slit-3 protein-mediated inhibition of cxcr4-induced ... · chemokines have recently been shown to...
TRANSCRIPT
1
Slit-3 protein-mediated inhibition of CXCR4-induced chemotactic and
chemoinvasive signaling pathways in breast cancer cells
Anil Prasad*, Aaron Z. Fernandis*, Yi Rao§ and Ramesh K. Ganju*
*Division of Experimental Medicine,
Beth Israel Deaconess Medical Center,
Harvard Medical School,
Boston, MA 02115
§ Department of Anatomy and Neurobiology,
Washington University School of Medicine,
St. Louis, Missouri 63110, USA.
Correspondence should be addressed to:
Dr. Ramesh K. Ganju, Ph.D.
Harvard Institutes of Medicine-BIDMC
4 Blackfan Circle, Room 343,
Boston, MA 02115
Tel: 617-667-0060; Fax: 617-975-5240
E-mail: [email protected]
Running Title: Slit-3 blocks CXCL12-mediated chemotaxis and chemoinvasion
Keywords: CXCR4, Slit, Robo, Chemotaxis, Chemoinvasion, Adhesion
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on November 26, 2003 as Manuscript M308083200 by guest on A
pril 30, 2020http://w
ww
.jbc.org/D
ownloaded from
2
ABSTRACT
Slit, which mediates its function by binding to the Roundabout (Robo) receptor,
has been shown to regulate neuronal and CXCR4-mediated leukocyte migration. Slit-2
was shown to be frequently inactivated in lung and breast cancers due to
hypermethylation of its promoter region. Furthermore, the CXCR4/CXCL12 axis has
recently been reported to be actively involved in breast cancer metastasis to target organs
such as lymph nodes, lung and bone. In this study, we sought to characterize the effect of
Slit on the CXCL12/CXCR4-mediated metastatic properties of breast cancer cells. We
demonstrate here that breast cancer cells and tissues derived from breast cancer patients
express Robo 1 and 2 receptors. We also show that Slit-3 treatment inhibits
CXCL12/CXCR4-induced breast cancer cell chemotaxis, chemoinvasion and adhesion,
the fundamental components that promote metastasis. Slit-3 had no significant effect on
the CXCL12-induced internalization process of CXCR4. In addition, characterization of
signaling events revealed that Slit-3 inhibits CXCL12-induced tyrosine phosphorylation
of focal adhesion components such as RAFTK/Pyk2 at residues 580 and 881, focal
adhesion kinase (FAK) at residue 576, and paxillin. We found that Slit-3 also inhibits
CXCL12-induced PI 3-kinase, p44/42 MAP kinase and Metalloproteinase 2 and 9
activities. However, it showed no effect on c-jun–N-terminal (JNK) and p38 MAP kinase
activities. To our knowledge, this is the first report to analyze in detail the effect of Slit
on breast cancer cell motility as well as its effect on the critical components of the cancer
cell chemotactic machinery. Studies of the Slit/Robo complex may foster new anti-
chemotactic approaches to block cancer cell metastasis.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
3
INTRODUCTION
Breast cancer metastasis is the main cause of treatment failure and death in
patients with breast cancer. Tumor cells migrate to other distant organs, invading blood
and lymphatic vessels, and leading to secondary tumor formation. A wide number of
molecules, including various cytokines, chemokines and growth hormones, have been
implicated to be responsible for the metastatic property of breast cancer cells (1-7).
Chemokines have recently been shown to play a key role in breast cancer
metastasis (5). Moreover, breast cancer cells have been demonstrated to express the
chemokine receptors, CXCR4 and CCR7, whereas organs such as the lymph nodes and
lungs, which represent important sites of breast cancer metastasis, have been reported to
express ligands of these receptors (5). Furthermore, in vitro neutralization of the
CXCL12/CXCR4 interaction led to a marked inhibition of lymph node and lung
metastasis. In other cancers, such as of the prostate and ovary, CXCL12 was shown to
promote tumor cell transendothelial chemotaxis (8, 9). Human melanoma cells have also
been shown to express functional CXCR4 receptor that may contribute to tumor cell
invasion and the growth of these tumors (10). Besides CXCL12, other α-chemokines
such as IL-8 and GROα have also been reported to stimulate the migration and adhesion
of prostrate carcinoma cells (11).
CXCL12-induced leukocyte chemotaxis was recently shown to be inhibited by a
secretory glycoprotein termed Slit (12). Slit was originally found to be expressed in
neurons and glial cells in the neuronal system and was later shown to play the role of a
multifunctional signaling molecule by acting as a silencer and a repellent, and perhaps as
a branching and elongation factor (13-19). Slit consists of a family of three genes, Slit-1,
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
4
Slit-2 and Slit-3 that have been cloned from different model systems (20-23). The
roundabout (Robo) receptor is a molecular target for Slit (22-25). Robo, which is highly
conserved from fruit flies to mammals, consists of a novel subfamily of immunoglobulin
(Ig) superfamily proteins (19). Identification of Robo mutations in genetic screens for
guidance defects has revealed the importance of Slit/Robo signaling in axon guidance and
cell migration (24, 26, 27).
