Fig. 5 | Oncogene

Fig. 5

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

Fig. 5

E2 signaling through astrocytes promotes cancer cell BDNF upregulation and blocking BDNF/TrkB signaling abolishes E2-induced BM. a BDNF expression in TNBC cells assessed by ELISA (n = 3). Cell lysates were obtained from cells cultured in 10% FBS-supplemented media. b Left: pro-BDNF and BDNF levels in 231BR cells expressing scramble control (shNC) or human BDNF targeting shRNAs. Right: Serum-starved shNC or hu-shBDNF-1 (1395) 231BR cells were treated with vehicle or CM-E2 as chemoattractants in scratch wound assays. Graphs show RWD ± SEM (4 wells/treatment). c Left: 4T1BR5 cells expressing sh NC or mouse BDNF targeting shRNAs. Right: Serum-starved shNC or mo-shBDNF-1-(5386) 4T1BR5 cells were treated with vehicle or CM-E2 as in b (6 wells/treatment). Data show one of 2 independent experiments; ****P < 0.0001 at 24 h using repeated measures ANOVA with Tukey’s multiple comparison test. d pro-BDNF and BDNF levels in 4T1BR5 cells treated with 50 ng/ml BDNF for indicated times. e Left: pro-BDNF and BDNF levels in 4T1BR5 cells treated for 24 h (WB) or 16 h (ELISA) with vehicle, 10 nM E2, 50 ng/ml BDNF, CM-OH, or CM-E2. Right: BDNF levels in 4T1BR5 cells treated with CM-E2 alone or in presence of 10 µg/ml Cycloheximide for 3 and 16 h (n = 3). f BDNF expression in shNC or shTrkB 4T1BR5 cells treated with CM-OH or CM-E2 for 16 h (n = 6) measured using ELISA. For experiments b-f, cells were cultured in hormone-deprived media (serum-free or 2% CS-FBS as indicated in Methods). g Double IF shows BDNF (green) expression in 4T1BR5 BM in brain sections of OVX or E2-treated mice injected with 4T1BR5 cells, 15 days after IC injection. Arrow indicates brain metastasis. Scale bar is 100 µm. For all WBs, numbers indicate ratio of protein/α-tubulin relative to control or time zero. For ELISAS, the data were analyzed using non-parametric ANOVA with Dunn’s multiple comparison test. h. 4T1BR5 cells were injected IC in OVX BALB/c female mice supplemented with E2 (n = 14) or placebo pellets (OVX, n = 15). E2-treated mice were administered daily IP injections of vehicle (E2) or ANA-12 (E2 + ANA12), and mice sacrificed 15 days later. Graph shows total number of brain metastatic clusters per mouse. The data were analyzed using Kruskal–Wallis ANOVA followed by Dunn’s multiple comparisons test

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