Osteoblast-derived Oxysterol Is a Migration-inducing Factor for Human Breast Cancer Cells*
Jeane Silva
,
Anke Beckedorf
and
Erhard Bieberich
¶ ||
From the
Institute of Molecular Medicine and
Genetics, ¶Department of Medicine, Medical
College of Georgia, Augusta, Georgia 30909 and
Westfälische Wilhelms
Universität-Münster (University of Muenster), Institute of Medical
Physics and Biophysics, Robert Koch Strasse 31, 48149 Muenster, Germany
Received for publication, February 4, 2003
, and in revised form, April 22, 2003.
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ABSTRACT
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Bone metastasis is the major reason for death caused by breast cancer. We
used human breast cancer (MCF-7) cells that are poorly metastatic but show
highly inducible migration to determine bone-derived factors that induce
migration of initially non-disseminating breast cancer cells. We have found
that a lipid fraction from human osteoblast-like MG63 cell-conditioned medium
(MG63CM) contains a migration-inducing factor for MCF-7 cells. In this
fraction, we have identified oxysterol (OS) as a lipid mediator for tumor cell
migration. In MCF-7 cells, insulin-like growth factor 1 elevates the
expression of OS-binding protein-related protein 7. Binding of OS to
OS-binding protein or OS-binding protein-related protein is known to trigger
elevation of sphingomyelin, a sphingolipid that organizes lipid microdomains
in the cell membrane. In MCF-7 cells, OS increases the intracellular
concentration of sphingomyelin and other phospholipids and induces the
translocation of the small GTPase p21Ras to GM1- and cholesterol-rich membrane
areas. The induction of migration by MG63CM is prevented by incubation of MG63
cells with mevinolin, a statin-type cholesterol biosynthesis inhibitor that
depletes the conditioned medium of OS. Osteoblast-derived OS may, thus, be a
yet unrecognized lipid mediator for bone metastasis of breast cancer and a new
target for anti-metastasis chemotherapy with statins.
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INTRODUCTION
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The analysis of cell signaling for bone metastasis of breast cancer cells
is crucial for the development of novel approaches for treatment of cancer. A
great body of work has been done on the identification of proteinogenic growth
and migration-inducing factors
(1,
2). Bone-derived
IGF-11 and chemokines
play central roles as trophic factors that attract breast cancer cells to bone
tissue
(35).
Only little is known, however, about the significance of lipid mediators in
this process. Numerous studies show that the response of cancer cells to
growth or migration-inducing factors critically depends on the lipid
microenvironment of their receptors
(6,
7). For example, incubation
with cholesterol has been found to significantly enhance IGF-1 and
chemokine-dependent migration of human breast cancer MCF-7 cells toward the
source of the growth or migration-inducing factor
(8). These studies have also
shown that the use of MCF-7 cells with a low degree of intrinsically active
cell migration allows for the sensitive determination of exogenous factors
that induce migration by alteration of the membrane lipid composition. The
potential of these factors to induce migration in poorly metastatic MCF-7
cells indicates that they are likely to turn a non-disseminating into a
disseminating tumor.
Lipids of the plasma membrane, in particular cholesterol and sphingolipids
(i.e. sphingomyelin, GM1) are clustered in lipid microdomains that
are known to associate with growth factor or chemokine receptors
(911).
The tight packing between cholesterol and sphingolipids has been shown to
organize the formation of membrane lipid microdomains
(12). This observation
indicates that up-regulation of intracellular cholesterol and sphingomyelin
may enhance lipid microdomain formation and in turn assist or amplify growth
factor or chemokine-dependent cell signaling for breast cancer cell migration
(6,
8). It has also been shown that
lipid domains in the plasma membrane recruit small GTP-binding proteins
(e.g. Ras, Rho) and growth factor or chemokine receptors to the
leading edge of migrating cancer cells
(8,
13,
14). The association of these
proteins in cholesterol/sphingolipid-rich membrane domains may, thus, form a
cell signaling platform or module that triggers or controls the induction of
tumor cell migration (3,
6,
8,
15).
The cholesterol and sphingomyelin biosynthesis is tightly regulated by
soluble lipids, in particular steroids. We have identified hydroxylated
cholesterol or oxysterol (OS) in medium that has been conditioned by
osteoblast-like MG63 cells. OS is a soluble and cell-permeable form of
cholesterol that has been reported to modulate both the intracellular
cholesterol and sphingomyelin concentration in a variety of cell species
(1621).
To our knowledge, however, this is the first study that reports OS-induced
migration of tumor cells. We have developed an improved drop agarose migration
assay that allows us to determine the expression of OS-binding proteins and
microdomain-associated lipids specifically in migrating tumor cells. This
assay has been used to visualize the effect of OS on lamellipodia formation at
the leading edge of MCF-7 cells. Our analyses show that osteoblast-derived OS
may be an important lipid mediator for the navigation of migrating breast
cancer cells to bone tissue. Furthermore, we discuss results that suggest a
therapeutic potential of cholesterol biosynthesis inhibitors for
anti-metastasis therapy.
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EXPERIMENTAL PROCEDURES
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MaterialsHuman breast cancer MCF-7 (ATCC HTB-22) and human
osteosarcoma MG63 (ATCC CRL-1427) cells were purchased from the American Type
Culture Collection (Manassas, VA). EMEM medium was from Invitrogen.
