randomly selected in each group at each experiment)]. **p < 0.01, ***p < 0.001 between the indicated groups (Kruskal–Wallis test followed by Dunn’s multiple comparison test).

Fig 5: VIP–VIPR2 signaling regulates the activity of PI3K and subsequent PI(3,4,5)P3 level. (A) HeLa cells stably expressing mCherry-tagged VIPR2 or control vector were used for experiments. The cell lysates were incubated with BODIPY FL-PI(4,5)P2 for 1 h at 37°C, and the lipids extracted from cells were distinguished by thin layer chromatography. The lower panel was obtained using a high sensitivity mode (high exposure). The intensities of BODIPY bands in the top panel were quantified using ImageJ software, and the data are presented in the graph as means ± SD (n = 3). **p < 0.01 (Mann–Whitney U test). (B) Cell lysates were analyzed by western blotting to detect the indicated proteins. The density of mCherry-VIPR2, p110α, p110β, p110γ and classIII were normalized against that of GAPDH. The data are presented as means ± SD (n = 3). ***p < 0.001 (Mann–Whitney’s U test). n.s.: not significant. (C) MDA-MB-231 cells transfected with EGFP-tagged AKT-PH plasmid and either VIPR2 siRNA1 or control siRNA were stimulated with 100 nM VIP for 10 min and then fixed; EGFP was detected by fluorescent microscopy. A set of representative images from 3 independent experiments is shown. The plasma membrane was defined as the region 3 μm from the cell periphery. Relative fluorescence intensity of AKT PH-EGFP (plasma membrane/whole cell) was calculated. The data are presented in the graph as means ± SD [n = 30 (30 cells in each group)]. **p < 0.01, between the indicated groups (Kruskal–Wallis test followed by Dunn’s multiple comparison test). n.s. indicates not statistically significant. (D) MDA-MB-231 cells transfected with EGFP-tagged AKT-PH plasmid and either empty vector or VIPR2-mCherry were stimulated with 100 nM VIP for 10 min and then fixed; EGFP and mCherry were detected by fluorescent microscopy. A set of representative images from 3 independent experiments is shown. Relative fluorescence intensity of AKT PH-EGFP (plasma membrane/whole cell) was calculated. The data are presented in the graph as means ± SD [n = 20 (20 cells in each group)]. *p < 0.05, ***p < 0.001, between the indicated groups (Kruskal–Wallis test followed by Dunn’s multiple comparison test) ns, not significant.