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. 2011 Sep;22(18):3344-54.
doi: 10.1091/mbc.E10-09-0739. Epub 2011 Jul 27.

S100A4-induced cell motility and metastasis is restricted by the Wnt/β-catenin pathway inhibitor calcimycin in colon cancer cells

Ulrike Sack  1 , Wolfgang Walther, Dominic Scudiero, Mike Selby, Jutta Aumann, Clara Lemos, Iduna Fichtner, Peter M Schlag, Robert H Shoemaker, Ulrike Stein
Affiliations

S100A4-induced cell motility and metastasis is restricted by the Wnt/β-catenin pathway inhibitor calcimycin in colon cancer cells

Ulrike Sack et al. Mol Biol Cell. 2011 Sep.

Abstract

The calcium-binding protein S100A4 is a central mediator of metastasis formation in colon cancer. S100A4 is a target gene of the Wnt/β-catenin pathway, which is constitutively active in the majority of colon cancers. In this study a high-throughput screen was performed to identify small-molecule compounds targeting the S100A4-promoter activity. In this screen calcimycin was identified as a transcriptional inhibitor of S100A4. In colon cancer cells calcimycin treatment reduced S100A4 mRNA and protein expression in a dose- and time-dependent manner. S100A4-induced cellular processes associated with metastasis formation, such as cell migration and invasion, were inhibited by calcimycin in an S100A4-specific manner. Calcimycin reduced β-catenin mRNA and protein levels despite the expression of Δ45-mutated β-catenin. Consequently, calcimycin inhibited Wnt/β-catenin pathway activity and the expression of prominent β-catenin target genes such as S100A4, cyclin D1, c-myc, and dickkopf-1. Finally, calcimycin treatment of human colon cancer cells inhibited metastasis formation in xenografted immunodeficient mice. Our results demonstrate that targeting the expression of S100A4 with calcimycin provides a functional strategy to restrict cell motility in colon cancer cells. Therefore calcimycin may be useful for studying S100A4 biology, and these studies may serve as a lead for the development of treatments for colon cancer metastasis.

