Loss of TGF-β signaling leads to reduced tumor differentiation. (A) The TSTSS is weakly anticorrelated with proliferation, as assessed using a metaPCNA index, in ER+ but not ER- breast cancers (TCGA cohort). (B) Expression of the TSTSS inversely correlates with tumor grade in ER+ breast cancer (n = 904 patients), as assessed using GOBO tool. (C) Correlation between the TSTSS and luminal differentiation, as assessed using a meta-differentiation index, in ER+ and ER- tumors in the TCGA cohort. (D) Immunohistochemical staining of cytokeratin 8 (CK8) and ER-α in primary tumors from M3 cells expressing shGFP, shSmad2 or shSmad3. Scale bars represent 100 μm. (E) Quantitation of % area occupied by structures with a differentiated glandular histology in M3 tumors expressing shGFP (control), shSmad2 or ShSmad3. Results are mean +/−SEM for five tumors/group. (F) Quantitation of CK8 and ER staining was performed using Image-Pro Plus software. Each datapoint represents the mean of five fields/tumor, and results are shown as mean +/−SEM for five tumors/group. *P <0.05 for one-way ANOVA with Dunnett’s multiple comparison test; ns, not significant; hpf, high power field. ER, estrogen receptor; GOBO, gene expression-based outcome for breast cancer online; TGF-β, transforming growth factor beta; TSTSS, TGF-β/Smad3 tumor suppressor signature.