Recently, Slit was also shown to act as a tumor suppressor, since overexpression
of Slit suppressed colony growth in breast tumor cell lines (28). Furthermore, the
importance of the Slit/Robo complex in breast cancer has been confirmed by genetic
studies showing that the Slit and Robo genes are frequently inactivated in breast cancer
patients due to hypermethylation of the promoter region (28). Although Slit has been
demonstrated to play an important role in regulating the movement of neurons and
leukocytes, the signaling pathways that mediate these effects are not well known.
In this study, we present a novel observation that Slit inhibits breast cancer cell
chemotaxis and chemoinvasion by down-modulating critical downstream molecules in
the CXCR4-mediated signaling pathway, including components of focal adhesion kinase.
It also inhibits MAP kinase and metalloproteinase 2 and 9 activities that may be involved
in chemoinvasion. Thus Slit, by blocking breast cancer cell movement, may inhibit
CXCL12-induced breast cancer metastasis.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
5
EXPERIMENTAL PROCEDURES
Cell Lines. DU4475 cells were obtained from ATCC. MDA-MB-231 cells were
generously provided by Hava Avraham (Beth Israel Deaconess Medical Center, Boston).
DU4475 cells were grown at 37oC in 5% CO2 in RPMI 1640 containing 10% fetal bovine
serum, according to the recommendations of the supplier. MDA-MB-231 cells were
maintained in DMEM with 10% fetal bovine serum and 1% Penicillin-Streptomycin at
37oC in 5% CO2.
RT-PCR Analysis. Total RNA was extracted from DU4475 and MDA-MB-231
breast cancer cells using a standard TRIZOL extraction protocol. 0.5 - 1 µg of total RNA
was reverse transcribed with extended PCR using the TITANIUMTM one step RT-PCR
kit according to the manufacturer’s instructions (BD Biosciences). The primers used
were:
Robo 1 forward: GCCGCCCCACACCCACTAT,
Robo 1 reverse: GGCCACACAGCTGAGGACGAAAG,
Robo 2 forward: CCCCAGCAGCCCAACAGTAGA and
Robo 2 reverse: TGGGCCGCAGTCCTCTTACA.
The PCR was carried out at the annealing temperature of 60oC, and the PCR products
were analyzed on 0.7% agarose gels.
Immunohistochemistry. The paraffin mounted normal and malignant breast tissues
slides obtained from the Co-operative Human Tissue Network (Philadelphia, PA) were
deparaffinized thrice with Xylene, followed by hydration with sequential treatment of the
slides twice each to 100%, 95% and 70% ethanol for 5 minutes per treatment. The
DU4475 cells or the MDA-MB-231 cells were mounted and fixed on slides with
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
6
paraformaldehyde for 15 min at room temperature, followed by washings with phosphate
buffered saline (PBS). The slides were subjected to immunohistochemistry by using the
DAB staining kit (Vector Labs, CA). Briefly, the slides were blocked for 10 min with
1.5% normal horse serum followed by incubation with either Robo 1 (1:20), Robo 2
(1:20), CXCR4 (1:100) or Control IgG (1:20) for 1 hour. The slides were then rinsed with
PBS and incubated with pan-specific universal secondary antibody for 10 min. Following
a wash with PBS, the slides were further incubated with streptavidin/peroxidase complex
reagent for 5 min. The slides were again washed and developed with 3,3’-
diaminobenzidine (DAB) as substrate according to the manufacturer’s instructions. The
slides were mounted with aqua mount and photographed.
Preparation of Slit-3 and Control Supernatant. Slit-3 was obtained from the
supernatants of myc-tagged Slit-3 transfected human embryonic kidney (HEK) cells,
according to published procedures (12, 16). Slit-3 expression was monitored by anti-c-
myc or anti-Slit-3 antibody. Control preparations obtained from the vector-transfected
cell lines were prepared using the same procedure as that employed for the Slit-3
preparations. This partially purified Slit was further enriched on a Superdex 200 gel
filtration column using the Pharmacia FPLC system. The column was run on PBS
containing 2M NaCl. The fractions were analyzed on 8% SDS-PAGE gels stained with
Silver stain or on immunoblots probed with anti-myc antibodies. The purified fractions,
which were found to be homogenous by silver staining and western blotting with c-myc
tag antibodies, were dialyzed with PBS, concentrated and used for the chemotaxis assays.
