S100A7 enhances invasion of human breast cancer MDA-MB-468 cells through activation of nuclear factor-κB signaling

Abstract

Background: S100A7 signaling plays a critical role in the pathogenesis and progression of human breast cancers but the precise role and mechanism of S100A7 for tumor invasion remains unclear. in the present study, we investigated whether S100A7 overexpression could be mechanistically associated with the up-regulation of NF-κB, VEGF and MMP-9, resulting in the promotion of breast cancer cell invasion and growth, and vice versa.

Methods: pcDNA3.1-S100A7 cDNA plasmid was constructed and transfected into the MDA-MB-468 cells. 4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect cell proliferation, Matrigel was used to detect cell mobility and invasion in vitro.The MMP-9 and VEGF expression and levels was detected by western blot and ELISA assay. NF-κB DNA binding activity was detected by Electrophoretic mobility shift assay.

Results: Up-regulation of S100A7 by stable S100A7 cDNA transfection increased cell invasion and proliferation, whereas downregulation of S100A7 by small interfering RNA in S100A7 cDNA-transfected MDA-MB-468 cells decreased cell invasion and proliferation. Consistent with these results, we found that the up-regulation of S100A7 increased NF-κB DNA-binding activity and MMP-9 and VEGF expression. Down-regulation of S100A7 in S100A7 cDNA -transfected decreased NF-κB DNA-binding activity and MMP-9 and VEGF expression.

Conclusions: Our data demonstrate that the S100A7 gene controls the proliferation and invasive potential of human MDA-MB-468 cells through regulation of NF-κB activity and its target genes, such as MMP-9 and VEGF expression. Down-regulation of S100A7 could be an effective approach for the down-regulation and inactivation of NF-κB and its target genes, such as MMP-9 and VEGF expression, resulting in the inhibition of invasion and growth.

Figure 1

Effect of S100A7 overexpression by transfection of S100A7-pcDNA3.1 plasmid on S100A7. Representative images showing expression of S100A7 in vector (pcDNA3.1), pcDNA3.1-S100A7 and transfected with S100A7 siRNA in MDA-MB-468 cells as analyzed by Western blot.

Figure 2

Effect of S100A7 overexpression on invasiveness of MDA-MB-468 cells transfected with recombinants in Matrigel invasion assay. Cells were stably transfected with empty vector pc.DNA3.1 or pc.DNA3.1-S100A7, or transfected with S100A7 siRNA. After 2 days, 1 × 10⁵ cells were allowed to invade through transwell inserts (8 μm) coated with Matrigel. The cells on the lower surface of the chambers were stained, counted, and photographed under a light microscope. Histogram shows invasive capability of transfected cells. Each bar represents mean ± SE (n = 3); ^*, P <0.05.

Figure 3

Effect of S100A7 overexpression on cytotoxicity of breast tumor MDA-MB-468 cell lines. MDA-MB-468 cells were stably transfected with pc.DNA3.1-S100A7 cells. Cell viability was determined by the MTT assay. The growth rate of the pc.DNA3.1-S100A7 transfected MDA-MB-468 cells was significantly increased in the MTT assay. Each bar represents mean ± SE (n = 3); ^**, P <0.01. MTT, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

Figure 4

S100A7 overexpression induced NF-κB in the MDA-MB-468 cells. A, NF-κB p65 expression in investigated MDA-MB-468 cells was analyzed by western blot. B, EMSA analysis was done for MDA-MB-468 cells. Nuclear extracts were prepared from control and transfected cells and subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. Retinoblastoma protein level served as the nuclear protein loading control. EMSA, electrophoretic mobility shift assay; NF-κB, nuclear factor-kappa B.

Figure 5

MMP-9 expression was up-regulated by S100A7 cDNA transfection and down-regulated by S100A7 siRNA transfection. A, Western blot analysis of MMP-9 protein expression in transfected MDA-MB-468 cells. B, MMP-9 activity assay showing that MMP-9 was up-regulated by S100A7 cDNA transfection and down-regulated by S100A7 siRNA transfection. Each bar represents mean ± SE (n = 3); ^*, P <0.01. MMP-9, matrix metalloproteinase 9.

Figure 6

VEGF expression was up-regulated by S100A7 cDNA transfection and down-regulated by S100A7 siRNA transfection. A, Western blot analysis of VEGF protein expression in transfected MDA-MB-468 cells. B, VEGF activity assay showing that VEGF was up-regulated by S100A7 cDNA transfection and down-regulated by S100A7 siRNA transfection. Each bar represents mean ± SE (n = 3); ^*, P <0.05. VEGF, vascular endothelial growth factor.

Figure 7

Down-regulation of MMP-9 or VEGF by siRNA transfection decreased tumor cell invasion. A, down-regulation of MMP-9 or VEGF by siRNA transfection showed low-expression of MMP-9 or VEGF protein in S100A7-transfected MDA-MB-468 cells as confirmed by Western blot analysis. B, invasion assay showing that MMP-9 or VEGF siRNA–transfected cells exhibited low penetration of cells through the Matrigel-coated membrane, compared with control siRNA-transfected cells. MMP-9, matrix metalloproteinase 9. VEGF, vascular endothelial growth factor.