Dextran-coated charcoal, LongR3-IGF-1 peptide analog, low melt agarose,
wortmannin, arachidonic acid, acetylsalicylic acid, filipin, sphingomyelin,
cholesterol, and 25-hydroxycholesterol were from Sigma. Polyclonal rabbit IgG
against IGF-1 receptor
-subunit and monoclonal mouse IgG against RhoA
IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse
monoclonal IgG against anti-phospho-Thr-202/Tyr-204-p42/44-MAPK and rabbit
polyclonal IgG against p42/44-MAPK were from Cell Signaling (Beverly, MA).
Y-27632, PD98059, prostaglandins, and rabbit polyclonal IgG against p21Ras was
from Calbiochem. Alexa 488-labeled goat anti-rabbit IgG and Alexa 594-labeled
cholera toxin B were from Molecular Probes (Eugene, OR). HPTLC plates were
obtained from Merck. All other chemicals were analytical grade or higher, and
organic solvents were freshly re-distilled before use.
Tumor Cell Culture and Medium ConditioningMCF-7 and MG63
cells were propagated in a humidified incubator at 5% CO2 using
EMEM medium with or without phenol red or fetal bovine serum. Medium was
conditioned by incubation of MG63 or MCF-7 cells with serum-free EMEM for 48 h
followed by filtration of the conditioned medium through a 0.2-µm filter
membrane. To remove lipids, the filtered medium was incubated with
dextran-coated charcoal for 2 h, and the supernatant was used for incubation
of MCF-7 cells as described
(22). In one series of
experiments, the MG63-conditioned medium was supplemented with 1 µg/ml
anti-IGF-1R antibody to prevent signaling via the IGF-1-induced pathway
following a previously published procedure
(23).
Migration AssaysThe soft agarose drop migration assay was
performed following the procedure introduced by Varani et al.
(24). MCF-7 cells
(104105 cells) in 10 µl of serum-free medium
were mixed with 10 µl of 0.6% low melting point agarose liquefied in
serum-free medium and then dropped onto the pre-cooled surface of a 6-well
dish. The agarose was allowed to solidify for 15 min at 4 °C and was then
covered with 0.5 ml of serum-free medium. After incubation at 37 °C for 5
h, growth or migration factors were added, and the assay mixture was incubated
for another 24 h at 37 °C. Cells that migrated out of the rim of the drop
were counted for quantitative determination of migrating cells. A modified
drop agarose migration assay (co-culture migration assay) was designed to
determine the trophic effect of growth or migration factors on MCF-7 cells
that were released from a local source over a period of time. A dialysis tube
containing MG63 cells was placed into a 60-mm dish before adding the agarose
drop with MCF-7 cells to the dish. Alternatively, the dialysis tube was filled
with medium that has been supplemented with migration or growth factors. After
incubation of the cells, the agarose was removed, and the tumor cells were
fixed and analyzed by immunocytochemistry. Statistically significant
differences in data sets (p < 0.05) were assessed from at least 10
independent experiments using one way analysis of variance.
Lipid Preparation and AnalysisLipids were extracted from
conditioned medium or agarose-embedded cells in 500 µl of deionized water
by thoroughly mixing with an equal volume of CHCl3/CH3OH
(1:1, by volume). The lower (organic) phase was evaporated to dryness with a
stream of nitrogen, and the residue was dissolved in
CHCl3/CH3OH (1:1, by volume) for further analysis by
HPTLC and followed by mass spectrometry or preparative chromatography of the
migration-inducing lipid fraction. HPTLC of cholesterol, ceramide, and
prostaglandins was performed in the solvent system
CH3OH/CH3COOH (9:1, by volume) followed by staining with
3% cupric acetate in 8% phosphoric acid and comparison to standard lipids as
described (25). Phospholipids,
including sphingomyelin, were resolved by HPTLC in
CHCl3/EtOH/H2O/triethylamine (35:30:7:35, by volume) and
specifically stained using a modified Dittmer's
(CuSO4/H2SO4/molybdate) reagent
(26). Cholesterol and
oxysterol were separated using HPTLC in CHCl3/CH3OH
(95:5, by volume) and visualized by staining with 5% ammonium molybdate in 10%
sulfuric acid as described
(27). For preparative HPTLC,
individual lipid fractions that co-migrated with cupric acetate-stained bands
were scraped of the silica plate and extracted with
CHCl3/CH3OH (1:1, by volume). After removal of the
silica particles by centrifugation, the supernatant was dried, and the residue
was redissolved in serum-free EMEM medium or organic solvent for incubation of
MCF-7 cells or analysis by mass spectrometry, respectively. Lipid extracts (in
CHCl3/CH3OH, 1:1 by volume) and matrix
(2,5-dihydroxybenzoic acid in H2O/EtOH, 9:1 by volume) were
co-crystallized on the target in a dried-droplet application. MALDI-TOF mass
spectra were acquired in positive ion mode on a Voyager DE STR (Applied
Biosystems, Foster City, CA) equipped with a UV laser (
= 337 nm). The
assignment of mass peaks to lipid species and their fragments followed
previously published procedures
(28).
SDS-PAGE and Proteomics AnalysisProtein was extracted from
free or agarose-embedded MCF-7 cells using SDS-sample buffer according to
Laemmli (29) for SDS-PAGE or
sample buffer for isoelectric focusing (8 M urea, 4% CHAPS, 10
mM dithiothreitol, 0.2% (w/v) Bio-Lytes 3/10). Two-dimensional gel
electrophoresis was performed with pre-cast immobilized pH gradient gels
following the procedures given by the manufacturer (Bio-Rad). The SDS gels
were stained with Coomassie, and stained bands/proteins were cut out for
tryptic digestion and proteomics analysis. Proteins were digested (trypsin,
sequencing grade, Promega, Madison, WI) in gel. The tryptic digests were
extracted from the gel, dried, and dissolved in 50% acetonitrile in
trifluoroacetic acid (0.1%), and an aliquot was mixed on target with matrix
(
-cyano-4-hydroxycinnamic acid in 50% acetonitrile in trifluoroacetic
acid (0.1%). MALDI-TOF mass spectra were acquired as described for the
analysis of lipids, and potential peptide sequences were identified using the
PROFOUND and MASCOT programs for mass fingerprinting.
