Fig 2: MUC1-C is upregulated in KRAS mutant PDAC cells and is necessary for self-renewal. (A) Analysis of the TCGA-PAAD/PDAC dataset comparing MUC1 expression in mutant (n = 108) and wild-type (n = 55) KRAS PDAC tumors. (B) Lysates from the designated PDAC cell lines were immunoblotted with antibodies against the indicated proteins. (C and D) HPAF-II (C) and AsPC-1 (D) cells stably expressing a tet-CshRNA or tet-MUC1shRNA were treated with vehicle or 500 ng/ml DOX for 6 days. Lysates were immunoblotted with antibodies against the indicated proteins. (E) Representative images are shown for the indicated HPAF-II (upper) and AsPC-1 (lower) cells suspended in tumorsphere medium in the presence of vehicle or DOX for 7 days. (F) Number of tumorspheres from three separate determinations of the indicated HPAF-II (blue bars) and AsPC-1 (red bars) cells treated with vehicle or DOX. The asterisk (*) denotes a significant difference from that obtained for DOX-treated tet-CshRNA cells.

Fig 3: MUC1-C is necessary for induction of RNA PRRs. (A–D) RNA-seq was performed in triplicate on BT-549 cells expressing a tet-MUC1shRNA, which were treated with vehicle or DOX for 7 days. Candidate pathway enrichment plots for the HALLMARK INTERFERON ALPHA RESPONSE (A), HALLMARK INFLAMMATORY RESPONSE (B), KEGG RIG-I LIKE RECEPTOR SIGNALING (C) and REACTOME DDX58 IFIH1 MEDIATED INDUCTION OF INTERFERON ALPHA BETA (D) gene signatures. (E,F) BT-549/tet-MUC1shRNA cells were treated with vehicle or DOX for 7 days. MUC1-C, RIG-I and MDA5 mRNA levels were analyzed by qRT-PCR (E). The results (mean ± SD of 4 determinations) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1). The asterisk (*) denotes a p-value < 0.05. Lysates were immunoblotted with antibodies against the indicated proteins (F, left). Lysates from BT-549/CshRNA and BT-549/MUC1shRNA#2 were immunoblotted with antibodies against the indicated proteins (F, right). (G,H). BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days and stimulated with poly I:C for 12 h (G) or IFN-β for 4 h (H) were analyzed for RIG-I and MDA5 mRNA levels by qRT-PCR. The results (mean ± SD of 4 determinations) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Uncropped Western Blots can be found at supplemental original images.

Fig 4: MUC1 enhances CCCP-induced mitophagy.A, C Western blotting was performed to detect proteins as indicated in MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells treated with DMSO, 5 μM CCCP (24 h) or 5 μM CCCP (24 h) + 10 μM Mdivi-1 (24 h). Quantification of mitochondrial proteins was calculated. B, D Western blotting was performed to detect proteins as indicated in BT549/g-CTL and BT549/g-MUC1 cells treated with DMSO, 10 μM CCCP (24 h) or 10 μM CCCP (24 h) + 10 μM Mdivi-1 (24 h). Quantification of mitochondrial proteins was calculated. E After treatment with DMSO, 30 μM CCCP (24 h) or 30 μM Mdivi-1 (24 h), western blotting was performed to detect proteins with the indicated antibodies in HeLa229/g-CTL, HeLa229/g-MUC1, and HeLa229/g-MUC1 cells treated with 1 μg/ml DOX for 48 h to induce MUC1 expression (DOX+) and without DOX treatment (DOX−) as a negative control. Quantification of mitochondrial proteins was calculated. F The ratio of mtDNA (COX1) to ncDNA (B2M) (mtDNA/ncDNA) in MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells treated with DMSO, 5 μM CCCP (24 h) or 10 μM Mdivi-1 (24 h). G The ratio of mtDNA (COX1) to ncDNA (B2M) (mtDNA/ncDNA) in BT549/g-CTL and BT549/g-MUC1 cells treated with DMSO, 10 μM CCCP (24 h) or 10 μM Mdivi-1 (24 h). H The ratio of mtDNA (COX1) to ncDNA (B2M) (mtDNA/ncDNA) in HeLa229/g-CTL, HeLa229/g-MUC1, and HeLa229/g-MUC1 cells treated with 1 μg/ml DOX for 48 h to induce MUC1 expression (DOX+) and without DOX treatment (DOX−) as a negative control. HeLa229 cells were treated with DMSO, 30 μM CCCP (24 h) or 30 μM Mdivi-1 (24 h). I, J Average length of mitochondria per cell (I) and the number of mitochondria per cell (J) in TEM micrographs from Fig. S1I. More than 16 cells in each graph were analyzed. K Representative fluorescence images from (Fig. S1J) with Mtphagy Dye (Red), Lysosome Dye (Green), and MitoBright (Purple) staining in MDA-MB-468 cells stimulated with DMSO, 5 μM CCCP and 500 nM rapamycin. The arrow indicates colocalization pots. Rapa: Rapamycin. Bars: 20 μm. L HeLa229/g-CTL and HeLa229/g-MUC1 cells were treated with 30 μM CCCP for the indicated times, and western blotting was performed to detect pSer65-Ub and total-Ub. The data represent the mean ± SD from three independent experiments. Differences between linked groups were evaluated by a two-tailed Student’s t test. *P < 0.05, **P < 0.01; ***P < 0.001; NS not significant.

