S100A4-induced cell motility and metastasis is restricted by the Wnt/β-catenin pathway inhibitor calcimycin in colon cancer cells.
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.
- SourceAvailable from: Dennis Kobelt[Show abstract] [Hide abstract]
ABSTRACT: We previously identified the gene metastasis-associated in colon cancer-1 (MACC1) and demonstrated its important role for metastasis prediction in colorectal cancer. MACC1 induces cell motility and proliferation in vitro as well as metastasis in several mouse models. Here we report non-invasive real time imaging of inhibition of colorectal tumor progression and metastasis in xenografted mice by MACC1 shRNA. First, we demonstrated reduction of tumors and liver metastases by endpoint imaging of mice transplanted with MACC1 endogenously high expressing colorectal cancer cells and treated with shRNAs acting on MACC1 or Met. Next, we generated a novel bicistronic IRES vector simultaneously expressing the reporter gene firefly luciferase and MACC1 to ensure a direct correlation of bioluminescence signal with MACC1 expression. We transfected MACC1 endogenously low expressing colorectal cancer cells with this luciferase-IRES-MACC1 construct, transplanted them intrasplenically, and monitored MACC1 induced tumor growth and metastasis by in vivo imaging over time. Transfection of an IRES construct harboring the firefly luciferase reporter gene together with MACC1 lacking the SH3-domain reduced tumor growth and metastasis. Finally, we counteracted the luciferase-IRES-MACC1 induced effects by shRNA targeting MACC1 and monitored reduced tumor growth and metastasis by in vivo imaging over weeks. In summary, the new bicistronic luciferase-IRES-MACC1 construct is suitable for in vivo imaging of tumor progression and metastasis, and moreover, for imaging of therapy response such as treatment with MACC1 shRNA. Thereby, we provide proof-of-concept for employment of this MACC1-based in vivo model for evaluating therapeutic intervention strategies aiming at inhibition of tumor growth and metastasis.Clinical and Experimental Metastasis 04/2012; 29(6):573-83. · 3.46 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The vast diversity of S100 proteins has demonstrated a multitude of biological correlations with cell growth, cell differentiation and cell survival in numerous physiological and pathological conditions in all cells of the body. This review summarises some of the reported regulatory functions of S100 proteins (namely S100A1, S100A2, S100A4, S100A6, S100A7, S100A8/S100A9, S100A10, S100A11, S100A12, S100B and S100P) on cellular migration and invasion, established in both culture and animal model systems and the possible mechanisms that have been proposed to be responsible. These mechanisms involve intracellular events and components of the cytoskeletal organisation (actin/myosin filaments, intermediate filaments and microtubules) as well as extracellular signalling at different cell surface receptors (RAGE and integrins). Finally, we shall attempt to demonstrate how aberrant expression of the S100 proteins may lead to pathological events and human disorders and furthermore provide a rationale to possibly explain why the expression of some of the S100 proteins (mainly S100A4 and S100P) has led to conflicting results on motility, depending on the cells used.Cellular and Molecular Life Sciences CMLS 06/2013; · 5.62 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The metastasis-inducing protein S100A4 was found to be a prognostic indicator for the development of metachronous metastases. S100A4 expression levels correlate with the formation of human colorectal cancer metastases and shorter patients' survival. Inhibition of S100A4 expression in patients might therefore result in decreased metastasis formation and prolonged survival. In the present study, we used shRNA expression plasmids to inhibit S100A4 expression in the colorectal cancer cell lines HCT116, SW620 and DLD-1. Cell lines with reduced S100A4 expression showed reduced cell migration and invasion in vitro. The knock-down of S100A4 expression also led to significantly diminished formation of liver metastases when intrasplenically transplanted in mice (P = 0.004). We then focused on the therapeutic potential of systemically applied shRNA expression plasmids acting on S100A4 via repeated hydrodynamics-based tail vein injection of plasmid DNA. Mice, intrasplenically transplanted with HCT116 cells and treated systemically with S100A4‑shRNA plasmids, showed a decrease of S100A4 and MMP9 expression levels, resulting in significantly reduced liver metastases (P = 0.005). In summary, we show for the first time the intratumoral knock down of S100A4 via systemic application of S100A4‑shRNA plasmid DNA, which restricts metastasis formation in a xenografted mouse model of colorectal cancer.Oncotarget 08/2012; 3(8):783-97. · 6.64 Impact Factor
3344 | U. Sack et al. Molecular Biology of the Cell
MBoC | ARTICLE
S100A4-induced cell motility and metastasis is
restricted by the Wnt/β-catenin pathway
inhibitor calcimycin in colon cancer cells
Ulrike Sacka, Wolfgang Waltherb, Dominic Scudieroc, Mike Selbyc, Jutta Aumanna, Clara Lemosa,
Iduna Fichtnera, Peter M. Schlaga,b,d, Robert H. Shoemakere, and Ulrike Steinb
aMax-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany; bExperimental and Clinical Research Center,
Charité University Medicine, Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany; cSAIC-Frederick,
National Cancer Institute at Frederick, Frederick, MD 21702; dCharité Comprehensive Cancer Center, Charité
University Medicine, 10117 Berlin, Germany; eScreening Technologies Branch, Developmental Therapeutics Program,
Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, Frederick, MD 21702
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E10-09-0739) on July 27, 2011.
Address correspondence to: Ulrike Stein (email@example.com).
