EMMPRIN Down-regulating miR-106a/b Modifies Breast Cancer Stem-like Cell Properties via Interaction with Fibroblasts Through STAT3 and HIF-1α

Abstract

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a heavily glycosylated protein and expresses in cancer cells widely, which plays important roles in tumor progression. However, the role of EMMPRIN in breast cancer stem-like cell properties by interaction with fibroblasts is not known. In the present study, we investigated the effects of fibroblasts on breast cancer stem-like cells. We found that fibroblasts activated by co-cultured breast cancer cells produced higher levels of EMMPRIN, which stimulated the stem-like cell specific, self-renewal and sphere-forming phenotype in breast cancer cells. Increased EMMPRIN expression in activated fibroblasts increased the expression of STAT3 and HIF-1α and showed cancer stem-like cell features in breast cancer cells. We also found that EMMPRIN could down-regulate miR-106a and miR-106b expression in breast cancer cells, which led to activating STAT3 and enhancing HIF-1α expression. Our results illustrated that EMMPRIN has an important role in breast cancer stem-like cells by activation STAT3/HIF-1α through interaction with cancer cells and fibroblasts. The study for the first time indicated that cancer cells and fibroblasts interaction promotes breast cancer cells showing stem-like cells through up-regulation EMMPRIN, and led to inhibiting miR-106a/b expression which targets both STAT3 and HIF-1α expression.

Figure 1. EMMPRIN expression increased in the breast cancer cells with fibroblasts interaction.

(A) EMMPRIN increased in the conditioned medium (CM) from six breast cancer cells co-cultured with Hs578Bst fibroblasts. EMMPRIN was examined by ELISA. (B) EMMPRIN mRNA was enhanced in six breast cancer cell lines with CM from co-cultured cancer cells with fibroblasts compared with CM from fibroblasts. (C) EMMPRIN protein levels increased in SUM102 and BT474 cells with CM. CM was collected from the medium of co-cultured breast cancer cells and Hs578Bst fibroblasts. The data presented were shown as means ± s.d. collected from three independent experiments. *p < 0.05, **p < 0.01.

Figure 2. Recombinant EMMPRIN increases breast cancer cells to show stem-like cell properties.

EMMPRIN increased the mammosphere formation of breast cancer cells. SUM102 (A) and BT474 (B) breast cancer cells were treated with hrEMMPRIN and the cells were cultured in stem cell specific medium for 10 days and then numbers of sphere were counted. (C) hrEMMPRIN increased CD44 expression and decreased CD24 expression in breast cancer cells. BT474 breast cancer cells were treated with human recombinant EMMPRIN, and then the cells were fixed for immunofluoresence assay. (D) hrEMMPRIN increased the percentage of CD44⁺CD24⁻ breast cancer cells. BT474 breast cancer cells were treated with human recombinant EMMPRIN, and then the cells were collected for flow cytometry analysis. (E) Data analysis from D. The data presented were shown as means ± s.d. collected from three independent experiments. *p < 0.05, **p < 0.01.

Figure 3. Knocking down of EMMPRIN decreases breast cancer cells to show stem- like cell properties.

(A,B) EMMPRIN knocking down decreased the percentage of CD44⁺CD24⁻ breast cancer cells. SUM102 and BT474 breast cancer cells were infected lentivirus mediated EMMPRIN knocking down, and the cells were treated with conditioned medium from Hs578Bst fibroblasts for 3 days, and then the cells were collected for mammosphere formation assay. The conditioned medium were collected from Hs578Bst fibroblasts or the breast cancer cells co-cultured with fibroblasts for 3–5 days. (C,D) Down-regulation of EMMPRIN decreased mammosphere formation in breast cancer cells. SUM102 and BT474 breast cancer cells with EMMPRIN knocking down or the control were treated with conditioned medium from 1068SK fibroblasts for 3 days and then the cells were digested and used for mammosphere formation assay. EMMPRIN knocking down induced changes of breast cancer stem cell markers. SUM102 (E,F) BT474 breast cancer cells were infected with LV-EMMPRIN shRNA, and the cells were treated with conditioned medium from fibroblasts 1068SK or Hs578Bst for 3 days, and then the protein were extracted from the cells for western blotting. The data presented were shown as means ± s.d. collected from three independent experiments. *p < 0.05, **p < 0.01.