Stimulation of Cells. Stimulation of cells was carried out as described earlier (29-
31). Briefly, DU4475 cells were washed twice with Hank’s buffered salt solution
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
7
(Cellgro), and then resuspended in the Hank’s buffered salt solution with a density of 107
cells/ml. The cells were subsequently starved of serum for 3 h at 37 °C followed with
either Slit supernatant or control supernatants for 1 h at 37 °C. The pretreated cells were
stimulated with 100 ng/ml CXCL12 (10 nM) (PeproTech, Inc) at 37 °C for various time
periods. At the end of the stimulation, cells were harvested by centrifugation and lysed in
modified radioimmune precipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 1%
Nonidet P-40, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin,
10 µg/ml leupeptin, 10 µg/ml Antipain, 10 µg/ml Chymostatin, 100 µg/ml TI, 10 µg/ml
pepstatin, 10 mM sodium vanadate, 10 mM sodium fluoride, and 10 mM sodium
pyrophosphate). The cell lysates were clarified by centrifugation at 10,000 × g for 10 min
at 4°C. Protein concentrations were determined by the BioRad detergent compatible
protein assay.
Immunoprecipitation, Immunodepletion and Western Blot Analysis. Equal
amounts of protein from the stimulated time points were clarified by incubation with
protein A-sepharose CL-4B or GammaBindΤΜ sepharose beads (both from Amersham
Pharmacia Biotech) for 1 h at 4°C. The sepharose beads were removed by brief
centrifugation, and the supernatants were incubated with different primary antibodies for
2 h at 4°C. Immunoprecipitation of the antibody-antigen complexes was performed by
incubation at 4°C overnight with 50 µl of protein A-Sepharose CL-4B or GammabindΤΜ
sepharose (50% suspension). Nonspecific interacting proteins were removed by washing
the beads thrice with modified RIPA buffer and once with phosphate-buffered saline
(PBS). Immune complexes were solubilized in 50 µl of 2X Laemmli buffer, boiled and
subjected to SDS-polyacrylamide gel electrophoresis. The proteins were transferred onto
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
8
nitrocellulose membranes. The membranes were then blocked in 5% nonfat milk protein
for 2 h at 37°C or overnight at 4°C and probed with primary antibody for 3 h at room
temperature (RT) or at 4°C overnight. Immunoreactive bands were visualized using
horseradish peroxidase-conjugated secondary antibody and the enhanced
chemiluminescence system (ECL, Amersham Pharmacia Biotech). For the
immunodepletion experiments, the Slit or control supernatant concentrates were
incubated with anti-myc antibodies for 1 h at 4οC, followed by overnight incubation with
Protein A Sepharose. The beads were removed by centrifugation and the supernatant was
used as the immunodepleted sample.
Flow Cytometry. For receptor expression, both DU4475 and MDA-MB-231 cells
(1 x 106) were washed twice with phosphate buffered saline (PBS), resuspended in 100 µl
of PBS with 5% fetal bovine serum (FBS) and Robo 1 or Robo 2 antibodies (Santa Cruz
Biotechnologies) or with goat IgG antibodies as control, and then incubated at 4οC. Cells
were washed thrice in PBS containing 5% FBS and incubated with anti-goat IgG labeled
with FITC for 2 h at 4οC. The cells were next washed three times with ice-cold PBS, 5%
FBS buffer, resuspended in 200 µl of PBS, and then analyzed by flow cytometry to
determine the surface expression levels of these receptors.
For receptor down-modulation, DU4475 cells were pretreated with Slit-3 or
control supernatant and then stimulated with CXCL12 at different time points. The
stimulation was terminated with ice-cold PBS, followed by 3 washes with PBS. The cells
were fixed in 4% paraformaldehyde for 15 min at RT. The CXCR4 receptor on the cells
was stained with phycoerythrin (PE)-coupled anti-CXCR4 antibody for 1 h at 4 οC. The
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
9
cells were next washed with PBS, suspended in 1% formaldehyde in PBS, and subjected
to flow cytometric analysis.
Chemotaxis and Chemoinvasion Assays. Migration and invasion were assayed in
24-well cell-culture chambers with 8 µm pore membranes, as described (5, 30).
Membranes were pre-coated with either fibronectin (FN) (2.5 µg/ml) for chemotaxis
studies or with Matrigel (BD Biosciences) for chemoinvasion studies of the MDA-MB-
231 cells. Breast cancer cells were resuspended in chemotaxis buffer (DMEM / 0.1%
BSA / 12 mM HEPES for the MDA-MB-231 cells and RPMI 1640 / 2.5% FBS for the
DU4475 cells) at 2.5 X 106 cells/ml. Cells were pretreated with Slit-3 or control
preparation for 30 min. 150 µl of cells from each sample was loaded onto the upper
chamber. 0.6 ml medium containing CXCL12 (5 nM) with Slit-3 or the control was then
loaded onto the lower chamber. The plates were incubated for 24 h at 37οC in 5% CO2.
