BMP-4 enhances epithelial mesenchymal transition and cancer stem cell properties of breast cancer cells via Notch signaling

Abstract

Bone morphogenetic protein (BMP) signaling and Notch signaling play important roles in tumorigenesis in various organs and tissues, including the breast. BMP-4 enhanced epithelial mesenchymal transition (EMT) and stem cell properties in both mammary epithelial cell line and breast carcinoma cell line. BMP-4 increased the expression of EMT biomarkers, such as fibronectin, laminin, N-cadherin, and Slug. BMP-4 also activated Notch signaling in these cells and increased the sphere forming efficiency of the non-transformed mammary epithelial cell line MCF-10A. In addition, BMP-4 upregulated the sphere forming efficiency, colony formation efficiency, and the expression of cancer stem cell markers, such as Nanog and CD44, in the breast carcinoma cell line MDA-MB-231. Inhibition of Notch signaling downregulated EMT and stem cell properties induced by BMP-4. Down-regulation of Smad4 using siRNA impaired the BMP-4-induced activation of Notch signaling, as well as the BMP-4-mediated EMT. These results suggest that EMT and stem cell properties are increased in mammary epithelial cells and breast cancer cells through the activation of Notch signaling in a Smad4-dependent manner in response to BMP-4.

Figure 1

BMP-4 induces the EMT and the activation of Notch signaling in MCF-10A cells. (a) Western blot analysis of phosphorylated Smad1/5/9 proteins (pSmad1/5/9) in extracts of MCF-10A cells treated with BMP-4 (0, 5, 10, 25, 50 ng/ml). α-Tubulin was used as an internal control. The full-length blots/gels are presented in Supplementary Fig. 1a,b. (b) Densitometric analysis of western blot data from (a). Data are presented as the mean ± SD and p-values were calculated using Student’s t-test (3 independent experiments). (c) Western blot analysis of pSmad1/5/9 in extracts of MCF-10A cells treated with BMP-4 (50 ng/ml) for the indicated times. α-Tubulin was used as an internal control. The full-length blots are presented in Supplementary Fig. 1c,d. (d) Real-time PCR analysis of Smad6 expression in MCF-10A cells treated with BMP-4 (50 ng/ml) for the indicated times. Human ribosomal protein S9 (RPS9) was used as an internal control. Data are presented as the mean ± SD and p-values were calculated using a Student’s t-test (3 independent experiments). (e) Representative images of MCF-10A cells treated with BMP-4 (50 ng/ml) or vehicle (CON) for 24 hours. Scale bar: 100 µm. (f) Immunocytochemistry of MCF-10A cells treated with BMP-4 (50 ng/ml) or vehicle (CON) for 24 hours. E-cadherin (E-cad) was stained either green or red. ZO-1 or N-cadherin (N-cad) were stained green. β-Catenin (β-Cat) was stained red. Actin (Act) was stained red with phalloidin and nuclei were stained blue with DAPI. Mer, merged images. Scale bar: 50 µm. (g) Real-time PCR analysis of EMT-associated genes and Notch target genes in MCF-10A cells treated with BMP-4 (50 ng/ml) or vehicle (CON) for 24 hours. Endogenous BMP-4 expression was analyzed at 5 hours post treatment of BMP-4. Human ribosomal protein S9 (RPS9) was used as an internal control. Data are presented as the mean ± SD and p-values were calculated using a Student’s t-test (3 independent experiments). (h) Western blot analysis of EMT-associated proteins and Notch target proteins in the MCF-10A cells treated with BMP-4 (50 ng/ml) or vehicle (CON) for 24 hours. The full-length blots are presented in Supplementary Fig. 4.

