Fig. 4 | Oncogene

Fig. 4

From: Estradiol induces BDNF/TrkB signaling in triple-negative breast cancer to promote brain metastases

Fig. 4

BDNF and CM-E2 activate TrkB and promote invasion and mammosphere formation in EGFR¯4T1BR5 cells. a 4T1BR5 cells were serum-starved overnight and pre-treated with vehicle or 10 µM ANA-12 2 h before treatment with 50 ng/ml BDNF for 5 min. Arrows indicate 140 and 110 kD TrkB isoforms. b Left: 4T1BR5 cells were plated in matrigel-coated plates, serum-starved for 16 h, and treated with vehicle or ANA-12 for 2 h. After creating the scratch wound, wells were coated with matrigel and vehicle or BDNF was added as a chemoattractant. Graphs show relative wound density (RWD) ± SEM (n = 6 wells/treatment). Right: 4T1BR5 cells were plated in Mammocult media at a density of 100 cells per well in a 96-well plate and treated with vehicle or 100 ng/ml BDNF alone or in combination with 1 µM ANA-12. Data show mammospheres at 10 days. c Signaling pathways activated in 4T1BR5 cells treated for 10 min with vehicle, CM-OH, or CM-E2. d Signaling pathways activated in serum-starved 4T1BR5 cells treated for 30 min with vehicle, 10 nM E2, 50 ng/ml BDNF, CM-OH, or CM-E2. Graphs shows fold change in levels of p-TrkB/tubulin, p-AKT/Total AKT, and p-PLCγ/Total PLCγ relative to vehicle-treated cells from at least 3 independent experiments. e 4T1BR5 cells were treated as in (b, left), and vehicle, CM-OH, or CM-E2 were used as chemoattractants in a scratch wound-modified invasion assay. f 4T1BR5 cells were treated as in (e), but CM-E2 was pre-incubated with a Trk-B Fc antibody (CM-E2- BDNF depleted) or an isotype IgG control (CM-E2). TrkB-Fc/BDNF or IgG complexes were depleted from CM-E2 using agarose beads. g 4T1BR5 cells were plated as in (b, right) and treated with vehicle, CM-OH, CM-E2 alone or in combination with 1 µm ANA-12. Graph shows number of mammospheres at day 10. h. Left: WB shows TrkB and downstream signaling in 4T1BR5 cells transduced with a shRNA lentiviral vector targeting mouse-TrkB (mo-shTrkB) or a non-targeting control (mo-shNC). Right: mo-shNC and mo-shTrkB cells were plated as in (b, left) and vehicle or CM-E2 were used as chemoattractants for the invasion assay. For WBs, numbers indicate ratio of protein/α-tubulin relative to control or time zero. For invasion assays, the data were analyzed using repeated measures ANOVA with post-hoc correction. Adjusted P values at 24 h are shown (*P < 0.05, **P < 0.01, ****P < 0.001). Graph represents one of at least two independent experiments. For mammosphere formation, graphs show # mammospheres/well (10 wells/treatment) in one of three independent experiments. The data were analyzed using Kurskal-wallis ANOVA with Dunn’s multiple comparison test

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