After incubation, the inserts were removed carefully and the MDA-MB-231 cells were
fixed, stained and counted using standard procedures. For the DU4475 line, cells were
counted directly by using a Neubar chamber. The results are expressed as the percent of
migrated cells as compared to the control.
Adhesion Assay. The wells of a 96-well tissue culture plate, pre-coated with 10
µg/ml FN (BD Biosciences) or Collagen IV (Col IV) (BD Biosciences) overnight at 4οC,
were washed with PBS and blocked with 0.5% BSA in DMEM for 1 h at 37οC. Plates
were again rinsed with PBS and air-dried. Cells were seeded at 5 X 104 cells / well in 200
µl of serum-free medium and allowed to attach for 1 h at 37οC in the presence or absence
of Slit-3 or the control. Non-adherent cells were removed by gentle washing with PBS.
The adherent cells were quantified by using the Roche MTT cell proliferation assay kit.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
10
Receptor Binding Assay: Binding of CXCL12 to its receptor CXCR4 was
assessed using 1 ng/ml of 125I-labelled CXCL12 (Amersham) in the presence of various
concentrations of purified Slit-3 or unlabelled CXCL12 (Pepro Tech)(32). Briefly,
DU4475 cells at 107/ml in RPMI 1640 containing 1% bovine serum albumin (w/v) and 25
mM/L HEPES were incubated in the presence of various concentrations of purified Slit-3
or unlabelled CXCL12 together with 1 ng/ml of 125I-labelled CXCL12 for 1 h at room
temperature and then washed thrice with cold RPMI 1640 containing 25 mM/L HEPES.
Cell-pellet associated radioactivity was determined in a gamma counter.
JNK and p38 MAP Kinase Assays. The JNK and p38 MAP kinase assays were
performed as described earlier (33, 34). Briefly, cell lysates were immunoprecipitated
with JNK antibody (Santa Cruz Biotechnology). The immune complexes were washed
twice with RIPA buffer and once in kinase buffer (50 mM HEPES, pH 7.4, 10 mM
MgCl2, 20 µM ATP). The complex was then incubated in kinase buffer containing
recombinant GST-c-Jun 0.2 µg/µl (1-79 amino acids) (Santa Cruz Biotechnology) and 5
µCi of [γ-32P]ATP for 10 min at RT. The reaction was terminated by adding 2X SDS
sample buffer and boiling the samples for 5 min. Proteins were separated on 12% SDS-
PAGE and detected by autoradiography. For the p38 MAP kinase assays, cell lysates
were immunoprecipitated with p38 MAP kinase antibody (Santa Cruz Biotechnology).
The immune complexes were washed twice with RIPA buffer and once in kinase buffer,
and then incubated in kinase buffer containing 7 µg of myelin basic protein (Upstate
Biotechnology, Lake Placid, NY) and 5 µCi of [γ-32P]ATP for 20 min at 30οC. Proteins
were separated on 15% SDS-PAGE and detected by autoradiography.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
11
c-Src Kinase Assay. The c-Src kinase assay was carried out as described
previously (30, 34). Briefly, the cell lysates were immunoprecipitated with c-Src
antiserum. The immune complexes were washed twice with RIPA buffer and once with
kinase buffer (10 mM HEPES [pH 7.4], 5 mM MnCl2, 10 µM Na3VO4). For the in vitro
kinase assays, the immune complexes were incubated for 30 min at 25οC in kinase buffer
containing acid-denatured rabbit muscle enolase (Sigma Chemical Co.) and 5 µCi of [γ–
32P]ATP. The reaction was stopped by adding 2x SDS sample buffer and boiling the
samples for 5 min. The samples were subjected to 10% SDS-PAGE and detected by
autoradiography.
PI 3-Kinase Assay. The Slit-3 and control pretreated DU4475 cells were
stimulated with CXCL12 (100 ng/ml) at different time points and lysed in lysis buffer (25
mM HEPES (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100,
1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml pepstatin, 10 µg/ml
leupeptin, 10 µg/ml Antipain, 10 µg/ml Chymostatin, 100 µg/ml TI, 10 mM sodium
vanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate). PI 3-Kinase
activity was immunoprecipitated from 500 µg of lysates using anti-p85 antibody (Santa
Cruz Biotechnology) and protein A-Sepharose CL-4B beads, as described above. The
immunoprecipitates were washed once with lysis buffer followed by three washes with
wash buffer containing 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2 and 0.2
mM EDTA. The beads were suspended in 35 µl of reaction buffer containing 25 mM
HEPES, pH 7.4, 5 mM MgCl2, 0.2 mM EDTA and 5 µl of sonicated phosphatidylinositol
(SIGMA). The reaction was initiated with the addition of 50 µM ATP containing 5 µCi
[γ-32P]ATP, and after 10 min, terminated with the addition of 300 µl Methanol: 1N HCl
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
12
(1:1). The lipids were extracted with 250 µl of CHCl3, and the organic phase containing
the labeled lipids was separated and vacuum-dried. The lipids were suspended in
chloroform and spotted on tartrate impregnated Silica gel tlc plates (Whatman Ltd,
Germany). The tlc was developed with CHCl3: CH3OH: H2O: NH4OH (90: 90: 20:7) and
the labeled lipids visualized by autoradiography. The labeled products were cut from the
plates and measured for radioactivity.