RT-PCRTotal RNA was prepared from free or agarose-embedded
MCF-7 cells using the Trizol method according to the manufacturer's
(Invitrogen) protocol. An aliquot (0.61.0 µg of RNA) was used for
RT-PCR with the ThermoScriptTM RT-PCR system following the supplier's
(Invitrogen) instructions. PCR was carried out by applying 35 cycles with
various amounts of first strand cDNA template (equivalent to 0.050.2
µg of RNA) and 20 pmol of OSBP-related protein 7 (ORP7)-specific sense
(ORP7s 5'-agcgctggatcgagcactatg-3') and antisense (ORP7a
5'-catgttcccatagcctggctc-3') oligonucleotide primers.
Immunofluorescence MicroscopyThe agarose drop migration
assay was performed on cover slips, and the drop was removed after incubation
with migration-inducing factors. Migrating cells attached to the cover slips
were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS).
Unspecific binding sites were saturated by incubation with 3% ovalbumin in PBS
for 1 h at 37 °C. The cover slips were then incubated with 5 µg/ml
primary antibody in 0.1% ovalbumin, PBS followed by incubation with the
appropriate fluorescence-labeled secondary antibody for 2 h at 37 °C.
After staining with antibodies, GM1 was stained by incubation with 1 µg/ml
Alexa 594-labeled cholera toxin subunit B in 0.1% ovalbumin, PBS for 1 h at 37
°C. Cholesterol was stained by incubation with filipin at a concentration
of 50 µg/ml in 0.1% ovalbumin, PBS for 1 h at room temperature as described
previously (30,
31).
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RESULTS
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MG63-conditioned Medium Induces Migration of MCF-7 CellsThe
observation that 50% of primary breast tumors metastasize to bone suggests the
activity of bone-derived growth factors on the migration of breast cancer
cells. We assayed the degree of inducible migration of human breast cancer
(MCF-7) cells using a soft agarose drop migration assay
(Fig. 1A). Medium was
conditioned by incubating human osteoblast-like MG63 cells with serum-free
EMEM. The conditioned medium (MG63CM) was added to MCF-7 cells that had been
embedded into agarose drops, and migration was determined after incubation for
24 h. As shown in Fig.
1A, cells migrating out of the agarose drop surrounded
its edge and were also attached to the dish surface beneath the drop
(Fig. 1B). The
surrounding cells were counted for quantitative determination of migration
(Fig. 2). The results were
consistent with the number of migrating cells that stayed attached to the dish
surface after the drop was removed (Fig.
1B).

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FIG. 1. Agarose drop migration assay with MCF-7 cells. MCF-7 cells were
embedded in low melting agarose, and drops were formed by spotting on a
precooled tissue culture dish surface. The agarose drops were incubated with
or without serum or IGF-1- or MG63-conditioned medium (MG63CM). Cells migrated
out of the drops (A) and remained attached to the surface after
removal of the drops (B).
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FIG. 2. IGF-1- and MG63-conditioned medium-induced migration of MCF-7 cells.
Agarose-embedded MCF-7 cells were incubated with or without serum or IGF-1 or
MG63CM. The bars have been calculated from counts for cells that
migrated out of the drops. 100% migration is equivalent to the value obtained
from counts with serum-incubated cells. All values were determined as the
averages from counts in at least 10 independent experiments with standard
variances indicated as line bars. The asterisks mark values
that were significantly different (p < 0.05) from those obtained
with cells that were incubated without serum as compared with IGF-1 or
MG63CM(*) or anti-IGF-1R and IGF-1 as compared with IGF-1 (**).
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The effects of serum or IGF-1, a well known migration-inducing factor for
breast cancer cells
(35),
on MCF-7 cell migration were determined as positive controls. Incubation of
the agarose drop in serum-free medium resulted in 30% of the migration
observed with serum-containing medium (Fig.
2). Supplementation of serum-free medium with LongR3-IGF-1 peptide
analog (100 ng/ml) restored migration to 55% of the serum value. MG63CM also
restored migration of MCF-7 cells to 60% of the serum value
(Fig. 2). This effect was
specific for MG63 cells since medium that was self-conditioned by MCF-7 cells
only slightly induced migration. We tested whether IGF-1 is a
migration-inducing factor in MG63CM since it has been reported that
osteoblasts secrete IGF-1 to the medium
(32). An antibody against
IGF-1R, however, did not suppress the migration-inducing effect of MG63CM,
whereas the effect of LongR3-IGF-1 was completely inhibited
(Fig. 2). These results
indicated that MG63CM contained a migration-inducing factor that was different
from IGF-1. Migration was also not affected by the presence of phenol red in
the conditioned medium or by the addition of estrogen (not shown), indicating
that the migration-inducing effect of MG63CM is independent of estrogen.
Oxysterol in MG63-conditioned Medium Is a Migration-inducing
FactorMG63-conditioned medium was treated with charcoal to
determine whether the migration-inducing factor was a lipid or protein. As
shown in Fig. 3, charcoal
treatment of MG63CM suppressed MCF-7 migration to the value obtained with
serum-free medium, which suggested that the MG63-derived migration-inducing
factor was a lipid. We isolated four different lipid fractions from MG63CM
using organic extraction and preparative HPTLC
(Fig. 4A). The
migration-inducing effect of each lipid fraction was determined after
back-addition to serum-free or charcoal-treated MG63CM in the agarose drop
assay. Only the lipid fraction termed L1
(Fig. 3 and lane 11,
Fig. 4A) restored
migration of MCF-7 cells to 85% of the value obtained with MG63CM.