Abbreivations used: ANOVA, one-way analysis of variance; DKK-1, dickkopf-1;
GAPDH, glycerin-aldehyde-3-phosphate dehydrogenase; G6PDH, glucose-6-
phosphate dehydrogenase; LOPAC, library of pharmacologically active com-
pounds; MMP, matrix metalloproteinase; NOD/SCID, nonobese diabetic/severe
immune deficient; TCF, T cell factor.
© 2011 Sack et al. This article is distributed by The American Society for Cell Biol-
ogy under license from the author(s). Two months after publication it is available
to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
ABSTRACT The calcium-binding protein S100A4 is a central mediator of metastasis forma-
tion in colon cancer. S100A4 is a target gene of the Wnt/β-catenin pathway, which is consti-
tutively 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 for-
mation, such as cell migration and invasion, were inhibited by calcimycin in an S100A4-specif-
ic 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
S100A4 is a ubiquitous small, calcium-binding protein that enables
cell migration and invasion to increase cell motility (Garrett et al.,
2006). Consequently, S100A4 expression is up-regulated during
wound healing, neurite outgrowth, fibrosis, or neovascularization—
all physiological processes that rely on increased cell motility. How-
ever, overexpression of S100A4 is often correlated with pathological
conditions such as epithelial–mesenchymal transition, tumor out-
growth, and metastasis formation (Schneider et al., 2008; Boye and
In colon cancer distant metastases are the major cause for cancer
death, rendering this disease the second-most-frequent cause of
cancer-related death in the Western world (Jemal et al., 2008).
S100A4 expression is significantly associated with metastasis forma-
tion in colorectal cancer patients, and the expression of S100A4 rep-
resents a significant prognostic marker in colorectal carcinoma
(Gongoll et al., 2002; Cho et al., 2005; Stein et al., 2006). Despite
S100A4 increasing the metastatic potential of the cell, S100A4
transgenic mice are not phenotypically changed (Ambartsumian
et al., 1996). However, when these are crossed with spontaneous
tumor-forming mice, S100A4 overexpression leads to highly
Josephine Clare Adams
University of Bristol
Received: Sep 2, 2010
Revised: Jul 18, 2011
Accepted: Jul 21, 2011
Volume 22 September 15, 2011 S100A4 and calcimycin | 3345
aggressive primary tumors and formation of metastasis (Davies
et al., 1996). Highly metastatic mouse mammary carcinoma cells in-
jected into S100A4 null mice are unable to form metastases (Grum-
Schwensen et al., 2005). These observations suggest that S100A4 is
not simply a marker for metastatic disease but rather has a causal
role in mediating this process.
Tumor growth and metastasis depend on increased cell migra-
tion, invasion, and angiogenesis. S100A4 is involved in all of these
processes by interaction with multiple proteins in the cytoplasm and
extracellular space. For instance, intracellular S100A4 increases cell
migration by rearranging proteins of the cytoskeleton. S100A4 binds
tropomyosin and nonmuscle myosin II in a Ca2+-dependent manner
and inhibits the actin-regulated ATPase activity of myosin II
(Kriajevska et al., 1994; Ford et al., 1997). It thus promotes the disas-
sembly of myosin filaments and inhibits their reassembly (Ford et al.,
1997; Li et al., 2003). S100A4 is located at the leading edge of mi-
grating cells (Kim and Helfman, 2003), where it induces the formation
of flexible protrusions. Moreover, in the presence of a chemoattrac-
tant, S100A4 enhances directed migration (Li and Bresnick, 2006).
Thus the S100A4–myosin II interaction not only increases cell motil-
ity, but it also enhances cell polarization and directed migration.
S100A4 expression is associated with reduced expression levels
of E-cadherin, thus loosening epithelial cell adhesion (Keirsebilck
et al., 1998). In addition, S100A4 reduces cell adhesion by interacting
with liprin-β1 (Kriajevska et al., 2002). S100A4 can access the extra-
cellular space by a yet-unknown mechanism, by which it interacts
with endothelial-bound annexin II and thus enhances angiogenesis
(Ambartsumian et al., 2001; Kriajevska et al., 2002). Extracellular
S100A4 can also bind to the receptor for glycation end products and
thereby activate matrix metalloproteinase-13 (MMP-13) expression
(Yammani et al., 2006). Furthermore, up-regulation of MMP-2 and
MMP-9 expression by extracellular S100A4 allows cell invasion and
thus promotes metastasis formation (Mathisen et al., 2003; Saleem
et al., 2006).
S100A4 gene transcription in colon cancer cells is regulated by
the canonical Wnt/β-catenin pathway, one of the most frequently
deregulated pathways in colon cancer (Stein et al., 2006). The path-
way strictly controls the cytoplasmic level of β-catenin via a destruc-
tion complex. In this complex β-catenin is phosphorylated and thus
marked for proteasomal degradation (Barker and Clevers, 2006).
Mutations within this pathway lead to intracellular accumulation of
β-catenin, which then translocates to the nucleus and activates
β-catenin/T cell factor (TCF) target gene transcription (Giles et al.,
2003; Stein et al., 2006).
Because S100A4 plays such a central role in the process of me-
tastasis formation, the inhibition of S100A4 expression provides a
promising strategy for novel antimetastastic treatments (Sack and
Stein, 2009). To identify potential S100A4 transcription inhibitors,
we performed a high-throughput screen for compounds that inhib-
ited S100A4-promoter activity. Thus we identified calcimycin as a
novel inhibitor of S100A4 transcription, which overcomes a constitu-
tively active Wnt/β-catenin pathway and significantly restricts
S100A4-induced cell motility in colon cancer cells.
