Ephrin signaling contributes to the tumor suppressive effects of TGF-β in ER+ breast cancer. (A) Pathway enrichment in the TSTSS assessed by Ingenuity Pathway Analysis. Fisher’s exact test with Benjamini-Hochberg (B-H) correction. The dotted line represents the P <0.05 significance threshold. (B) Correlation of TSTSS with meta-EFNA index in ER+ tumors of the TCGA cohort. (C) Association of meta-EFNA index with distant metastasis-free survival (DMFS) in ER+ breast cancer (n = 856 patients) using the GOBO tool. (D) Smad3 ChIP-QPCR at the EFNA1 locus in M3 and M4 cells. (E) ChIP-QPCR for H3AcK9/14 at the EFNA1 locus to identify active chromatin. (F) Time course of EFNA1 mRNA induction by TGF-β (2 ng/ml) in M3 and M4 cells. Results are mean +/−SEM of three determinations. *P <0.05. (G) Western blot of effect of TGF-β treatment of M3 cells on Ephrin A1 (EFNA1) expression and oncogenic signaling through phosphorylation of the EphA2 receptor on S897. EFNA-Fc was used as a positive control for activation of the EphA2 signaling path. (H) Western blot showing knockdown of EFNA1 in M3 cells. (I) Knockdown of EFNA enhances tumorigenesis in M3 cells. n = 8 to 10 mice/group. *P >0.05 one-way ANOVA, Tukey’s multiple comparison test. ChIP, chromatin immunoprecipitation; ER, estrogen receptor; GOBO, Gene expression-based Outcome for Breast cancer Online; H3AcK9/14, histone H3 acetylated on lysine 9 or 14; QPCR, quantitative polymerase chain reaction; TGF-β, transforming growth factor beta; TSTSS, TGF-β/Smad3 tumor suppressor signature.