Fig 2: Downregulating MMP1 inhibits tumor growth and induces tam sensitivity in xenografted mice. (A) Immunohistochemical analysis of MMP1 expression was conducted on xenografted tumor tissues containing MCF-7 and MCF-7/tamR cells. Three tissue sets were analyzed, and the protein expression is depicted in the bar graph (B). Representative images are shown. Scale bar, 100 μm. MCF-7/tamR cells stably transfected with shMMP1 or shNC were subcutaneously injected into BALB/c nude mice. The tumor volume was examined every week for eight weeks. Corn oil (C) or Tam (E) was administered according to intraperitoneal method after 3 weeks of cell transplantation. At week 8, mice were sacrificed to extract untreated (D) and tam-treated tumor tissues (F), of which size is denoted in a bar graph (G). n = 5 for each group. * p < 0.05 versus shNC control; # p < 0.05 versus shNC tam; ± p < 0.05.

Fig 3: MMP1 is hypomethylated and upregulated in tamR breast cancer compared to that in tamS breast cancer. (A) Heatmap of the 20 most hypomethylated genes in MCF-7/tamR cells compared to those in parental MCF-7 cells. Data of two arrays are presented. Methylation level of the CpG site at the MMP1 promoter (B) and mRNA level (C) were analyzed by MSP and qRT-PCR in breast cancer cells. The data are presented as mean ± SE of three independent experiments. (D) Western blot analysis was performed to detect MMP1 protein level in cultured cells. (E) Hypomethylation and upregulation of MMP1 were identified by MSP and qPCR in breast cancer tissue of patients. n: number of samples. (F) The association between the CpG methylation and mRNA expression of MMP1. (G) Kaplan–Meier survival curve of MMP1 level in breast cancer patients. Samples (n = 1379) were categorized into tertiles based on MMP1 expression. Distant metastasis-free survival (DMFS) was compared for all tumor samples using the log-rank test (p < 0.001). * p < 0.05, ** p < 0.01.

Fig 4: Effects of MMP1 on apoptosis and necrosis of MCF-7/tamR cells. Flow cytometry was performed after downregulating MMP1 with a stably expressed shRNA lentiviral vector. Representative flow cytometry images at 0 μM (A), 1 μM (B), and 2 μM (C) of tam are shown. Top and bottom images are for shNC and shMMP1, respectively. Cells in each quadrate of the FACS image represent, clockwise from the upper left, necrosis (green), late apoptosis (blue), early apoptosis (red), and live cells (black). Ratio of live cells (D), apoptotic cells (E), and necrotic cells (F) are shown in a bar graph. All assays were performed for three independent experiments, and data presented as mean ± SE. shMMP1, short hairpin RNA against MMP1; shNC, negative control shRNA. * p < 0.05, ** p < 0.01; ## p < 0.01 versus shMMP1 control.