Figure 2

Noggin inhibits the BMP-4-induced EMT and increases the expression of Notch target genes in MCF-10A cells. (a) Western blot analysis of phosphorylated Smad1/5/9 proteins (pSmad1/5/9) in extracts of MCF-10A cells treated with 200 ng/ml Noggin for 30 minutes prior to BMP-4 (50 ng/ml) treatment for the indicated times. α-Tubulin was used as an internal control. The full-length blots are presented in Supplementary Fig. 5. (b) Real-time PCR analysis of Smad6 expression in MCF-10A cells treated with 200 ng/ml Noggin for 30 minutes prior to BMP-4 (50 ng/ml) treatment for 24 hours. Human ribosomal protein S9 (RPS9) was used as an internal control. (c) Representative images of MCF-10A cells treated with 200 ng/ml Noggin for 30 minutes prior to BMP-4 (50 ng/ml) treatment for 24 hours. Scale bar: 100 µm. (d) Real-time PCR analysis of EMT-associated genes and Notch target genes in MCF-10A cells treated with 200 ng/ml Noggin for 30 minutes prior to BMP-4 (50 ng/ml) treatment for 24 hours. Human ribosomal protein S9 (RPS9) was used as an internal control. Data are presented as the mean ± SD and p-values were calculated using the Student’s t-test (3 independent experiments). (e) Western blot analysis of fibronectin, laminin, and Jagged-1 in MCF-10A cells treated with 200 ng/ml Noggin for 30 minutes prior to BMP-4 (50 ng/ml) treatment for 24 hours. α-Tubulin was used as an internal control. The full-length blots are presented in Supplementary Fig. 6.

Figure 3

γ-Secretase inhibitor prevents the BMP-4-induced EMT and the increase in expression of Notch target genes in MCF-10A cells. (a,b) Real-time PCR analysis of Jagged-1 or Hey1 in MCF-10A cells treated with γ-secretase inhibitor X (GSIX; 5 μM) for 30 minutes prior to BMP-4 (50 ng/ml) treatment for 24 hours. Human ribosomal protein S9 (RPS9) was used as an internal control. (c) Representative images of MCF-10A cells treated with GSIX (5 μM) for 30 minutes prior to BMP-4 (50 ng/ml) treatment for 24 hours. Scale bar: 100 µm. (d) Real-time PCR analysis of fibronectin, laminin, N-cadherin, and Slug genes in MCF-10A cells treated with GSIX (5 μM) for 30 minutes prior to BMP-4 (50 ng/ml) treatment for 24 hours. Human ribosomal protein S9 (RPS9) was used as an internal control. Data are presented as the mean ± SD and p-values were calculated using a Student’s t-test (3 independent experiments). (e) Western blot analysis of fibronectin, laminin, and Jagged-1 in MCF-10A cells treated with GSIX (5 μM) for 30 minutes prior to BMP-4 (50 ng/ml) treatment for 24 hours. α-Tubulin was used as an internal control. The full-length blots are presented in Supplementary Fig. 7.

Figure 4

Smad4 knockdown prevents the BMP-4-induced EMT and the increase in expression of Notch target genes in MCF-10A cells. (a,b) mRNA and protein levels of Smad4 in MCF-10A cells transfected with Smad4 siRNAs (siSmad4 #1 or siSmad4 #2) or a control siRNA (siCON). Human ribosomal protein S9 (RPS9) was used as an internal control for real-time PCR and α-tubulin was used as an internal control for Western blot analysis. The full-length blots are presented in Supplementary Fig. 8. (c) Real-time PCR to analyze Smad6 mRNA expression in MCF-10A cells transfected with Smad4 siRNAs (siSmad4 #1 or siSmad4 #2) or a control siRNA (siCON). (d) Representative images of MCF-10A cells transfected with a Smad4 siRNA (siSmad4 #1) or a control siRNA (siCON) prior to BMP-4 (50 ng/ml) treatment for 24 hours. Scale bar: 100 µm. (e,f) Real-time PCR analysis of fibronectin, laminin, N-cadherin, and Hey1 mRNA expression in MCF-10A cells transfected with Smad4 siRNAs (siSmad4#1 in e or siSmad4#2 in f) or a control siRNA (siCON) prior to BMP-4 (50 ng/ml) treatment for 24 hours. Human ribosomal protein S9 (RPS9) was used as an internal control. Data are presented as the mean ± SD and p-values were calculated using a Student’s t-test (3 independent experiments).