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FIG. 3. MG63-conditioned medium- and oxysterol (OS)-induced migration of MCF-7
cells. Agarose-embedded MCF-7 cells were incubated with or without serum
or MG63CM or lipid fractions isolated from MG63CM. 100% migration has been
calculated from serum-incubated cells as described in the legend for
Fig. 2. All values were
determined as the averages from counts in at least 10 independent experiments
with standard variances indicated as line bars. The
asterisks mark values that were significantly different (p
< 0.05) from those obtained with cells that were incubated with MG63CM or
OS as compared with serum-free medium (*), charcoal-treated MG63CM as compared
with untreated MG63CM (**), or MG63CM lipid fraction L1 (MG63CM L1) from
mevinolin-treated MG53 cells as compared with MG63CM L1 from untreated cells
(***).
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FIG. 4. HPTLC analysis of the lipid fraction L1 from MG63-conditioned
medium. Lipids were isolated by partitioning with organic solvent from
MG63CM and separated by HPTLC in CHCl3/CH3COOH (9:1, by
volume (A)) or CHCl3/CH3OH (95:5, by volume
(B)). A, fractions co-migrating with stained bands (cupric
acetate reagent) were scraped off the HPTLC plate, eluted from the silica gel,
and analyzed for their migration-inducing effect on MCF-7 cells. Lane
11 shows lipid fraction L1 that was found to induce migration (see
Fig. 3). Lane 1,
standard gangliosides; lanes 2 and 3, standard neutral
glycolipids; lane 4, sphingomyelin; lane 5,
dihydrosphingosine and dimethylsphingosine; lane 6, phosphatidic
acid; lane 7, sphingosine 1-phosphate; lane 8, fatty acids;
lane 9, cholesterol; lane 10, lipid extract from
MG63-conditioned medium (MG63CM); lane 11, lipid fraction L1 from
MG63CM. B, oxysterol and cholesterol species from MG63CM were
visualized with ammonium molybdate reagent and quantified by comparison to the
staining intensity obtained with known OS (25-hydroxycholesterol)
concentrations in serum-free medium. Lane 1, MG63CM; lane 2,
cholesterol; lane 3, 10 µM OS; lane 4, 5
µM OS; lane 5, 2 µM OS; lane 6,
serum-free medium without OS.
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A mass spectrometric (MALDI-TOF) analysis of L1 revealed mass peaks that
corresponded to the sodium salts of prostaglandins and cholesterol derivatives
(Fig. 5A). The
respective mass peaks were also found with prostaglandin, cholesterol, or OS
standards, suggesting that these compounds were present in L1. The
biologically active prostaglandins PGE2 (0.5 µM),
PGB2 (0.5 µM), and PGD2 (0.5
µM, not shown) or their precursor arachidonic acid (30
µM), however, did not induce migration of MCF-7 cells
(Fig. 6). These results were
consistent with the observation that incubation of MG63 or MCF-7 cells with
200 µM cyclooxygenase inhibitor acetylsalicylic acid did not
reduce migration that was induced by MG63CM
(Fig. 6).

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FIG. 5. MALDI-TOF mass spectrometric analysis of lipid fraction L1 from
MG63-conditioned medium. Lipid fraction L1 was isolated by HPTLC from
MG63CM as shown in Fig. 4.
Lipids were co-crystallized with 2,5-dihydroxybenzoic acid, used as a matrix,
and MALDI-TOF mass spectra were acquired in positive ion mode. Labeled peaks
correspond to the masses of sodium salts of the lipids indicated in the
figure. A, without mevinolin treatment of MG63 cells. B,
with mevinolin treatment of MG63 cells.
PGE2/D2,
prostaglandins E2/D2.
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FIG. 6. Effects of prostaglandins on migration of MCF-7 cells.
Agarose-embedded MCF-7 cells were incubated with or without prostaglandin
PGB2, PGE2, arachidonic acid, or acetyl salicylic acid.
100% migration has been calculated from serum-incubated cells as described in
the legend for Fig. 2. All
values were determined as the averages from counts in at least 5 independent
experiments with standard variances indicated as line bars.
PGB2/E2, prostaglandins
B2/E2.
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A mass peak at 425 indicated the presence of the sodium salt of OS in L1.
The concentration of OS extracted from MG63CM was quantified by comparison to
the mass peak (not shown) or staining intensity of 25-hydroxycholesterol, a
biologically active OS species that was added to serum-free medium at
different concentrations and then re-extracted with organic solvent for mass
spectrometry and quantitative HPTLC (Fig.
4B). The OS concentration in MG63CM was 0.8 ± 0.2
µM. The exact structure of the hydroxylated cholesterol was not
determined yet. A migration assay, however, showed that 110
µM 25-hydroxycholesterol in serum-free medium restored migration
of MCF-7 cells to 100% that of the value obtained with MG63CM
(Fig. 3). The significance of
OS as a migration-inducing factor in L1 was verified by incubation of MG63
cells with mevinolin, an inhibitor of cholesterol biosynthesis. Mevinolin
incubation resulted in the disappearance of the 425 mass peak in the L1
fraction (Fig. 5B).