Identification of S100A4 transcription inhibitor calcimycin
Calcimycin was identified as an inhibitor for S100A4 expression in a
high-throughput screen of the Library of Pharmacologically Active
Compounds (LOPAC) 1280, a collection of 1280 pharmacologically
active compounds that have shown effects on major drug target
classes such as kinases, G proteins, and ion channels. HCT116/
S100A4pLUC cells expressing S100A4-promoter-driven firefly lu-
ciferase were exposed to 20 twofold dilutions of calcimycin. After
24 h, luciferase activity was determined as a read-out for S100A4-
promoter activity (Figure 1A). From the high-throughput data it was
found that calcimycin inhibited luciferase activity in a concentration-
dependent manner, with an EC50 of 2.7 μM (95% confidence interval
[CI], 1.0–4.0 μM). At the same time growth inhibition was assessed
from the high-throughput data, yielding an IC50 of ∼80 μM (95% CI,
To define cytotoxic side effects potentially affecting further ex-
periments, the effect of calcimycin on cell viability of HCT116 cells
was analyzed more precisely by manual pipetting. Exposure of
HCT116 cells to calcimycin concentrations ranging from 2 nM to
300 μM for 24 h resulted in a reduction of cell viability in a concen-
tration-dependent manner with an IC50 of 2.1 μM (95% CI,
1.5–2.2 μM; Figure 1B).
We next analyzed the ability of calcimycin to reduce endogenous
S100A4 expression in HCT116 cells. Exposure of HCT116 cells to
increasing concentrations of calcimycin for 24 h resulted in a con-
centration-dependent reduction of S100A4 mRNA and protein.
Treatment with 1 μM calcimycin reduced S100A4 expression to 30%
of that for the solvent-treated control (Figure 1C). Cell viability was
not affected by this concentration of calcimycin after 24 h. Therefore
we chose the calcimycin concentration of 1 μM for use in the follow-
S100A4 expression is inhibited in a time-dependent manner
To assess the kinetics of the calcimycin effect, HCT116 cells were
exposed to 1 μM calcimycin, and S100A4 expression was analyzed
every 6 h. S100A4 mRNA was reduced in a time-dependent manner.
After 12 h of treatment a basal reduction level of ∼30% of the solvent-
treated control was observed (Figure 1D). After 18 h of treatment the
S100A4 protein levels were also reduced. S100A4 expression inhibi-
tion continued for 5 consecutive days after a single dose of 1 μM
calcimycin (Figure 1E). Similar to HCT116 cells, HCT116/vector cells
showed reduced S100A4 mRNA to ∼30% of solvent-treated control
cells after 1 μM calcimycin treatment for 24 h (Figure 1F). In contrast,
HCT116/S100A4 cells showed no significant reduction in S100A4
mRNA or protein upon calcimycin treatment. These cells overex-
pressed S100A4 cDNA under the control of a cytomegalovirus (CMV)
promoter, which was not targeted by calcimycin treatment.
S100A4-induced cell migration and invasion is inhibited
We previously showed that S100A4 expression in colon tumors sig-
nificantly correlates with metastasis formation (Stein et al., 2006).
Moreover, S100A4 drives metastasis formation by increasing cell mi-
gration and invasion (Garrett et al., 2006). Therefore we next exam-
ined S100A4-induced cell motility in the presence of calcimycin.
Exposure of HCT116 cells to calcimycin significantly reduced the
number of migrated cells to 20% of that for solvent-treated HCT116
cells (Figure 2A). Similarly, calcimycin treatment inhibited cell migra-
tion of HCT116/vector cells to 16% of that for the control cells. In
contrast, HCT116/S100A4 cells showed no significant reduction in
cell migration upon calcimycin treatment. Cell invasion of calcimy-
cin-treated HCT116 and HCT116/vector cells was inhibited to <30%
of that for the solvent-treated control cells (Figure 2B). In contrast,
cell invasion of HCT116/S100A4 cells was not significantly changed
upon calcimycin treatment. Directed migration analyzed by wound-
healing assay revealed that calcimycin clearly inhibited wound clo-
sure in HCT116 and HCT116/vector cells but not in HCT116/S100A4
cells (Figure 2C). In summary, calcimycin displayed an antimigratory
3346 | U. Sack et al. Molecular Biology of the Cell
and anti-invasive effect in HCT116 cells, and this effect was over-
come by the exogenous overexpression of S100A4.
Calcimycin inhibits anchorage-dependent and
anchorage-independent cell proliferation
We next analyzed cell proliferation under calcimycin treatment. Ex-
posure of HCT116, HCT116/vector, and HCT116/S100A4 cells to a
single dose of 1 μM calcimycin resulted in a clear inhibition of an-
chorage-dependent proliferation after day 3
(Figure 3A). Calcimycin further inhibited an-
chorage-independent growth as analyzed
by colony formation. On exposure to calci-
mycin the number of colonies formed by
HCT116, HCT116/vector, and HCT116/
S100A4 cells was significantly reduced to
<5% of that for solvent-treated control cells
(Figure 3B). Furthermore, as shown in the
insets of the microphotographs, the size of
colonies was clearly reduced by calcimycin
treatment. Proliferation of cells that exoge-
nously overexpress S100A4 was also af-
fected by calcimycin treatment. Thus the
antiproliferative effect of calcimycin on an-
chorage-dependent and anchorage-inde-
pendent growth was not overcome by an
increased S100A4 expression level.