Measurement of PTEN activity. The Slit-3 and control pretreated DU4475 cells
were stimulated with CXCL12 (100 ng/ml) at different time points and lysed in lysis
buffer without sodium vanadate. PTEN activities were measured using the PTEN
Malachite Green Assay Kit according to the manufacturer’s protocol (Upstate
Biotechnology, Inc).
MMP assay. MMPs were assayed either by gelatin zymography or by using MMP
ELISA assay kits. The gelatin zymography was performed with slight modifications, as
described (35). Briefly, the cell culture was maintained to 80% confluency in serum-
supplemented media. The monolayer was rinsed two times with PBS. The cells were then
pretreated with Slit-3 or control preparation for 30 min and stimulated with CXCL12
under serum-free conditions. After 24 h incubation at 37oC in 5% CO2, cell supernatants
were collected and concentrated using centricon units (Millipore). After standardization,
samples were resolved on 10% SDS-PAGE containing 0.3% gelatin. Following
electrophoresis, gels were washed twice in wash buffer containing 2.5% Triton X-100, 50
mM Tris-HCl, pH 7.0, 6.5 mM CaCl2, 5 µM ZnCl2 and 0.5 g per L of NaN3. Gels were
then rinsed briefly in the same buffer without Triton X-100. The gels were incubated for
24 h at 37οC in the same buffer containing 1% Triton X-100. Subsequently, the gels were
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
13
fixed and stained with Coomassie Brilliant Blue. The MMP-2 and -9 quantikine ELISA
assays were performed according to the manufacturer’s instructions (R & D Systems,
MN).
Statistical Analysis. The results are expressed as the mean ± SD of data obtained
from three or four experiments performed in duplicate or triplicate. Statistical
significance was determined using the Student’s t test.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
14
RESULTS
Expression of Robo receptors in breast cancer cells and in normal and malignant
breast tissues. - Slit-3 mediates its effect by binding to Robo receptors, which are highly
conserved from fruit flies to mammals (19). In mammals, four Robo genes (Robo 1,
Robo 2, Robo 3 and Robo 4) have been identified (16, 22, 23, 36). The extracellular
domain of Robo 1 contains five immunoglobulin (Ig) domains and three fibronectin type
III repeats, whereas the intracellular region contains four identifiable conserved motifs
designated CC0, CC1, CC2 and CC3. Robo 2 and Robo 3 lack the CC2 and CC3 motifs
(25, 37). Total cellular RNA isolated from the DU4475 and MDA-MB-231 breast cancer
cell lines was analyzed for the expression of Robo 1 and Robo 2 receptors by reverse
transcriptase (RT)-PCR. As shown in Fig. 1A, both DU4475 cells and MDA-MB-231
cells expressed Robo receptors. The results were further confirmed by
immunohistochemistry with Robo 1 and Robo 2 antibodies (data not shown). We further
confirmed expression of the Robo receptors in breast cancer cell lines by FACS analysis.
We found that 45% of DU4475 cells expressed Robo 2, while 20% of the cells expressed
Robo 1. In contrast, 35% of MDA-MB-231 cells expressed Robo 1, while 21% of the
cells expressed Robo 2 (Fig. 1B).
We also examined the expression of Robo 1 and Robo 2 by
immunohistochemistry of various breast cancer tissues obtained from the Co-operative
Human Tissue Network (Philadelphia, PA). As shown in Fig. 2, Robo 1 and Robo 2 were
expressed by the breast cancer tissues. We also observed Robo expression in several
other breast cancer samples (data not shown). Some of these tissues showed a variation
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
15
in the level of Robo 1 and Robo 2. For example, some sections showed more of Robo 2
than of Robo 1 and vice versa (data not shown).
Slit-3 inhibits CXCL12-induced breast cancer cell migration, chemoinvasion and
cell adherence. - Since CXCL12 has been shown to induce breast cancer cell chemotaxis,
we analyzed whether Slit-3-mediated Robo receptor activation could modulate this
CXCL12-induced chemotaxis. The breast cancer cells were pre-incubated with Slit-3 or
control preparation and then analyzed for chemotaxis towards CXCL12. As shown, the
chemotactic response of the DU4475 (Fig. 3A) and MDA-MB-231 (Fig. 3B) breast
cancer cells was inhibited significantly in the presence of Slit-3 as compared to the
control sample. Furthermore, Slit-3 was also able to block the CXCL12-induced
chemoinvasion of MDA-MB-231 cells (Fig. 3C). We also studied the effect of Slit-3 on
the CXCL12-induced adhesive property of breast cancer cells. As shown in Fig. 3D, Slit-
3 pretreatment significantly inhibited the CXCL12-mediated adhesion of breast cancer
cells to fibronectin- and collagen-coated plates.