Consistently, the L1 fraction from mevinolin-treated MG63 cells (MG63CM L1
mevinolin-treated) did not induce migration of MCF-7 cells
(Fig. 3). We also tested for
the migration-inducing effect of cholesterol following a procedure that has
previously been reported to induce lamellipodia protrusion and migration in
MCF-7 cells (8). However, we
were not able to detect a direct effect of cholesterol added to serum-free
medium on cell migration of agarose-embedded MCF-7 cells (not shown).
IGF-1 Elevates the Expression of ORP7 in MCF-7 CellsTo
identify a potential alteration of proteins by incubation with OS we
determined the protein expression patterns in migrating MCF-7 cells that were
incubated with IGF-1, serum, or MG63CM. As shown in
Fig. 7, protein was extracted
from cells that migrated out of the agarose drop and analyzed by
two-dimensional gel electrophoresis. Coomassie-stained protein fractions
between 40 and 90 kDa were cut out of the gel and digested with trypsin, and
the tryptic fragments were analyzed by mass spectrometry. As shown in
Table I, of 17 tryptic
fragments from a 85-kDa protein fraction 8 could be assigned to ORP7 and 9 to
heat shock 70-kDa protein 8 isoform 1 (HSP8). This fingerprint was only found
when MCF-7 cells were incubated with serum or IGF-1
(Fig. 7A) but not
without serum or with MG63CM (Fig.
7B). In addition to ORP7 and HSP8 we observed the
enhanced expression of cytokeratin, which is commonly found in metastatic
breast cancers (33,
34). Up-regulation of the ORP7
expression by incubation with IGF-1 was also demonstrated by RT-PCR with RNA
prepared from migrating MCF-7 cells (Fig.
7C).

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FIG. 7. IGF-1-induced protein expression in MCF-7 cells. Agarose-embedded
MCF-7 cells were incubated with or without IGF-1 or MG63CM. Protein was
isolated from cells that migrated out of the agarose drops and separated by
two-dimensional gel electrophoresis (A, with IGF-1; B, with
MG63CM). Labels indicate potential protein candidates that were identified by
MALDI-TOF/mass spectroscopy of tryptic fragments from Coomassie-stained
protein spots (see Table I for
fragments of ORP7 and HSP8). C, RT-PCR with RNA isolated from MCF-7
cells after incubation with IGF-1. Lane 1, cells that were not
embedded in agarose; lane 2, agarose-embedded cells; lane 3,
migrating cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
CK8, cytokeratin 8; -Tub, -tubulin;
Act, actin; BiP (GRP78), binding protein.
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TABLE I MALDI-TOF analysis of tryptic fragments from ORP7 and heat shock 70-kDa
protein 8
Protein from agarose-embedded and IGF-1-treated MCF-7 cells was separated
by two-dimensional gel electrophoresis as shown in
Fig. 7B.
Coomassie-stained protein spots were subjected to tryptic digestion, and the
peptide fragments were analyzed by MALDI-TOF/mass spectroscopy. The table
shows the assignment of the tryptic fragments of the ORP7 HSP8 spot
(Fig. 7) to known peptide
sequences in protein databases using the MASCOT and PROFOUND programs for
fingerprint analyses.
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IGF-1 and OS Induce Migration via Different Cell Signaling
PathwaysCell signaling for the migration of breast cancer cells
relies critically on signal transduction via the small GTPase families Ras and
Rho (14,
35). Membrane recruitment and
activation of Ras is required for the induction of the IGF-1R-to-MAPK
signaling pathway (35). Rho
activates the Rho-associated protein kinase (Rock) pathway for rearrangement
of actin and myosin filaments
(3537).
As shown in Fig. 8A,
inhibition of MAPK phosphorylation by the MAPK kinase inhibitor PD98059 or the
phosphatidylinositol 3-kinase inhibitor wortmannin prevented IGF-1-induced
migration of MCF-7 cells. In contrast to IGF-1, the migration-inducing effect
of MG63CM was not significantly affected by PD98059 or wortmannin. These
results indicated that IGF-1 and OS induced migration by two independent cell
signaling pathways. The IGF-1 signal was transduced by the IGF-1R-to-MAPK and
phosphatidylinositol 3-kinase pathway, whereas OS appeared to activate a
different pathway. This assumption was corroborated by the observation that
serum and IGF-1, but not MG63CM, elevated the degree of p42/44-MAPK
phosphorylation in MCF-7 cells (Fig.
8B). Migration induced by IGF-1 as well as MG63CM and OS,
however, was completely inhibited by Y-27632, a specific inhibitor of p160Rock
and Rock II (Fig. 8A).
This result suggested that IGF-1 as well as OS triggered RhoA-to-Rock
signaling. Fig. 8B
verifies that p21Ras as well as RhoA were expressed in MCF-7.

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FIG. 8. Effect of protein kinase inhibitors on tumor cell migration and MAPK
phosphorylation. Agarose-embedded MCF-7 cells were incubated with or
without serum or IGF-1 or MG63CM. Media were supplemented with MAPK kinase
inhibitor PD98059, phosphatidylinositol 3-kinase inhibitor wortmannin, or
Rho-associated protein kinase inhibitor Y-27632. A, agarose drop
migration assay. 100% migration has been calculated from serum-incubated cells
as described in the legend for Fig.
2. All values were determined as the averages from counts in at
least 10 independent experiments with standard variances indicated as line
bars. The asterisks mark values that were significantly
different (p < 0.05) from those obtained with cells that were
incubated with IGF-1 as compared with serum-free medium (*) or with inhibitors
as compared with medium without inhibitors (**, ***). B,
phosphorylation assay with antibodies specific for the phosphorylated
(pp42/44-MAPK) and both the phosphorylated and unphosphorylated form
(p42/44-MAPK) of MAPK. Immunostaining was also performed with antibodies
against p21Ras or RhoA. Lane 1, with serum; lane 2, without
serum; lane 3, with IGF-1; lane 4, with MG63CM.
