S100A4 accelerates tumorigenesis and invasion of human prostate cancer through the transcriptional regulation of matrix metalloproteinase 9

Abstract

We previously showed that the calcium-binding protein S100A4 is overexpressed during the progression of prostate cancer (CaP) in humans and in the TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse model. We tested a hypothesis that the S100A4 gene plays a role in the invasiveness of human CaP and may be associated with its metastatic spread. We observed that siRNA-mediated suppression of the S100A4 gene significantly reduced the proliferative and invasive capability of the highly invasive CaP cells PC-3. We evaluated the mechanism through which the S100A4 gene controls invasiveness of cells by using a macroarray containing 96 well characterized metastatic genes. We found that matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor (TIMP-1) were highly responsive to S100A4 gene suppression. Furthermore, S100A4 suppression significantly reduced the expression and proteolytic activity of MMP-9. By employing an MMP-9-promoter reporter, we observed a significant reduction in the transcriptional activation of the MMP-9 gene in S100A4-siRNA-transfected cells. Cells overexpressing the S100A4 gene (when transfected with pcDNA3.1-S100A4 plasmid) also significantly expressed MMP-9 and TIMP-1 genes with increased proteolytic activity of MMP-9 concomitant to increased transcriptional activation of the MMP-9 gene. S100A4-siRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. Our data demonstrate that the S100A4 gene controls the invasive potential of human CaP cells through regulation of MMP-9 and that this association may contribute to metastasis of CaP cells. We suggest that S100A4 could be used as a biomarker for CaP progression and a novel therapeutic or chemopreventive target for human CaP treatment.

Fig. 1.

S100A4 gene knockdown by siRNA transfection in PC-3 cells. (A) Photomicrographs showing transfection of fluorescein-labeled siRNA in PC-3 cells. (Magnification: ×100.) (B and C) Representative images showing expression of S100A4 protein and mRNA in nonsilencing siRNA control and S100A4-siRNA-transfected cells as analyzed by Western (B) and Northern (C) blotting. Equal loading of protein was confirmed by stripping the blots and reprobing with β-actin antibody, and equal loading of RNA was confirmed by measuring the 28S rRNA. Densitometric measurements of the bands in Western and Northern blot analyses were performed by using the digitizing software UN-SCAN-IT (Silk Scientific, Orem, UT). (D) Photomicrographs showing soft agar colony formation and histogram showing number of colonies formed by PC-3 cells transfected with siRNA. (E) Representative photomicrographs (Inset) and histogram showing invasive capability of PC-3 cells transfected with S100A4 siRNA in a chemoinvasion chamber. (Magnification: ×100.) Each bar represents mean ± SE; ∗, P < 0.05. All experiments were repeated three times with similar results.

Fig. 2.

Effect of S100A4 gene knockdown on 96 well characterized metastatic genes and on the expression level, gelatinolytic activity, and transcriptional activation of MMP-9 and TIMP-1. (A) Autoradiographic image of control (Left) and S100A4-siRNA-transfected cells (Right). Encircled tetra spots indicate the position of genes. Black arrow, TIMP-1; white arrow, MMP-9. (B) Representative images showing the expression of MMP-9 mRNA as determined by RT-PCR and Northern blot analysis. (C) Representative image showing gelatinolytic activity of MMP-9 in cells. Densitometric measurements of the bands in RT-PCR and Northern blot analysis were performed by using the digitizing software UN-SCAN-IT. (D) Representative images showing expression of TIMP-1 as determined by RT-PCR and Northern blot analysis. Equal loading of PCR products and RNA was confirmed by using constitutively expressed GAPDH and by measuring the 28S rRNA. All experiments were repeated three times with similar results. (E) Histogram showing the activity of MMP-9 promoter (in terms of relative luciferase activity) in transfected cells.

Fig. 3.

Effect of S100A4 overexpression by transfection of S100A4-pcDNA3.1 plasmid on MMP-9, its tissue inhibitor, and on the invasive capability of PC-3 cells. (A and B) Representative images showing expression of S100A4 in vector (pcDNA3.1) and pcDNA3.1-S100A4 transfected cells as analyzed by Western (A) and Northern (B) blot analysis. (C) Histogram showing invasive capability of transfected cells. Each bar represents mean ± SE (n = 3); ∗, P < 0.05. (D) Autoradiographic images showing the expression of MMP-9 mRNA in transfected cells as determined by Northern blot analysis. (E) Representative image showing gelatinolytic activity of MMP-9 in transfected cells. (F and G) Representative images showing expression of TIMP-1 in transfected cells as determined by RT-PCR (F) and Northern blot (G) analysis. Equal loading of protein was confirmed by stripping the blots and reprobing with anti-β-actin antibody. Equal loading of PCR products and RNA was confirmed by using constitutively expressed GAPDH and by measuring the 28S rRNA, respectively. Densitometric measurements of the band in RT-PCR and Northern blot analysis were performed by using the digitizing scientific software UN-SCAN-IT. All experiments were repeated three times with similar results.

Fig. 4.

Effect of S100A4 overexpression on transcriptional activation of MMP-9 in PC-3 cells. Histogram represents the relative luciferase activity in cells cotransfected with pGL3-MMP9-Luc, pcDNA3.1, or pcDNA3.1-S100A4.

Fig. 5.

Effect of S100A4 gene knockdown in PC-3 cells on the tumorigenecity in a nude mouse xenograft model. (A) Growth of tumors of control and S100A4-siRNA-transfected cells in terms of average volume of tumors as a function of weeks on test. (B) Tumor-free survival as assessed by Kaplan–Meier plot.