To further confirm that Slit-3 inhibits CXCL12-induced chemotaxis, Slit-3 was
immunodepleted (Slit-3 I.D.) from the concentrated Slit-3 supernatants using anti-myc
antibody. As a control, the control supernatants were also immunodepleted (control I.D.).
The immunodepleted supernatants were then analyzed for their inhibitory activities. We
found that the Slit immunodepleted supernatants were not able to inhibit the chemotaxis
of breast cancer cells in response to CXCL12 (Fig. 4A). We next determined the anti-
chemotactic activity of highly purified Slit-3. Slit-3 was purified using the Superdex 200
FPLC system, and the purity of the sample was determined by silver staining and
immunoblotting with c-myc antibody (data not shown). We observed that purified Slit-3
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
16
was able to block CXCL12-induced chemotaxis in a dose dependent manner, with
maximum inhibition obtained at 250 ng/ml (1.3 nM) of Slit-3 (Fig. 4B). Since Slit acts as
a non-competitive antagonist to CXCL12-mediated chemotaxis, the Ki value is equal to
the IC50 value, the concentration of antagonist that inhibits the response by 50% (38).
The IC50 of Slit-3 was found to be 111 ng/ml based on linear interpolation. Therefore,
the Ki value of Slit-3 is ~111 ng/ml. We also studied the effect of purified Slit-3 on the
chemotaxis induced by different concentrations of CXCL12. As shown in Fig. 4C, we
observed that Slit-3 blocks chemotaxis induced by higher CXCL12 concentrations (500
ng/ml). These results indicate that Slit-3 could induce inhibition of chemotaxis,
chemoinvasion and adhesion through the down-regulation of the CXCR4 receptor or by
inhibiting the binding of CXCL12 to the CXCR4 receptor.
We therefore next examined CXCR4 receptor expression on DU4475 breast
cancer cells after exposure to CXCL12, Slit-3, or the combination of CXCL12 and Slit-3.
As shown in Fig. 5A, Slit-3 pretreatment had no effect on CXCR4 expression.
Furthermore, Slit-3 pretreatment did not alter the down-modulation of the CXCR4
receptor induced by CXCL12. These results suggest that Slit-3 has no effect on CXCR4
receptor levels in DU4475 cells.
Moreover, we did not observe any significant change in 125I-CXCL12 binding to
CXCR4 in DU4475 cells in the presence of different concentrations of Slit-3. However,
unlabelled CXCL12 (100 ng/ml) did inhibit 125I-CXCL12 binding to CXCR4 (Fig. 5B).
This suggests that Slit-3 does not actively compete with CXCL12 for binding to the
CXCR4 receptor.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
17
Slit-3 inhibits the phosphorylation of CXCL12/CXCR4-mediated focal adhesion
components. - We next examined which signaling pathways might be inhibited following
treatment with the Slit-3 protein. Currently, the signaling pathways that modulate
chemotactic pathways remain imprecisely defined. One possible mechanism whereby
Slit-3 could inhibit chemotaxis is through modulation of the components of focal
adhesion complexes, which are involved in cell shape, movement and invasion. The focal
adhesion components, FAK and RAFTK/Pyk2, have been shown to interact with various
signaling molecules such as Src kinases, adaptor molecules, PI 3-kinase and SH2-
containing phosphatases through various tyrosine-containing motifs (39-41). We
determined the effect of Slit-3 on the phosphorylation of various tyrosine motifs of these
proteins using tyrosine site-specific antibodies. As shown, Slit-3 treatment specifically
blocks the tyrosine phosphorylation of RAFTK at 580 and 881 residues (Fig. 6A) and of
FAK at the 576 residue (Fig. 6B). Equal amounts of RAFTK and FAK proteins were
present in each sample (Fig. 6A and B, bottom panels). In addition, we observed that Slit-
3 treatment inhibits the CXCL12-induced tyrosine phosphorylation of paxillin, a major
cytoskeletal component of focal adhesions (Fig. 6C).