Figure 5

BMP-4 enhances the mammosphere formation ability of MCF-10A cells. (a) Mammospheres formed by MCF-10A cells treated with BMP-4 (50 ng/ml) and/or Noggin (200 ng/ml) for 9 days. Scale bar: 100 µm. (b) Quantitative data showing the fold difference in the mammosphere forming ability of MCF-10A cells treated with BMP-4 and/or Noggin. (c) Mammospheres formed by MCF-10A cells treated with BMP-4 (50 ng/ml) and/or γ-secretase inhibitor X (GSIX; 5 μM) for 9 days. Scale bar: 100 µm. (d) Quantitative data showing the fold difference in the mammosphere forming ability of MCF-10A cells treated with BMP-4 and/or GSIX. Data are presented as the mean ± SD and p-values were calculated using a Student’s t-test (3 independent experiments).

Figure 6

BMP-4 enhances EMT, Notch signaling, cancer stem cell capacity, and tumorigenesis in MDA-MB-231 cells. (a) Phosphorylated Smad1/5/9 proteins (pSmad1/5/9) detected in MDA-MB-231 cells treated BMP-4 (50 ng/ml) for the indicated time durations. The full-length blots have been presented in Supplementary Fig. 9. (b,c) Real-time PCR analysis (b) and Western blot analysis (c) of the expression of Slug, Jagged-1, Hes1, and CD44 in MDA-MB-231 cells treated with BMP-4 (50 ng/ml) for 72 hours. The full-length blots have been presented in Supplementary Fig. 10. (d) Real-time PCR analysis of the expression of Slug, Jagged-1, Hes1, CD44, and Nanog in MDA-MB-231 cells treated with Noggin for 30 minutes prior to BMP-4 treatment for 72 hours. The red lines indicate the mean values. (e) Mammospheres formed by MDA-MB-231 cells treated with BMP-4 (0, 50 or 100 ng/ml) for 10 days (Scale bar: 100 µm, n = 6 wells, 2 independent experiments). (f,g) Colony formation efficiency of MDA-MB-231 cells treated with Noggin or GSIX for 30 minutes prior to BMP-4 treatment for 72 hours. (n = 6 wells, 3 independent experiments). (h,i) Colony formation efficiency of MDA-MB-231 cells transfected with a Jagged-1 siRNA or a control siRNA prior to BMP-4 treatment for 72 hours (n = 6 wells, 3 independent experiments). (j) Migration of bone marrow-derived mesenchymal stem cells toward indirectly co-cultured MDA-MB-231 cells. For the co-culture, MDA-MB-231 cells were cultured at 3 different densities (CON; no cells, Low; 1 × 10⁴ cells/cm², Medium; 2 × 10⁴ cells/cm², High; 3 × 10⁴ cells/cm²). MDA-MB-231 complete culture media including 10% FBS was used as a positive control (2 independent experiments). (k) Migration of bone marrow-derived mesenchymal stem cells toward MDA-MB-231 cells pretreated or not with BMP-4 for 72 hours (2 independent experiments). (l,m) The expression of p21 (l) and cyclin D1 (m) in MDA-MB-231 cells transfected with a Jagged-1 siRNA or a control siRNA prior to BMP-4 treatment for 72 hours. (n) Growth of the MDA-MB-231 xenografts in nude mice (2 independent experiments, 10~16 mice/group; p < 0.0001, ANOVA).

Figure 7

Jagged-1 expression is associated with the expression of BMP-4, EMT associated genes, and cancer stem cell marker genes in human breast cancer. (a) The expression of Jagged-1, BMP-4, and Slug in breast cancer compared to that in normal tissue. (b–d) Samples with the highest 18% (green dots) and the lowest 18% (gray dots) expression of Jagged-1 (b), the highest 26% (green dots) and the lowest 26% (gray dots) expression of BMP-4 (c), and the highest 13% (green dots) and the lowest 13% (gray dots) expression of Slug (d) were selected for analysis and p-values were calculated using Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant). Mean values are shown as red lines. (e) Spearman’s rank-order correlation test analysis for BMP-4, Jagged-1, Slug, ALDH, and CD44 in breast cancer. Numerical values of correlation coefficients are presented in the boxes.