Calcimycin inhibits S100A4 expression,
cell motility, and proliferation in other
human colon cancer cell lines
We next analyzed calcimycin effects in other
human colon cancer cell lines. Exposure of
SW620, LS174T, and SW480 cells to 1 μM
calcimycin for 24 h reduced the S100A4
mRNA level to <40% of that for the respec-
tive solvent-treated controls (Figure 4A).
S100A4 protein in these cells was clearly re-
duced upon calcimycin treatment. In DLD-1
cells no S100A4 mRNA and protein expres-
sion was detected, and therefore no further
effect by calcimycin was possible.
Exposure of SW620, LS174T, and SW480
cells to 1 μM calcimycin for 24 h inhibited
cell migration to <25% of that for the re-
spective solvent-treated controls (Figure 4B).
DLD-1 cells presented the lowest migration
rate, which was not further affected by calci-
mycin treatment. Cell invasion of SW620,
LS174T, and SW480 cells was reduced to
<30% of that for the respective solvent-
treated controls by calcimycin treatment
(Figure 4C). The low cell invasion rate of
DLD-1 cells was not significantly changed
upon calcimycin treatment. In the wound-
healing assay solvent-treated
LS174T, and SW480 cells were able to close
the wound by day 4 after wound insertion
(Figure 4D). In contrast, in solvent-treated
DLD-1 cells wound closure was not com-
pleted by day 4, which was consistent with
their decreased migration and invasion
rates. Following a single dose of 1 μM calci-
mycin, wound closure, as measurement for directed migration,
was impaired in all four colon cancer cell lines for 4 d post–wound
Anchorage-dependent cell proliferation was inhibited upon cal-
cimycin treatment in all four colon cancer cell lines as measured on
day 4 (Figure 4E). In a colony formation assay solvent-treated
SW620, LS174T, and SW480 cells formed large colonies by day
7 (Figure 4F). Colony formation was inhibited in all four colon cancer
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.
Volume 22 September 15, 2011 S100A4 and calcimycin | 3347
cell lines following calcimycin treatment. Colonies of solvent-treated
DLD-1 cells were smaller than colonies of other solvent-treated co-
lon cancer cells lines. However, the size of DLD-1 colonies was still
reduced upon calcimycin treatment. Quantification of formed colo-
nies revealed that calcimycin treatment significantly reduced the
number of colonies in all four colon cancer cell lines to <10% of the
respective-solvent treated controls (Figure 4G). In summary, calci-
mycin inhibited cell migration and invasion of various colon cancer
cell lines in relation to their S100A4 expression level. Cell prolifera-
tion was inhibited independent of the S100A4 expression level of
S100A4 in these cells.
Calcimycin inhibits constitutively active Wnt/β-catenin
We previously reported that S100A4 is a β-catenin target gene (Stein
et al., 2006). Therefore we investigated Wnt/β-catenin pathway ac-
tivity using the TOP/FOPflash reporter assay to assess the mecha-
nism underlying calcimycin inhibition of S100A4 expression. We
used HCT116 cells that are heterozygous for mutated β-catenin.
The mutated β-catenin lacks serine 45, the initial phosphorylation
site needed for proteasomal degradation (Amit et al., 2002). Consis-
tent with this mutation, in these cells expression of the TOPflash
reporter is increased. Treatment of HCT116 cells with calcimycin re-
duced the TOPflash reporter activity to <30% upon calcimycin treat-
ment (Figure 5A). In HAB68mut cells, which are homozygous for this
β-catenin mutation, calcimycin treatment reduced TOPflash reporter
activity to <10% of that for solvent-treated HCT116 cells. TOPflash
reporter activity of solvent-treated HAB92wt cells, which are homozy-
gous for wild-type β-catenin, was <30% of the activity found in cells
that bear a constitutively active Wnt/β-catenin pathway due to mu-
tated β-catenin. Moreover, calcimycin treatment of HAB92wt cells
further reduced this TOPflash reporter activity to 12%.
We next analyzed the expression level of β-catenin, which repre-
sents the central player of this pathway. In calcimycin-treated
HCT116, HAB68mut, and HAB92wt cells the β-catenin mRNA levels
were reduced to <50% of that for solvent-treated HCT116 cells
(Figure 5B). Consistent with this result, the β-catenin protein level
was diminished upon calcimycin treatment of these cells.
Reduced β-catenin levels should result in reduced target gene
transcription. Therefore we analyzed the expression levels of promi-
nent β-catenin/TCF target genes such as cyclin D1 (Shtutman et al.,
1999), c-myc (He et al., 1998), and dickkopf-1 (DKK-1; Gonzalez-
Sancho et al., 2005). In calcimycin-treated HCT116 cells the expres-
sion of cyclin D1, c-myc, and DKK-1 was inhibited to 35, 25, and 2%
of the solvent-treated controls, respectively (Figure 5C).
