Fig 2: AF9 mRNA Level in Clinical Samples Correlates Inversely with miR-5694 Expression and Serves as a Biomarker of Malignant Breast Cancer(A and B) Boxplots of AF9 and miR-5694 expression in primary breast tumors and adjacent normal tissues in TCGA-BRCA cohort. (C and D) Quantitative analysis of AF9 and miR-5694 expression among four breast cancer subtypes in TCGA-BRCA cohort. Boxplots represent the median and interquartile range (IQR). (E) Correlation between AF9 and miR-5694 expression in clinical BLBC samples. (F) AF9 expression level in tumors prolongs breast cancer patients’ survival time. The data were mined from the Kaplan-Meier plotter database. (G) IHC analysis of breast cancer patients using anti-AF9 antibodies were performed. Images of four representative specimens (scale bar represents 50 μm). We divided the specimens into the following two groups according to the IHC scores: High-AF9 and Low-AF9. (H) Primary tumor tissues were collected from patients with or without metastasis. AF9 mRNA levels were tested in these tissues. (I) Schematic diagram of this study. Generally, we discovered a suppressor of breast cancer metastasis, AF9, which presented extremely low expression in BLBC cells due to miR-5694. In luminal A breast cancer cells or forced expression of AF9 in BLBC cells, AF9 interacts with Snail and hampers Snail transcriptional activity. Then, AF9 reconstitutes an activated state for EMT-related gene expression. (A–D and H) Two-tailed Student’s t test, ∗∗∗p < 0.001. (E) Pearson’s correlation test. Error bars represent the standard deviation (SD).

Fig 3: Snail represses 4E-BP1 expression at both the protein and mRNA levels. a HEK293, T47D, MCF7, and HCT116 cells with stable expression of Snail or vector control were analyzed by western blotting for the indicated proteins. b mRNA expression of the indicated genes was analyzed by quantitative RT-PCR in T47D, MCF7, and HCT116 cells with stable expression of Snail or vector control. The indicated gene expression was normalized against GAPDH and presented as a percentage of the expression level found in vector control cells. c HCT116, MDA-231, and SUM149 cells with stable expression of two different sets of Snail shRNAs (ShSnail_1 and ShSnail_2) or control shRNA (ShCtrl) were analyzed by western blotting for the indicated proteins. d mRNA expression of the indicated genes was analyzed by quantitative RT-PCR in HCT116, MDA-231, and SUM149 cells with stable expression of ShSnail_1, ShSnail_2, or ShCtrl. The indicated gene expression was normalized against GAPDH and presented as a fold increase over the expression level found in ShCtrl cells. All graphic data are presented as mean ± SEM (n = 3 technical replicates per condition). *P < 0.001 for Snail vs. vector or ShSnail vs. ShCtrl using Student's t-test

Fig 4: Snail influences mTOR/4E-BP1-controlled translation and polysome formation. a, b Snail-expressing a or -knockdown b cells and their respective controls were transfected with a bicistronic luciferase reporter that detects cap-dependent translation of the Renilla luciferase gene and cap-independent translation of the firefly luciferase gene. After 24 h, cells were treated with mTOR kinase inhibitors AZD8055 (100 nM), INK128 (100 nM), or DMSO for 12 h, followed by measurement of luciferase activities. The ratio of Renilla/firefly luciferase activities was calculated and presented as a percentage of the cap-dependent translation activity found in the DMSO-treated control cells. c Cap-dependent translation analysis of Snail wild-type (WT) and knockout (KO) HCT116 cells expressing 4E-BP1 shRNA or control shRNA treated with 100 nM INK128 for 12 h. d, e Snail-expressing T47D cells d or Snail-knockdown HCT116 cells e and their respective controls were treated with 100 nM AZD8055, 100 nM INK128, or DMSO for 12 h. Cell lysates were precipitated with m7GTP sepharose beads or immunoprecipitated with eIF4E antibody followed by western blot analysis for the indicated proteins. f–i Snail-expressing or vector control MCF7 cells f, g and Snail WT or KO HCT116 cells h, i were treated with 100 nM INK128 or DMSO as control for 12 h, followed by polysome analysis. The vertical blue line separates the polysomal (P) and monosomal (M) fractions. The P/M ratio, an index of translational efficiency, was calculated by comparing areas under the polysome and monosome peaks using NIH image J. The results are expressed as a percentage of the P/M ratio relative to the DMSO-treated vector g or Snail WT i controls. j, k Nascent protein synthesis was measured for Snail-expressing or vector control T47D cells j and Snail WT or KO HCT116 cells k treated with 100 nM INK128 or DMSO for 12 h. The results are expressed as a percentage of methionine analog incorporation relative to the DMSO-treated control cells. All graphic data are presented as mean ± SEM (n = 3 technical replicates per condition). *P < 0.01; # P < 0.001; NS, not significant using Student’s t-test

Fig 5: Transient and Sustained Snail-1-Induced Chromatin Marks Associated with Gene-Expression Clusters(A and B) ChIP-chip log2 heatmap of Snail-1, H3K4Ac, H3K27Ac, H3K4Me1, H3K4Me2, H3K4Me3, and H3K27Me3 immunoprecipitates at 6, 48, and 120 hr compared with 0 hr after Snail-1 induction. Heatmap results are shown for -2 kb to +2 kb promoter regions of genes whose expression is altered by Snail-1: early- (cluster I), late- (cluster II), and transiently (cluster III) repressed transcripts; and early- (cluster IV), late- (cluster V), and transiently (cluster VI) induced transcripts.See also Figures S3 and S4.