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OS Alters the Sphingomyelin and Cholesterol Composition in MCF-7
CellsOS has been reported to regulate the intracellular
concentration of cholesterol and sphingomyelin
(1621),
two membrane lipids that critically affect the formation of lipid microdomains
on the cell surface
(911).
The effect of serum, IGF-1, MG63CM, or OS on the lipid composition and of
MCF-7 cells was determined by organic extraction and quantitative HPTLC. As
shown in Fig. 9A,
extraction and analysis of intracellular lipids from cells embedded in agarose
drops revealed that after incubation with serum (lane 5), MG63CM
(lane 7), or OS (lane 8), the level of MCF-7-bound
sphingomyelin was almost 2-fold higher than with serum-free medium (lane
4). Elevation of sphingomyelin was less when MCF-7 cells were incubated
with IGF-1 (lane 6). As shown in
Table II, however, these
effects were not specific for sphingomyelin since the concentration of
sphingomyelin relative to that of other phospholipids, in particular
phosphatidylcholine, was not altered by incubation with serum, MG63CM, or
OS.

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FIG. 9. Analysis of lipids from MCF-7 cells. Agarose-embedded MCF-7 cells
were incubated with or without serum or MG63CM or OS. Cells remaining in the
agarose drops were isolated by extraction with organic solvent, analyzed by
HPTLC, and stained for phospholipids (A, lipid amount equivalent to
100,000 cells) or cholesterol (B, lipid amount equivalent to 20,000
cells). A, phospholipids. Lane 1, standard phospholipids
group I (PLI, from bottom phosphatidylserine (PS),
phosphatidylethanolamine (PE)); lane 2, standard
phospholipids group II (PLII, from bottom to top,
sphingomyelin (SM), phosphatidylcholine (PC),
phosphatidylinositol (PI), and cardiolipin (CL)); lane
3, standard sphingomyelin (SM); lane 4, phospholipids
from agarose-embedded MCF-7 cells incubated without serum; lane 5,
with serum; lane 6, with IGF-1; lane 7, with MG63CM;
lane 8, with OS. B, cholesterol. Lane 1, standard
cholesterol; lane 2, cholesterol from agarose-embedded MCF-7 cells
incubated without serum; lane 3, with serum; lane 4, with
IGF-1; lane 5, with MG63CM; lane 6, with OS.
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TABLE II Relative concentration of phospholipids in MCF-7 cells
Agarose-embedded MCF-7 cells were incubated with or without serum, IGF-1,
MG63CM, or OS, and lipids were isolated, separated by HPTLC, and stained for
phospholipids as described in the legend for
Fig. 9. The relative
concentrations of different phospholipid species was calculated from a
densitometric analysis of the visualized lipid bands. The data were obtained
from three independent experiments. SM, sphingomyelin; PC,
phosphatidylcholine; PI, phosphatidylinositol; PE, phosphatidylethanalamine;
CL, cardiolipin.
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We then determined the effect of serum, IGF-1, MG63CM, or OS on the
concentration of cholesterol in agarose-embedded MCF-7 cells using
quantitative HPTLC. The cholesterol concentration was elevated from 5 ±
1 nmol/106 cells in serum-free medium (lane 2 in
Fig. 9B) to 22
± 4 nmol/106 cells in cells incubated with serum (lane
3). The elevation of cholesterol was lower in OS-incubated cells (11
± 2 nmol/106 cells, lane 6), and it was only
slightly elevated when cells were incubated with IGF-1 (7 ± 2
nmol/106 cells, lane 4). These results suggested that
incubation with serum, conditioned medium, or OS elevated sphingomyelin and/or
cholesterol, whereas IGF-1 did not significantly affect the lipid composition
of MCF-7 cells. Alteration of the lipid composition, however, was not specific
for sphingomyelin or cholesterol but involved a variety of phospholipids as
well.
OS Attracts MCF-7 Cells and Promotes Membrane Translocation of
p21RasThe OS-induced elevation of intracellular cholesterol and
sphingomyelin prompted us to investigate the formation of lipid-enriched
domains on the surface of migrating MCF-7 cells. Cholesterol, sphingomyelin,
and GM1 are known to cluster in microdomains or lipid rafts and have been
shown to co-localize with IGF-1R, other growth factor and chemokine receptors,
and small GTPases
(810).
We analyzed the formation of GM1/cholesterol-enriched membrane areas and their
co-localization with p21Ras after OS incubation using fluorescence microscopy.
We used a co-culture migration assay with MG63 and MCF-7 cells to determine
the relative position of the leading edge to the source of the migration
factor (Fig. 10A).
This assay was also used to determine the effect of a localized OS source on
the formation of lipid-enriched membrane domains and their co-localization
with p21Ras. Hoechst staining of migrating MCF-7 cells surrounding the agarose
drop revealed that the number of cells on the side of the local OS source was
higher than on the opposite side. In these cells binding of
fluorescence-labeled cholera toxin B subunit to GM1 was used for the
identification of GM1-rich membrane domains
(8).