We analyzed the effect of calcimycin on S100A4 expression in
the presence of mutated or wild-type β-catenin and found that
S100A4 mRNA was reduced to 35% in HAB68mut and HCT116 cells
and was hardly detectable in HAB92wt cells (Figure 5D). In parallel,
calcimycin treatment reduced S100A4 protein levels in HCT116 and
HAB68mut cells, whereas no S100A4 protein was detectable in
HAB92wt cells. Cell migration in HCT116 and HAB68mut cells was
inhibited to the extent of HAB92wt cells, which was <25% of that for
solvent-treated HCT116 cells (Figure 5E). These results provide evi-
dence that calcimycin restricts the Wnt/β-catenin pathway–driven
S100A4 expression and thus S100A4-induced cell motility.
Calcimycin inhibits the metastatic potential of human colon
cancer cells in xenografted mice
To test the effect of calcimycin on the ability of cells to form me-
tastasis in vivo, we used HCT116/LUC cells, which stably ex-
pressed the firefly luciferase protein and therefore allowed the
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.
3348 | U. Sack et al. Molecular Biology of the Cell
From the reduced liver luminescence
signal in mice of the calcimycin group, we
hypothesized that the calcimycin treatment
hampered HCT116/LUC cells from forming
metastases in the liver tissue. We therefore
performed immunohistological staining for
human cytokeratin-19 in liver cryosections
of mice injected with control and calcimycin-
treated cells. In the livers of control mice the
incidence of micrometastases was clearly
higher than in those of mice of the calcimy-
cin group (Figure 6D). Moreover, the size of
the micrometastases was reduced in livers
of mice injected with calcimycin-treated
We next quantified the amount of hu-
man DNA in the liver sections of control and
calcimycin mice by quantitative PCR amplifi-
cation of human satellite DNA as previously
described (Becker et al., 2002). In livers from
mice of the calcimycin group the amount of
human DNA was reduced to ∼25% of that of
control mice, indicating that the calcimycin
treatment indeed inhibited human colon
cancer cells from forming liver metastases
(Figure 6E). Analysis of the human-specific
S100A4 expression in liver metastases of the
xenografted mice revealed that in livers
from mice of the calcimycin group the
S100A4 mRNA was hardly detectable, in
contrast to the control mice (Figure 6F).
From these data we conclude that the treat-
ment of colon cancer cells with calcimycin
restricted their potential to form liver metas-
tases in vivo.
Intensive research has demonstrated the
central role of S100A4 in the process of
cancer metastasis, which qualifies S100A4 as a potentially promis-
ing target for therapeutic intervention against metastasis (Sherbet,
2009; Boye and Maelandsmo, 2010). Most of the work concerning
S100A4 has concentrated on the elucidation of the mechanism by
which this molecule drives metastasis. Less work has focused on
the inhibition of S100A4 to reduce S100A4-induced cell motility.
Here we report the calcium ionophore calcimycin as an inhibitor of
S100A4 transcription in colon cancer cells. We show that calcimy-
cin treatment inhibits a constitutively active Wnt/β-catenin path-
way, thereby inhibiting S100A4 expression and leading to re-
stricted S100A4-induced cell migration and invasion in vitro and
In a high-throughput screen we identified calcimycin as one of
the most effective inhibitors of S100A4-promoter activity. In this
screen we used HCT116 cells in which the S100A4-promoter was
highly active. Inhibition of S100A4-promoter activity by calcimy-
cin indicated that calcimycin targets S100A4 at the transcription
level. Calcimycin reduced S100A4 mRNA levels in a concentra-
tion- and time-dependent manner in colon cancer cell lines with
increased basal S100A4 expression levels. In line with our obser-
vation in human colon cancer cells, calcimycin has been reported
to reduce the S100A4 mRNA level in mouse mammary adenocar-
cinoma cells, as well as in human monocytes and lymphocytes
detection of metastases via noninvasive in vivo luminescence
imaging. Cells were treated with 1 μM calcimycin or solvent for
24 h and subsequently inoculated intrasplenically into nonobese
diabetic/severe immune-deficient (NOD/SCID)–IL2R− mice. Six
days posttransplantation, luminescence signals were detected in
the lateral and ventral abdominal cavity, where the spleen and
liver are situated, respectively (Figure 6A). In control mice, the
ventral luminescence signal was stronger than in mice from the
To investigate the origin of luminescence, the spleen and the
liver were dissected. All animals developed tumors in the spleen,
which were clearly visible in the bright-field analysis (Figure 6B). A
strong luminescence signal originated from the spleen tumor, indi-
cating that the tumor was formed by HCT116/LUC cells. Bright-field
analysis of the resected livers revealed no clear differences between
mice of the control and calcimycin groups. However, the lumines-
cence signal from livers of control mice was clearly stronger than
that from livers of calcimycin mice.
Quantification of the luminescence signal revealed no significant
differences in the signal intensity of spleen tumors from mice of the
control and calcimycin groups (Figure 6C). In contrast, the lumines-
cence signal of livers was reduced to 30% in mice of the calcimycin
group compared with control mice.
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.
Volume 22 September 15, 2011 S100A4 and calcimycin | 3349
(Grigorian et al., 1994). We further showed that S100A4 protein
levels were also suppressed in a concentration- and time-depen-
dent manner following calcimycin treatment in several colon can-
cer cell lines.
Calcimycin is an ionophorous, polyether
antibiotic isolated from Streptomyces char-
treusensis. Calcimycin facilitates the trans-
port of divalent cations across the mem-
brane, which makes it a useful tool to study
calcium signaling (Pressman, 1976). Because
calcimycin elevates intracellular calcium lev-
els (Gwak et al., 2006), one would expect
that calcimycin treatment would increase
S100A4 protein activity, which depends on
calcium ions (Santamaria-Kisiel et al., 2006).