Fig 5: Targeting MUC1-mitophagy signaling inhibits tumor malignancy.A MDA-MB-468 cells were treated with 5 μM CP2 (48 h) and 5 μM GO203 (48 h) and then treated with DMSO, 5 μM CCCP (24 h) or 10 μM Mdivi-1 (24 h). Western blotting was performed to detect proteins as indicated. Mitochondrial proteins were quantified. GO-203, a cell-penetrating peptide containing a polyArg cell transduction domain linked to the CQCRRKN sequence ([R]9-CQCRRKN; all D-amino acids), can bind directly to the MUC1-C cytoplasmic domain. In contrast, CP2 is a GO203 analog (CP2: [R]9-AQARRKN) that is ineffective in inhibiting MUC1-C. CP2 acted as a control for GO203. B BT549 cells were treated with 5 μM CP2 (48 h) and 5 μM GO203 (48 h) and then treated with DMSO, 10 μM CCCP (24 h) or 10 μM Mdivi-1 (24 h). Western blotting was performed to detect proteins as indicated. Mitochondrial proteins were quantified. C T47D cells were treated with 10 μM CP2 (48 h) and 10 μM GO203 (48 h) and then treated with DMSO, 10 μM CCCP (24 h), or 10 μM Mdivi-1 (24 h). Western blotting was performed to detect proteins as indicated. Mitochondrial proteins were quantified. D SKBR3 cells were treated with 10 μM CP2 (48 h) and 10 μM GO203 (48 h) and then treated with DMSO, 10 μM CCCP (24 h) or 10 μM Mdivi-1 (24 h). Western blotting was performed to detect proteins as indicated. Quantification of mitochondrial proteins was calculated. E The cell viability assay was performed in MDA-MB-468 cells treated with 5 μM GO203 and 10 μM Mdivi-1 alone or in combination. F The mammosphere formation assay was performed in MDA-MB-468 cells treated with 5 μM GO203 and 10 μM Mdivi-1 alone or in combination. The number of mammospheres was calculated. GO + Md: GO203 + Mdivi-1. G The cell viability assay was performed in BT549 cells treated with 5 μM GO203 and 10 μM Mdivi-1 alone or in combination. H A mammosphere formation assay was performed in BT549 cells treated with 5 μM. GO203 and 10 μM Mdivi-1 alone or in combination. The number of mammospheres was calculated. I MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells were transfected with shCTL, shPink1-1, and shPink1-2 lentiviral vectors for 48 h and then treated with DMSO and 5 μM CCCP (2 h). The cell viability assay was performed. J MDA-MB-468/g-CTL and MDA-MB-468/g-MUC1 cells were transfected with shCTL, shPink1-1, and shPink1-2 lentiviral vector for 48 h, then treated with DMSO and 5 μM CCCP (2 h). Cells were subjected to the mammospheres formation assay. The number of mammospheres >50 μm in diameter was calculated. The data represent mean ± SD from three independent experiments. K–M Tumor sizes (K, L) and weights (M) were compared in mice that received four different treatments (PBS, GO203, Mdivi-1 and G0203 + Mdivi-1). G + M: GO203 + Mdivi-1. Differences between linked groups were evaluated by a two-tailed Student’s t test. *P < 0.05, **P < 0.01; ***P < 0.001; NS not significant.