Effect of Slit-3 on Src and PI 3-kinase activities.- Src kinase and PI 3-kinase have
been shown to associate with FAK and RAFTK/Pyk2 (39, 40). It is now well established
that Src and PI 3-kinase play a crucial role in the signaling pathways implicated in
cellular migration and adherence (42-44). Src and PI 3-kinase have been shown to
regulate the phosphorylation and activation of various signaling molecules including
FAK and RAFTK/Pyk2. We observed that Slit-3 pretreatment inhibits CXCL12-induced
Src kinase (Fig. 7A) and PI 3-kinase activities (Fig. 7B). Since PI 3-kinase activity is
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
18
regulated by PTEN (45-46), we further analyzed if Slit-3 blocks PI 3-kinase by inhibiting
the activity of PTEN. As shown in Fig. 7C, Slit-3 treatment had no effect on PTEN
activity. This data suggests that Slit-3 does not regulate PI 3-kinase by modulating PTEN
activity.
Slit-3 blocks p44/42 but not p38 and JNK kinase activities. - MAP kinases are
known to play a pivotal role in regulating the proliferation and chemotaxis of many cells
(47, 48). We investigated the effect of Slit-3 on different MAPK pathways, namely
p44/42 (ERKs), SAPK2/p38 and SAPK1/JNK. As shown in Fig. 8, Slit-3 treatment
abrogated the p44/42 activation induced by CXCL12, whereas under similar conditions,
it had no noticeable effect on JNK or p38 kinase activities.
Slit-3 inhibits the activities of matrix metalloproteinases-2 and -9. - Matrix
metalloproteinases (MMPs) are a family of structurally related zinc dependent neutral
endopeptidases that are involved in the extracellular matrix degradation required for
migration (49, 50). A recent investigation suggests that chemokine-mediated regulation
of MMP activity plays an important role in transmigration (49). Moreover, MMPs have
been shown to actively contribute towards cancer progression and metastasis. We
observed an increase in the expression of MMP-2 upon stimulation of MDA-MB-231
cells with CXCL12. This increase in the level of MMP-2 expression was seen to be
blocked by Slit-3 in comparison to the control (Fig. 9A). CXCL12-induced MMP-2 and
MMP-9 activities were also considerably inhibited in cells pretreated with Slit-3 as
compared to the control-treated cells (Fig. 9B and C). This data was further confirmed by
using the MMP quantikine ELISA assay kit. Fig. 9 (D and E) shows that the CXCL12-
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
19
induced expression of MMP-9 and MMP-2 was inhibited by Slit pretreatment, whereas
control pretreatment had no effect on MMP-9 or MMP-2 activity.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
20
DISCUSSION
In this study, we demonstrate that the secreted protein Slit-3 down-modulates the
CXCL12/CXCR4-mediated chemotaxis, chemoinvasion and adhesion of breast cancer
cells. These processes are critical steps in the intricate process leading to metastasis.
Previously, Slit was known to be involved in axon guidance decisions and to play a
critical role in determining the pattern of commissural neurons (13-19). However,
evidence is growing that supports the involvement of Slit family members in
carcinogenesis (28, 51). Recently, Slit-2 was shown to be frequently inactivated in lung
and breast cancers due to hypermethylation of its promoter region (28, 52). Furthermore,
Slit-2 was demonstrated to act as a tumor suppressor since the ectopic expression of Slit-
2 protein in several breast cancer cell lines inhibited cell growth and reduced colony
formation abilities (28).
Thus, inactivation of Slit could enhance the CXCL12/CXCR4-mediated
chemotaxis of breast cancer cells. It has been reported that more motile tumor cells may
have increased metastatic potential (53). Furthermore, the CXCL12/CXCR4 axis has
recently been shown to play an important role in the homing of breast cancer cells to sites
of metastasis such as bone, lung and lymph nodes (5). Moreover, a marked reduction of
lymph node and lung metastasis was observed in mice injected with neutralizing antibody
against CXCR4 (5). In addition, the CXCL12/CXCR4 complex has been shown to be
involved in prostate and ovarian cancer metastasis (8, 9).
Slit mediates its function by binding to the Robo receptors. Robo defines a novel
subfamily of immunoglobulin superfamily proteins and is highly conserved from fruit
flies to mammals (19). Four different Robo family members have been reported (16, 22,
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
21
23, 36). In the present study, we observed differences in expression patterns between
Robo receptor family members in breast cancer cells. MDA-MB-231 cells were shown to
express more of the Robo 1 receptor, whereas DU4475 cells expressed more of the Robo
2 receptor. We also observed Robo 1 and Robo 2 expression in breast cancer tissues
derived from cancer patients. Robo gene inactivation due to hypermethylation of its
promoter region has also been reported in breast cancer patients, suggesting the
importance of Robo in the development of breast cancer (28).