However, in our study, we did not see an in-
creased migratory or invasive phenotype in
exogenously S100A4-overexpressing cells
that were treated with calcimycin. Although
we cannot completely exclude that calcimy-
cin caused increased S100A4 protein activ-
ity, we definitely show here that reducing the
overall expression level of S100A4 signifi-
cantly inhibits S100A4-induced cell motility.
S100A4 protein drives metastasis by in-
teraction with a multitude of partner pro-
teins, leading to increased cell migration and
invasion (Belot et al., 2002; Stein et al., 2006).
Consequently, down-regulation of S100A4
expression by calcimycin restricted cell mi-
gration in colon cancer cells. This is in line
with observations from RNA interference ex-
periments in which knockdown of S100A4
mRNA was shown to reduce cell migration
(Gao et al., 2005; Tabata et al., 2009). Of in-
terest, calcimycin was not able to suppress
cell migration or invasion in cells that exog-
enously overexpressed S100A4. From these
observations we conclude that the antimi-
gratory and anti-invasive effect of calcimycin
was caused by the down-regulation of
S100A4 expression from its native promoter.
Knockdown of S100A4 mRNA levels with
short hairpin RNA (shRNA) was shown to in-
hibit cell proliferation in vitro and to reduce
tumor growth and metastasis in vivo (Shi
et al., 2006). Moreover, in gastric cancer
cells, shRNA knockdown of S100A4 in-
creased the occurrence of apoptosis (Hua
et al., 2009). In our study, knockdown of
S100A4 expression by treatment with calci-
mycin was accompanied by reduced cell
proliferation. Exogenous overexpression of
S100A4 was ineffective in overcoming the
antiproliferative effect of calcimycin, which
suggests that this effect is independent of
the S100A4 expression level.
S100A4 is a target gene of the Wnt/β-
catenin pathway (Stein et al., 2006). We
found that calcimycin treatment inhibits
β-catenin transcription, which leads to the
inhibition of the Wnt/β-catenin pathway ac-
tivity in colon cancer cells and explains its inhibitory effect on S100A4
expression. Of interest, calcimycin was able to overcome a constitu-
tively active Wnt/β-catenin pathway, since it reduced β-catenin also
in cells that are heterozygous or even homozygous for Δ45-mutated
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.
3350 | U. Sack et al. Molecular Biology of the Cell
β-catenin. In line with our findings, calcimycin treatment also abol-
ished Wnt/β-catenin pathway activity in HEK293 cells that stably
expressed the TOPflash reporter plasmid (Gwak et al., 2006).
In line with reduced Wnt/β-catenin pathway activity, we found
that S100A4 and other β-catenin/TCF target genes such as cyclin D1,
c-myc, and DKK-1 were down-regulated following calcimycin treat-
ment. Cyclin D1 and c-myc are known oncogenes, and their overex-
pression causes increased cell proliferation (He et al., 1998; Shtutman
et al., 1999). Knockdown of β-catenin expression levels by shRNA
was shown to result in reduced levels of cyclin D1 and c-myc, and this
reduction was associated with reduced cell proliferation (Huang et
al., 2007). Consistent with this observation, in our study calcimycin
reduced the cyclin D1 and c-myc mRNA levels, which might explain
the observed inhibition of anchorage-dependent and anchorage-in-
dependent cell proliferation following calcimycin treatment.
Intrasplenic inoculation of S100A4 overexpressing cells was
shown to induce liver metastases in xenografted mice (Stein et al.,
2006). Because S100A4 is a major regulator of colon cancer metas-
tasis, its inhibition should result in restricted metastasis formation.
Indeed, we found in this study that calcimycin treatment of human
colon cancer cells restricted their potential to form liver metastases
in vivo. The number and size of liver metastases formed by calcimy-
cin treated cells were significantly reduced. Bioluminescence imag-
ing, as well as immunohistochemistry, visualized larger and more
frequent micrometastases in livers of control animals than in livers of
mice from the calcimycin group. The amount of human satellite
DNA was reduced in livers of calcimycin mice, supporting the obser-
vation that fewer human colon cancer cells invaded the liver tissue.
Thus calcimycin treatment restricted the metastatic potential of co-
lon cancer cells in vivo.
We are aware that by interfering with the Wnt/β-catenin pathway,
calcimycin will have several different functions and actions within the
cell. Among these functions we show that calcimycin inhibits S100A4
expression and that this inhibition leads to reduced cell motility. We
showed that HCT116/S100A4 cells migrated and invaded despite
the calcimycin treatment. The inhibitory effect of calcimycin on cell
migration and invasion was overcome by exogenous overexpres-
sion of S100A4. Thus we provide evidence that the antimigratory
and anti-invasive effect of calcimycin was specific to the calcimycin-
mediated S100A4 expression inhibition.
In conclusion, our study reports calcimycin as a novel inhibitor of
S100A4-promoter activity, which leads to reduced S100A4 expression
and thus impairs S100A4-induced cell motility and metastasis. Be-
cause metastasis is the major cause of colon cancer death, there is an
urgent need for antimetastatic treatment. S100A4, as a mediator of
this disease progression, provides a promising therapeutic target. We
provide evidence that targeting S100A4 expression by application of
calcimycin restricts S100A4-induced cell migration and invasion in
vitro and in vivo. Therefore we show that calcimycin is a useful com-
pound not only to study S100A4 biology, but also to form the basis
for the development of treatments against colon cancer metastasis.