Fig. 10B shows that
in cells incubated with serum-free medium, GM1 was predominantly localized in
vesicles throughout the cytosol or in the perinuclear region. Membrane
protrusions or lamellipodia in migrating cells, however, did not show enhanced
staining for GM1. In contrast, overnight incubation with a local bolus of OS
resulted in distinct staining of GM1-rich areas in the plasma membrane at the
leading edge of migrating tumor cells. We determined whether OS also induced
the enrichment of cholesterol or p21Ras in or at the membrane. Indirect
immunofluorescence microscopy showed that after incubation with OS a portion
of p21Ras was distributed to the leading edge of the cell that also stained
for GM1 (with cholera toxin subunit B) and cholesterol (with filipin,
Fig. 10B). The
membrane translocation of GM1 or p21Ras was not found when MCF-7 cells were
treated with mevinolin (not shown). Taken together, these results suggest that
the chemoattraction induced by OS is concurrent with the translocation of
p21Ras to cholesterol/sphingolipid-rich membrane areas at the leading edge of
migrating tumor cells.

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FIG. 10. OS-induced chemoattraction and lamellipodia formation in MCF-7
cells. A, co-culture migration assay. Agarose-embedded MCF-7
cells were incubated overnight with a dialysis bag (3,500-dalton exclusion
size) that contained MG63 cells or serum-free medium supplemented with MG63CM
or 10 µM OS. Migrating MCF-7 cells (M) were visualized
by brightfield (2) or fluorescence microscopy (3) after
staining of the nuclei with Hoechst dye. B, cells migrating out of
the agarose drop were stained for cholesterol (with filipin), GM1 (with Alexa
594-cholera toxin B subunit), and p21Ras (with rabbit anti-p21 Ras and Alexa
488-anti-rabbit IgG). Arrows indicate cholesterol/GM1-rich membrane
domains that co-localized with p21Ras. COMA, co-culture migration
assay.
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|
 |
DISCUSSION
|
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The analysis of trophic signals that control bone metastasis of breast
cancer is crucial for the identification of new molecular targets for
anti-metastasis therapy. We provide evidence for the first time that
osteoblast-derived OS may play an important role for the migration of breast
cancer cells toward bone tissue. The significance of OS for the regulation of
sphingolipid and cholesterol metabolism has long been investigated and
pharmacologically applied
(16). The importance of this
regulation for the induction of migration in cancer cells, however, has not
been studied yet. Our results suggest that IGF-1 and OS may interact by
promoting migration in tumor cells in a two-step process. In Step 1, systemic
and bone-derived IGF-1 mobilizes the tumor cells and sustains the expression
of OSBPs or ORPs in migrating cancer cells. This assumption is supported by
the observation that IGF-1 elevates the ORP7 expression in migrating MCF-7
cells. Interestingly, a recent study shows that an OS-binding protein
homologue (ORP4) is specifically up-regulated in metastasizing tumor cells
(38,
39). In Step 2, an activated
complex of OS and OSBP or ORP modulates lipid biosynthesis in the migrating
tumor cells, which induces or enhances the formation of
cholesterol/sphingolipid-rich membrane areas. These membrane domains may
trigger or amplify the Ras-dependent cell signaling pathways for tumor cell
migration toward bone tissue.
Our results indicate that this two-step signaling for tumor cell migration
involves the IGF-1-dependent activation of MAPK and phosphatidylinositol
3-kinase. In Step 1, activation of MAPK may trigger or sustain the elevation
of ORP7 or other OSBPs and ORPs. It is likely that in mammalian cells, various
OSBPs or ORPs are elevated, which depends on the cell type and the binding
specificity for particular OS isomers
(39). This assumption is
supported by studies showing that in yeast the expression of ORP homologues is
up-regulated by MAPK and as part of the cell stress response
(39). In MCF-7 cells, cell
stress signals during agarose-embedding may trigger ORP7 elevation that is
sustained in migrating cells by incubation with IGF-1. This suggestion is
consistent with the observation that the expression of heat shock proteins, in
particular HSP8, is also up-regulated in migrating cells.
Phosphatidylinositol 3-kinase, on the other hand, may participate in the
IGF-1-to-MAPK cell signaling pathway or in membrane anchoring of ORPs.
Phosphatidylinositol 3-phosphates, in particular phosphatidylinositol
3,4-diphosphate and phosphatidylinositol 3,4,5-trisphosphate, are known to
bind to the pleckstrin homology domain of ORPs, which may anchor these
proteins to the Golgi membrane
(4045).
In Step 2, osteoblast-derived OS will directly bind to and activate ORPs. The
direct effect of OS on ORPs is consistent with the observation that OS-induced
migration is not suppressed by MAPK kinase or phosphatidylinositol 3-kinase
inhibitors. Binding of OS to ORPs has been shown to participate in the Golgi
translocation/anchoring and activation of ORPs
(4245).
It has also been found that an activated, Golgi-localized OS-ORP complex
modulates the biosynthesis or transport of sphingomyelin and cholesterol
(1621,
4245).
Our results are consistent with these reports in that OS elevates
sphingomyelin. It should be noted, however, that in contrast to the previous
reports our analyses revealed an OS-dependent elevation of cholesterol and a
variety of phospholipids. This result indicates that the effect of OS on the
sphingomyelin and cholesterol concentration may be cell-specific. In MCF-7
cells, OS appears to elevate phospholipids and cholesterol with less
specificity for sphingomyelin than reported for other cell types
(4245).
The effect of OS on cholesterol was verified by filipin staining of
OS-incubated MCF-7 cells, which showed accumulation of cholesterol in
lamellipodia or at the membranes of adjacent cells (not shown).
Hydroxylated cholesterols, in particular 25-hydroxycholesterol, have been
described to trigger the biosynthesis of other lipid second messengers that
may act as migration-inducing factors on breast cancer cells. In rat kidney
and coronary artery endothelial cells, OS enhances eicosanoid production and
prostaglandin synthesis
(4648).
