Fig 2: Targeting MUC1-C inhibits HPAF-II and AsPC-1 tumorigenicity. (A) Six-week-old female nude mice were injected subcutaneously in the flank with 3.5 × 106 HPAF-II/tet-MUC1shRNA cells. Mice were pair-matched into two groups when tumors reached approximately 150 mm3 and were fed without and with DOX. Tumor volumes are expressed as the mean ± SD for six mice. (B) Lysates from HPAF-II/tet-MUC1shRNA tumors obtained on day 18 were immunoblotted with antibodies against the indicated proteins. (C) Six-week-old female nude mice were injected subcutaneously in the flank with 2 × 106 HPAF-II cells. Mice were pair-matched into two groups when tumors reached approximately 100 mm3 and were treated intraperitoneally each day with phosphate-buffered saline or GO-203 at a dose of 12 μg/gm body weight. Tumor volumes are expressed as the mean ± SD for four mice. (D) Lysates from control and GO-203-treated tumors obtained on day 28 were immunoblotted with antibodies against the indicated proteins. (E) Six-week-old female nude mice were injected subcutaneously in the flank with 3.5 × 106 AsPC-1/tet-MUC1shRNA cells. Mice were pair-matched into two groups when tumors reached approximately 150 mm3 and were fed without and with DOX. Tumor volumes are expressed as the mean ± SD for six mice. (F) Lysates from AsPC-1/tet-MUC1shRNA tumors obtained on day 24 were immunoblotted with antibodies against the indicated proteins.

Fig 3: Silencing MUC1-C induces DARs with changes in accessibility that associate with biological processes. A, Concordance of closing (left) and opening (right) DARs in MUC1-C–silenced DU-145 and BT-549 cells. B and C, Associations of closing DARs in DU-145 (B) and BT-549 (C) cells with the indicated GO BIOLOGICAL PROCESSES. D and E, Associations of opening DARs in DU-145 (D) and BT-549 (E) cells with the indicated GO BIOLOGICAL PROCESSES.

Fig 4: MUC1-C binds directly to JUN and regulates AP-1 target gene signatures. A, Schema of the MUC1-C protein with the amino acid sequence of the 72 aa cytoplasmic domain (above). Schema of the JUN 331 aa protein highlighting regions of the transactivation (aa 1-194) and DNA binding (aa 195-331) domains (below). B, GST and GST-MUC1-CD (FL; 1-72 aa) were incubated with purified full-length JUN (1-331). C, GST, GST-MUC1-CD, GST-MUC1-CD (1–45) and GST-MUC1-CD (45-72) were incubated with purified JUN (1-331). The adsorbates and input were immunoblotted with anti-JUN. Input of the GST proteins was assessed by Coomassie blue staining. D, GST, GST-JUN (1-331), GST-JUN (1-194), and GST-JUN(195-331) were incubated with purified MUC1-CD (1-72). The adsorbates and input were immunoblotted with anti-MUC1-CD. E, GST, GST-MUC1-CD, and GST-MUC1-CD (mutation of CQC to AQA) were incubated with purified JUN (1-331). The adsorbates and input were immunoblotted with anti-JUN. F and G, RNA-seq was performed in triplicate on DU-145/tet-MUC1shRNA (F) and BT-549/tet-MUC1shRNA (G) cells treated with vehicle or doxycycline (DOX) for 7 days. The datasets were analyzed with GSEA using the AP-1 Q4 target gene signature.

Fig 5: MUC1, PBRM1, and IRF1 regulate similar gene sets that are shared with TNBC tumors. A, RNA-seq was performed in triplicate on BT-549 cells silenced for MUC1, PBRM1, and IRF1. The datasets were analyzed for effects of MUC1-C silencing on repressed and activated genes as depicted by the Volcano plots. B, Venn diagram depicting the overlap of 196 downregulated genes in BT-549 cells silenced for MUC1, IRF1, and PBRM1 (Supplementary Table S3). C, RNA-seq datasets from BT-549 cells silenced for MUC1, IRF1, and PBRM1 were analyzed with GSEA for enrichment distribution using the indicated IFN-related gene signatures. D, Heatmaps of 72 common genes between the HALLMARK INTERFERON GAMMA RESPONSE and HALLMARK INTERFERON ALPHA RESPONSE signatures in BT-549 cells silenced for MUC1, IRF1, and PBRM1. The row indicator shows the 45 common genes from the 196 DEGs identified in B (yellow, Supplementary Table S4). E, Common up- and downregulated IFN response genes (detectable 66 genes out of 72 genes) in MUC1-high versus MUC1-low TNBC tumors from the METABRIC and TCGA-BRCA datasets. The outline (gold/gray) represents common DEGs in BT549 cells identified in D. The red/gray/blue dot indicates significantly up/downregulated genes (MUC1high vs. MUC1low) in one or both of the TCGA-BRCA/METABRIC cohorts.