MATERIALS AND METHODS
Cell lines and treatments
The human colon cancer cell line HCT116 and its derivatives
HAB-68mut and HAB-92wt were kindly provided by Todd Waldman
(Georgetown University, Washington, DC). HCT116 cells are heterozy-
gous for gain-of-function mutated Δ45-β-catenin. In HAB-68mut and
HAB-92wt the wild-type or mutated allele of β-catenin was deleted,
respectively, by homologous recombination (Kim et al., 2002). These
cell lines were expanded briefly in culture and cryopreserved in mul-
tiple replicate vials. The cell banks were tested by PCR and culture
methods and found to be free of mycoplasma. To authenticate the
HAB-68mut and HAB-92wt cell lines as HCT116 derivatives, short tan-
dem repeat (STR) genotyping was performed in August 2010 using
the ABI Identifier Kit (Life Technologies, Darmstadt, Germany). The
STR genotypes were consistent with published genotypes for
HCT116. All cells were tested for the correct β-catenin genotype by
restriction fragment length polymorphism as previously described
(Stein et al., 2006). Cells were cultured in RPMI-1640 medium supple-
mented with 10% fetal calf serum (PAA Laboratories, Pasching, Aus-
tria) in a humidified incubator at 37°C and 5% CO2. HCT116 cells
were transfected to express S100A4-promoter controlled firefly lu-
ciferase (HCT116/S100A4pLUC cells), CMV promoter–controlled
firefly luciferase (HCT116/LUC), CMV promoter–controlled S100A4
cDNA (HCT116/S100A4 cells), or the empty vector as control
(HCT116/vector cells). The S100A4 promoter was a kind gift from
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.
Volume 22 September 15, 2011 S100A4 and calcimycin | 3351
start master mix (Roche) was used. Cyclin D1 and c-myc cDNAs
were quantified with SYBR green (Roche). For quantification of the
housekeeping gene glucose-6-phosphate dehydrogenase (G6PDH)
cDNA the hG6PDH Roche Kit (Roche) was used.
Western blot was performed with the following antibodies: a
polyclonal rabbit anti–human S100A4 antibody (Dako, Glostrup,
Denmark), a polyclonal goat anti–human glycerin-aldehyde-
3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz
Biotechnology, Santa Cruz, CA), and a monoclonal mouse
anti–human β-catenin antibody. Immunoblotting for GAPDH
served as loading control.
Immunohistochemistry was performed as previously described
(Stein et al., 2011). Briefly, cryosections were stained with rabbit
anti–human-specific cytokeratin-19 antibody (dilution 1:50; Acris
Antibodies, Herford, Germany)
dase–coupled anti-rabbit antibody (dilution 1:1000; Promega,
and horseradish peroxi-
Cell migration, cell invasion, and wound-healing assay
For the Boyden chamber assay (Boyden, 1962), 2.5 × 105 cells were
seeded into transwell filter membrane chambers (pore size, 12.0 μm;
Millipore, Billerica, MA) and allowed to accommodate for 15 h. Cells
were treated with 1 μM calcimycin for 18 h. The number of cells that
migrated to the lower chamber was counted in a Neubauer cham-
ber. For the invasion assays, transwell membranes were coated with
1:3 diluted Matrigel (BD Biosciences, Heidelberg, Germany). For
the wound-healing assay, 2.5 × 105 cells were seeded at 60% conflu-
ence 24 h before a wound of ∼300 μM width was made with a pi-
pette tip. Medium was exchanged daily, and microphotographs
were taken on day 4.
David Allard (University of Exeter and Plymouth, Exeter, United King-
dom) and comprised the sequence from −1487 to +33 base pairs
around the S100A4 transcription start site (Hernan et al., 2003). The
S100A4 cDNA was kindly provided by Claus Heizmann (University of
Zurich, Zurich, Switzerland; Engelkamp et al., 1992).
Substances from the LOPAC 1280 and additional samples of cal-
cimycin (both Sigma-Aldrich, St. Louis, MO) were dissolved in dim-
ethylsulfoxide. Dilutions were performed with RPMI-1640 medium.
High-throughput screening and cell cytotoxicity assay
A BIOMEK2000 automatic pipetting system (Beckman Coulter,
Brea, CA) was used to seed 2.5 × 103 HCT116/S100A4pLUC cells/
well into 384-well plates. Cells were exposed to dilutions of each
compound of the LOPAC 1280 for 24 h. Luciferase expression was
determined using Britelite reagent in a Wallac Victor reader (both
PerkinElmer, Waltham, MA). In parallel, cell cytotoxicity of the com-
pounds was measured by Alamar blue cytotoxicity assay. Briefly, af-
ter 24 h of incubation with cells, plates were treated with Alamar
blue (Sigma-Aldrich) solution for 4 h and then read on a fluores-
cence plate reader at excitation and emission wavelengths of 530
and 590 nm, respectively. Compounds showing inhibition in the
S100A4 promoter screen were counterscreened using an HIF-1 pro-
moter screen to establish selective activity (Rapisarda et al., 2002).