Interestingly, prostaglandins are known to be secreted by osteoblasts and have
been discussed to promote breast cancer metastasis
(49). The most prominent
prostaglandins (PGE2, PGB2, and PGD2),
however, did not induce migration of MCF-7 cells. We also investigated the
possibility of OS-induced migration due to enhanced prostaglandin biosynthesis
in MCF-7 cells. Incubation of neither MG63 nor MCF-7 cells with the
cyclooxygenase inhibitor acetylsalicylic acid prevented MG63CM from inducing
migration. These results suggest that the migration-inducing effect of MG63CM
or its active ingredient, OS, is not due to altered eicosanoid/prostaglandin
biosynthesis but may result from altered membrane lipid composition in MCF-7
cells.
In MCF-7 cells OS-induced or enhanced formation of
cholesterol/sphingolipid-rich membrane areas may amplify the small
GTPase-dependent activation of the Rho-associated kinase (Rock) cell signaling
pathway for actin re-arrangement and lamellipodia protrusion at the leading
edge of migrating tumor cells. This assumption is supported by the observation
that the Rock inhibitor Y-27632 prevents OS-dependent lamellipodia formation
and tumor cell migration. It has not been thoroughly investigated yet how the
metabolic regulation of cholesterol or sphingolipids modulates the formation
of membrane domains that facilitate lamellipodia protrusion or migration of
tumor cells. It is also not clear how the membrane lipid metabolism may affect
the activity of other Rho-related GTPases, e.g. Rac1, that are
essential for membrane protrusion
(5052).
MCF-7 cells have been shown to respond to exogenous cholesterol by formation
of lamellipodia at the leading edge of the cell
(8). It is, thus, likely that
elevation of intracellular sphingomyelin and cholesterol by incubation with OS
will also result in enhanced protrusion of lamellipodia. Induction of
migration may rely on the increased translocation of GTPases to GM1 and
cholesterol-rich membrane areas at the leading edge of the cell. This
assumption is supported by the observation that serum-starved MCF-7 cells,
although phenotypically similar to those incubated with MG63CM or OS, do not
show increased translocation of p21Ras to GM1 and cholesterol-rich membrane
areas.
We have shown that the migration-inducing effect of MG63CM is obliterated
by treatment of MG63 cells with mevinolin, an inhibitor of
hydroxymethylglutaryl-CoA reductase. Mevinolin treatment results in the
disappearance of mass peaks for cholesterol and OS in the migration-inducing
lipid fraction of MG63CM. These results strongly suggest that mevinolin or
other statins may be pharmacologically useful to inhibit the cholesterol or OS
biosynthesis and in turn may reduce OS-induced tumor cell migration or
metastasis. This suggestion is consistent with a recent study showing that
cerivastatin inhibits Rho-mediated migration of highly metastatic breast
cancer cells (53). The authors
discuss that this effect is caused by the inhibition of Rho
farnesylation/prenylation, which is necessary for Rho translocation from the
cytosol to the plasma membrane. Alternatively, statins may reduce tumor cell
migration by the inhibition of cholesterol/sphingolipid-dependent membrane
formation, which will also affect the translocation and activity of small
GTPases. In future studies, we will further characterize OS-induced membrane
domains and determine whether they are equivalent to detergent-insoluble
membrane fractions that are known as lipid rafts. We will also determine the
exact structure of migration-inducing, bone-derived OS-isomers, analyze the
gene expression of various OSBP/ORP species in breast cancer cells, and
investigate the significance of statins for the inhibition of OS-induced
migration and metastasis in animal studies. Taken together, our results
strongly suggest that OS is a novel migration-inducing factor and that an
understanding of the mechanism for OS-induced breast cancer cell migration
will be of significance for the development of new anti-cancer drugs.
 |
FOOTNOTES
|
---|
* This work was supported in part by National Institutes of Health Grant
MH61934-04 (to E. B.) and NS11853 (to R. K. Yu). The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
||
To whom correspondence should be addressed: Institute of Molecular Medicine
and Genetics, Medical College of Georgia, 1120 15th St., Rm. CB-2803, Augusta,
GA 30909.
1 The abbreviations used are: IGF-1, insulin-like growth factor 1; IGF-1R,
IGF-1 receptor; EMEM, minimum essential Eagle's medium with Earle's salts;
HPTLC, high performance thin layer chromatography; MALDI-TOF, matrix-assisted
laser desorption ionization-time-of-flight; MAPK, mitogen-activated protein
kinase; OS, 25-hydroxycholesterol; OSBP, oxysterol-binding protein; ORP,
oxysterol-binding protein-related protein; Rock, Rho-associated protein
kinase; HSP8, heat shock 70-kDa protein 8 isoform 1; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; RT, reverse
transcription; CM, conditioned medium; PBS, phosphate-buffered saline; GM1,
Gal
1,3GalNAc
1,4(NeuAc
2,3)Gal
1,4Glc
1,1-ceramide. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Rhea Beth-Markovitz, Institute of Molecular Medicine and
Genetics, Medical College of Georgia, for critically reading parts of the
manuscript. We also thank Dr. Somsankar Dasgupta, Institute of Molecular
Medicine and Genetics, Medical College of Georgia, for help with the lipid
analyses and Dr. Jasna Peter-Katalinic, Institute of Molecular Medicine and
Genetics, Medical College of Georgia and Westfälische Wilhelms
Universität-Münster (University of Muenster), Muenster, Germany, for
valuable discussion of the mass spectrometric analyses. We are grateful to the
imaging and mass spectrometry core facility of the Medical College of Georgia
and thank Dr. Robert K. Yu, Institute of Molecular Medicine and Genetics,
Medical College of Georgia for ongoing institutional support.
 |
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