Fig 1.
MMP1 is upregulated in MCF-7/ADR cells compared to the parental MCF-7 cells.
A. Heat map of the top 50 upregulated genes (left) and 50 downregulated genes (right) in MCF-7/ADR cells compared to the parental MCF-7 cells. Red: up-regulation; Blue: down-regulation. Raw microarray data was obtained from GEO dataset (accession: GDS4084). B. The protein-protein interaction (PPI) network of the 100 most upregulated and the 100 most downregulated genes analyzed by using the (STRING) (http://string-db.org/) database. Only experimentally validated interactions with a high confidence score ≥ 0.70 were included.
Fig 2.
High MMP1 expression is associated with worse survival outcomes in breast cancer patients after systematic therapy.
A-D. The OS curves (A and C) and RFS curves (B and D) of all breast cancer patients (A and B) and in ER+ breast cancer patients (C and D) with high or low MMP1 expression. Data was retrieved from the Kaplan Meier plotter (http://kmplot.com/analysis/). E-G. Kaplan-Meier survival analysis showing the relationship between MMP1 expression and AE-free survival (E and G) and MR-free survival (F and H) in breast cancer patients using bc-GenExMiner 4.0. ERm: mixed ER status.
Fig 3.
MMP1 upregulation results in enhanced MDR in MCF-7 cells.
A. Western blotting of MMP1 expression in MCF-7/ADR and MCF-7 cells. B-C. QRT-PCR analysis of MMP1 mRNA (up) and western blotting (down) of MMP1 protein expression in MCF-7 cells 24 h after transfection of MMP1 expression vector or the empty control (B) and in MCF-7/ADR cells 24 h after transfection of MMP1 siRNA or the empty control (C). D-I. Adriamycin, vincristine and paclitaxel IC50 in MCF-7 cells with or without MMP1 expression (D-F) and in MCF-7/ADR cells with or without MMP1 knockdown (G-I). J-K. Representative flow cytometric images of cells with active caspase-9 in MCF-7 cells with or without MMP1 overexpression and in MCF-/ADR cells with or without MMP1 knockdown (J) and in the cells with combined adriamycin treatment (20 nM for MCF-7 cells and 1 μM for MCF-7/ADR cells) for 48 h (K). L-M. The proportion of cells with active caspase-9 showed in J (L) and K (M). ** p<0.01.
Fig 4.
Slug modulates MMP1 expression in MCF-7/ADR cells.
A. Western blotting of E-cadherin, N-cadherin, Slug and MMP1 expression in MCF-7 cells and in MCF-7/ADR cells. B. Immunofluorescent staining of N-cadherin and Slug in MCF-7 and in MCF-7/ADR cells. Green: N-cadherin; Green: Slug; Blue: DAPI. C-H. QRT-PCR analysis of Slug (C and F) and MMP1 (D and G) mRNA expression and western blotting of Slug and MMP1 protein expression (E and H) in MCF-7 cells with or without Slug overexpression (C-E) and in MCF-7/ADR cells with or without Slug knockdown (F-H). ** p<0.01.
Fig 5.
Slug upregulation leads to aberrant MMP1 expression via directly binding to the promoter of MMP1.
A-B. Slug binding positions (A) and the predicted Slug binding sites in the promoter region of MMP1 (B). C. The luciferase reporter constructs of the truncated MMP1 promoters were introduced into HEK-293 cells transfected with Slug cDNA ORF clone or the empty control. Luciferase activity was measured 48 h post-transfection. D. Fold-enrichment of Slug binding at the MMP1 promoter relative to background in MCF-7 and MCF-7/ADR cells were measured by qRT-PCR. Upon normalization to GAPDH, results were expressed as n-fold compared to IgG. Data are expressed as mean ± SD from three independent experiments. ** p<0.01.