Quantitative real-time PCR, Western blot analysis,
Quantitative real-time (qRT) PCR was carried out using the LightCy-
cler480 (Roche, Mannheim, Germany) as described previously (Stein
et al., 2006) with the primers and probes summarized in Table 1. For
β-catenin, S100A4, and DKK-1 cDNA quantification HybProbe Fast-
Forward primergtg cta tct gtc tgc tct agt a
ctt cct gtt tag ttg cag cat c
agg act tca cct gac aga tcc aag tca-FITC
LCRed640-cgt ctt gtt cag aac tgt ctt tgg act ctc-phosphate
ctg ttt ggc gtt tcc cag agt cat c
agc ctc ctc ctc aca cct cct c
acc ctt gcc gca tcc acg aaa c
cgt agt cga ggt cat agt tcc tgt tgg
tag cac ctt gga tgg gta ttc
ata ttt cta gtc cat gag agc c
gtc tcc ggt cat cag act gtg cc-FITC
LCRed640-agg att gtg ttg tgc tag aca ctt ctg g-phosphate
ctc agc gct tct tct ttc
ggg tca gca gct cct tta
tgt gat ggt gtc cac ctt cca caa gt-FITC
LCred640-tcg ggc aaa gag ggt gac aag t-phosphate
Human satellite DNA Forward primer
ggg ata att tca gct gac taa aca g
aaa cgt cca ctt gca gat tct ag
ctt cac ata aaa act aca cag atg cat tct cag g-FITC
LCred640-ctt ttt ggt gat gtt tgt att caa ctc cca g-phosphate
FITC, fluorescein isothiocyanate.
TAbLE 1: Primers and probes used for qRT-PCR.
3352 | U. Sack et al. Molecular Biology of the Cell
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
Volume 22 September 15, 2011 S100A4 and calcimycin | 3353
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.
culated by sigmoidal dose–response curve fit of × = log(x) trans-
formed data. IC50 and EC50 values were given as geometric means
with 95% confidence interval. All significance tests were two-sided.
p < 0.05 was defined as statistically significant.
Cell proliferation and colony formation assay
For determination of cell proliferation, 8 × 103 cells were treated
daily for 4 d with 1 μM calcimycin. For determination of viable
cells 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide
(MTT; Sigma-Aldrich) was added to a final concentration of
0.5 mg/ml, incubated for 3 h, and dissolved by 10% SDS in 10 mM
HCl. The optical density was measured at 560 nm. For colony forma-
tion assays 1 × 103 cells were resuspended in 1 μM calcimycin- or
solvent-containing medium supplemented with 0.33% (wt/vol) aga-
rose and seeded as single cells. After incubation in a humidified in-
cubator at 37°C and 5% CO2 for 7 d, colonies were analyzed by light
microscopy. Colonies were counted if they consisted of more than
TOP/FOPflash reporter assay
Transfection of 8 × 105 cells with TOPflash or FOPflash plasmids
(Promega) occurred 24 h before the cells were treated with 1 μM
calcimycin. After 18 h of treatment luciferase activity was measured
by the Steady Glow Luciferase Assay System (Promega). TOPflash
reporter gene expression (representing the Wnt/β-catenin pathway
activity) was normalized to FOPflash reporter gene expression (rep-
resenting basal reporter gene expression).
In vivo bioluminescence imaging of xenograft mice
All experiments were performed in accordance with the United
Kingdom Coordinating Committee for Cancer Research guidelines
and approved by the State Office of Health and Social Affairs,
Berlin, Germany. HCT116/LUC cells were treated with 1 μM calcimy-
cin or solvent 24 h before 2 × 106 cells were intrasplenically injected
into eight NOD/SCID-IL2R− mice per group. Mice were anesthe-
tized by intraperitoneal injection of 35 mg/kg Hypnomidate (Jassen-
Cilag, Neuss, Germany) and received 150 mg/kg d-luciferin (Bio-
synth, Staad, Switzerland) intraperitoneally for bioluminescence
imaging. Imaging was performed with the NightOWL LB 981 sys-
tem (Berthold Technologies, Bad Wildbad, Germany). ImageJ, ver-
sion 2.3, was used for color coding of signal intensity (presenting a
256 grayscale) and for quantification of the luminescence signal.
Mice were killed 6 d posttransplantation, when a clear liver signal
was detected. The spleen (the tumor implantation site) and the liver
(the metastasis target organ) were shock frozen in liquid nitrogen,
and cryosections were performed for isolation of genomic DNA
(Qiagen, Hilden, Germany) and mRNA (Roboklon, Berlin, Germany)
and for immunohistochemistry.
Statistical analyses were performed with GraphPad Prism, version
4.01. Comparison of two groups was done by Student’s t test. Com-
parison of a control versus several treated groups was performed by
one-way analysis of variance (ANOVA) and Bonferroni post hoc mul-
tiple comparison. The inhibiting concentration 50 (IC50) was defined
as the concentration that reduced cell viability to 50% of solvent-
treated control cells. The effective concentration 50 (EC50) was de-
fined to be the concentration at which reporter activity was reduced
to 50% of solvent-treated control cells. The IC50 and EC50 were cal-
We are very grateful to Pia Hermann and Margit Lemm for technical
assistance and to Franziska Siegel and Dennis Kobelt for method-
ological and scientific advice. This work was supported by the
German Research Association (STE 671/8-1, to U.S. and P.M.S.), the
Alexander von Humboldt Foundation (to U.S. and W.W.), and a
Max-Delbrück-Center for Molecular Medicine Helmholtz Associa-
tion Fellowship (to U.S.).
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