Figure 4. HIF-1α involves in promoting breast cancer stem-like cell properties by EMMPRIN-STAT3 signal pathway.

(A) SUM102 and BT474 breast cancer cells were exposed to hrEMMRPIN for 24 h and total protein was extracted from the cells for western blotting. (B) STAT3 knocking down induced changes of breast cancer stem-like cell markers. STAT3 were knocked down in SUM102 and BT474 breast cancer cells by siRNA, and the cells were treated with conditioned medium from fibroblasts 1068SK or Hs578Bst for 3 days, and then the protein were extracted from the cells. The conditioned medium were collected from fibroblasts or the breast cancer cells co-cultured with fibroblasts for 3–5 days. (C) Knocking down of STAT3 decreased the percentage of CD44⁺CD24⁻ breast cancer cells of BT474 treated with human recombinant EMMPRIN by flow cytometry analysis. (D) Data analysis from C. (E) HIF-1α knocking down induced changes of breast cancer stem cell markers. HIF-1α were knocked down in SUM102 and BT474 breast cancer cells by siRNA. (F) HIF-1α knocking down induced changes of breast cancer stem cell markers by immonofluoresence. (G) HIF-1α knocking down decreased mammosphere formation in breast cancer cells. The data presented were shown as means ± s.d. collected from three independent experiments. **p < 0.01.

Figure 5. EMMPRIN attentuates miR-106a/b expression in breast cancer cells.

(A) The predicted target genes of STAT3 and HIF-1α genes contain the binding sites for miRNAs by bioinformatic analysis. (B) EMMPRIN decreased miR-106a and miR-106b expression in SUM102 and BT474 breast cancer cells. (C,D) breast cancer cells to CM from the co-cultured Hs578Bst and BT474, BT474-STAT3(−), BT474-HIF-1α(−) or SUM102, SUM102-STAT3(−), SUM102-HIF-1α(−) cells. The total RNA was extracted for miRNA expression. The data presented were shown as means ± s.d. collected from three independent experiments. **p < 0.01.

Figure 6. miR-106a/b suppresses both STAT3 and HIF-1α expression in breast cancer cells.

(A) The 3′-UTR of the STAT3 and HIF-1α genes contains binding sites for miR-106a/b according to bioinformatic analysis. (B,C) miR-106a/b suppressed the expression of a luciferase reporter gene harbouring the 3′-UTR of STAT3 or HIF-1α. The pGL3 plasmid was modified by adding the human 3′-UTR or the 3′-UTR with mutations in regions complementary to miR-106a/b seed regions behind the firefly luciferase gene. BT474 cells were transiently co-transfected with miR-control or miR-106a or miR-106b together with the indicated luciferase constructs, and luciferase activity was analysed 48 h later. (D,E) miR-106a or miR-106b restoration down-regulated STAT3 mRNA in BT474 and SUM102 cells. Cells were transfected with miR-106a, miR-106b or miR-control for 48 hours, then collected for real-time PCR. (F,G) miR-106a or miR-106b restoration down-regulated HIF-1α mRNA in BT474 and SUM102 cells. Cells were transfected with miR-106a, miR-106b or miR-control for 48 hours, then collected for real-time PCR. (H) miR-106a or miR-106b restoration down-regulated STAT3 and HIF-1α in breast cancer cells. Cells were transfected with miR-106a or miR-control for 48 hours, then collected for western blot analysis. The data presented are shown as means ± s.d. collected from three independent experiments. *p < 0.05, **p < 0.01.

Figure 7. miR-106a/b attenuates breast cancer stem-like cell properties by inhibition of STAT3 and HIF-1α.

(A) SUM102 cells were tranfected with miR-106a or miR-106b and STAT3 siRNA or HIF-1α in the present of CM from the co-cultured with fibroblasts and cancer cells. Sphere formation was assayed. (B) SUM102 cells were tranfected with miR-106a or miR-106b and STAT3 siRNA or HIF-1α in the present of CM from the co-cultured with fibroblasts and cancer cells. The second sphere formation was assayed. (C) SUM102 cells were tranfected with miR-106a or miR-106b and STAT3 siRNA or HIF-1α in the present of CM from the co-cultured with fibroblasts and cancer cells. Stem cell markers were examined by western blotting. (D) Protein bands from (C) were quanlified. The data presented are shown as means ± s.d. collected from three independent experiments. *p < 0.05, **p < 0.01.