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Figures

FIGURE 1:
FIGURE 1:
S100A4-promoter activity is inhibited by calcimycin in a concentration- and time-dependent manner. Luciferase activity and cell viability of calcimycin-treated HCT116/S100A4pLUC cells were determined after 24 h. S100A4/G6PDH mRNA ratios were determined by qRT-PCR. S100A4 and GAPDH protein levels were measured by Western blot. (A) High-throughput screening data of calcimycin inhibiting S100A4 promoter–driven reporter activity and cell viability. (B) Cell viability of HCT116 cells treated with increasing concentrations of calcimycin for 24 h is reduced in s concentration-dependent manner (C) S100A4 mRNA and protein levels are reduced in HCT116 cells treated with increasing concentrations of calcimycin. (C, D) S100A4 expression is reduced in HCT116 cells treated with a single dose of 1 μM calcimycin for the time indicated. Data represent mean ± SE (n = 4). Statistical significance was analyzed by two-sided ANOVA and Bonferroni post hoc multiple comparison test. (E) CMV promoter–driven S100A4 expression is not affected by calcimycin treatment. Data represent mean ± SE (n = 3). Statistical significance was analyzed by Student's t test.
FIGURE 2:
FIGURE 2:
S100A4 induced cell motility is inhibited by calcimycin. Cell migration was determined with Boyden chamber and wound-healing assay. Cell invasion was measured with a Matrigel-covered Boyden chamber assay (A) Cell migration is inhibited in HCT116 and HCT116/vector cells but not in HCT116/S100A4 cells when treated with calcimycin. (B) Cell invasion is inhibited in calcimycin-treated HCT116 and HCT116/vector cells but not in HCT116/S100A4 cells. Data represent mean ± SE (n = 5). Statistical significance was analyzed with Student's t test. (C) Directed migration is inhibited in calcimycin-treated HCT116 and HCT116/vector cells but not in HCT116/S100A4 cells. Microphotographs were taken with 10× magnification 4 d after wound was entered.
FIGURE 3:
FIGURE 3:
Calcimycin inhibits anchorage-dependent and anchorage-independent cell growth. Anchorage-dependent growth was determined by MTT assay; anchorage-independent growth was measured in a soft-agar colony formation assay. (A) Anchorage-dependent cell proliferation of HCT116, HCT116/vector, and HCT116/S100A4 cells was reduced upon calcimycin treatment (, solvent-treated HCT116; , calcimycin-treated HCT116; , solvent-treated HCT116/vector; , calcimycin-treated HCT116/vector; , solvent-treated HCT116/S100A4; , calcimycin-treated HCT116/S100A4 cells). (B) Anchorage-independent cell proliferation of HCT116, HCT116/vector, and HCT116/S100A4 cells was inhibited by calcimycin. Number of colonies was counted on day 7, when microphotographs were taken with 10× and 40× (insets) magnification. Data represent mean ± SE (n = 3). Statistical significance was determined by Student's t test.
FIGURE 4:
FIGURE 4:
Calcimycin inhibits S100A4 expression, cell motility, and proliferation in human colon cancer cell lines. S100A4/G6PDH mRNA ratios were determined by qRT-PCR. S100A4 and GAPDH protein levels were measured by Western blot. Cell migration was determined with Boyden chamber and wound-healing assay. Cell invasion was measured with a Matrigel-covered Boyden chamber assay. Anchorage-dependent growth was determined by MTT assay; anchorage-independent growth was measured in a soft-agar colony formation assay. (A) S100A4 expression was reduced upon calcimycin treatment. (B) Calcimycin treatment inhibited cell migration. (C) Cell invasion was inhibited upon calcimycin treatment. (D) Calcimycin inhibited directed migration. (E) Calcimycin inhibited cell proliferation. (F, G) Calcimycin reduced the size and number of colonies formed, respectively. Number of colonies was counted on day 7, when microphotographs were taken with 10× and 40× (insets) magnification. Data represent mean ± SE (n = 3). Statistical significance was determined by Student's t test.
FIGURE 5:
FIGURE 5:
Calcimycin inhibits constitutively active Wnt/β-catenin pathway. Activity of the Wnt/β-catenin pathway was analyzed by TOP/FOPflash reporter assay. Quantification of mRNA was performed with qRT-PCR; protein expression was determined by Western blot. (A) Wnt/β-catenin pathway was reduced in calcimycin-treated HCT116 cells and in derivative cell lines HAB68mut and HAB92wt. (B) The expression of β-catenin mRNA and protein was reduced in calcimycin-treated in HCT116, HAB68mut, and HAB92wt cells. (C) mRNA levels of β-catenin/TCF transcription target genes cyclin D1, c-myc, and DKK-1 were reduced upon calcimycin treatment. (D) S100A4 expression was inhibited in HCT116, HAB68mut, and HAB92wt cells treated with calcimycin. (E) Cell migration of HCT116, HAB68mut, and HAB92wt cells was decreased upon calcimycin treatment. Data represent mean ± SE (n = 3). Statistical significance was analyzed by Student's t test.
FIGURE 6:
FIGURE 6:
Calcimycin reduces the metastatic potential of human colon cancer cells in xenografted mice. Mice were intrasplenically injected with calcimycin- or solvent-treated HCT116/LUC cells. Two representative mice per group are shown. (A) Bioluminescence was measured 6 d posttransplantation in the region of spleen and liver (metastases target organ). (B) Spleen tumor and liver metastases are visualized by bioluminescence imaging. (C) No significant difference in the luminescence signal of spleen tumors was found between mice injected with control or calcimycin-treated cells. The liver luminescence signal was significantly reduced in the calcimycin group. Data represent mean ± SE (n = 6). Statistical significance was analyzed by Student's t test. (D) Immunohistochemistry for human cytokeratin-19 identified smaller and fewer micrometastases in the calcimycin group. (E) The amount of human satellite DNA was reduced in livers from the calcimycin group. (F) The S100A4 mRNA expression normalized to hG6PDH was absent in livers of mice of the calcimycin group. Data represent mean ± SE (n > 3). Statistical significance was analyzed by Student's t test.
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