Most of the work on Slit-mediated inhibition of chemotaxis has been done in the
neuronal system. Furthermore, not much is known about Slit-induced signaling pathways
that block chemotaxis. We entertained a different hypothesis, namely that Slit could
abrogate CXCL12/CXCR4-induced chemotaxis, chemoinvasion and adhesion
mechanisms. Because down-modulation of the CXCR4 receptor could lead to inhibition
of its activity, we considered whether CXCR4 receptor expression might be reduced by
Slit treatment. We did not detect down-regulation of the CXCR4 receptor upon Slit-3
treatment. This suggests that alteration in CXCR4 expression may not regulate Slit-3-
mediated inhibition of chemotactic mechanisms in breast cancer cells.
Slit-3 may regulate chemotaxis by modulating the Protein Tyrosine Kinase (PTK)
pathway. The PTK pathway regulates cell motility by modulating focal adhesions, which
consist of a complex assembly of cytoskeletal proteins (54). These structures are
important for cell migration (54). We observed that Slit-3 blocks the activity of Src
kinases, which are known to be involved in focal adhesion dynamics. The Src substrates
include FAK, RAFTK (Pyk2) and paxillin, which are components of focal adhesions (39,
40). In our study, Slit-3 treatment inhibited the phosphorylation of RAFTK at residues
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
22
580 and 881, FAK at residue 576, as well as paxillin. Tyrosine residue 580 of RAFTK
and 576 of FAK are present in the catalytic loop of these molecules and enhance their
function. Src has been shown to phosphorylate the 576 site of FAK. This suggests that
Src inhibition leads to decreased tyrosine phosphorylation of RAFTK and FAK at these
residues. c-Src has been shown to play a key role in regulating focal adhesions (41). It
has been reported that c-Src transformed cells have morphologically abnormal focal
adhesions and are defective in cell substrate adhesion (55). RAFTK, which is a novel
member of the focal adhesion kinase family, acts as a “platform kinase” and links
chemokine receptors to the nucleus via the MAP kinase pathway and cytoskeleton in
blood cells (40). It has been shown that RAFTK mediated the activation of MAP kinase
in PC12 cells (56). RAFTK and FAK have also been shown to associate with paxillin
through their proline-rich C-terminal domains (40). In neuronal cells, Slit has been shown
to affect actin cytoskeleton organization through the family of GTPase activating proteins
and N-Cadherins (57, 58).
In our study, pretreatment with Slit-3 inhibited CXCL12-induced PI 3-kinase
activity. Accumulating data have shown that multiple signaling mechanisms exist to
regulate cell migration. PI 3-kinase, the tyrosine phosphatase-SHP2 and adaptor protein
Cbl have been shown to regulate CXCL12-induced chemotaxis in T-cells (29, 42, 59-61).
The role of PI 3-kinase in cell migration has been confirmed in studies of cells obtained
from the knock-out mice of these proteins (59). Src kinases are known to activate PI 3-
kinase, which has been shown to associate with many signaling molecules including
RAFTK and FAK (62). Decreased Src kinase activation, followed by reduced
phosphorylation of RAFTK and FAK, may be involved in the Slit-3-mediated inhibition
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
23
of chemotaxis. Moreover, PI 3-kinase activity is regulated by PTEN (45-46). However,
Slit-3 had no effect on PTEN activity. This suggests that Slit-3-mediated inhibition of PI
3-kinase is not regulated through the PTEN pathway.
We also investigated the effect of Slit-3 on the downstream pathways that are
known to mediate transcriptional activation. Slit-3 was shown to selectively inhibit
p44/42 MAP kinase, but had no effect on p38 MAP kinase or JNK. Previous studies have
reported that CXCL12 selectively induces the activity of p44/42 MAP kinase in the pre B
cell line, L1.2 (63). Cell adhesion has been shown to activate p44/42 MAP kinase (64).
The p44/42 MAP signaling pathway can inhibit cell motility either by regulating gene
expression or by its inability to activate myosin light chain kinase (64). However, in the
case of T-cells, the p44/42 MAP kinase does not regulate CXCL12-induced chemotaxis
(29).
Slit-3 was also seen to influence the interaction of breast cancer cells with the
matrix environment by regulating the secretion of matrix-metalloproteinases (MMPs)-2
and -9. These enzymes are known to play a pivotal role in tumor metastasis and invasion
by the proteolytic degradation of ECM (extracellular matrix) (49). Recently, CXCL12
was shown to increase MMP-2 and MMP-9 in normal hematopoietic CD34+ cells, and
MMP-2 activity in rhabdomyosarcoma cells (65, 66).
Taken together, our results provide new information regarding Slit-3-mediated
reductions in CXCL12-induced processes, such as chemotaxis, adhesion, chemoinvasion
and MMP-2 and -9 secretion that are related to metastatic/invasive behaviors in breast
cancer cells. Furthermore, our results also provide new information about how Slit may
act at a molecular level to regulate CXCL12/CXCR4-mediated signaling pathways.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
24
These findings add a new dimension to our understanding of chemokine-mediated
metastasis and also open up a new era of potential therapeutic interventions.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
25
ACKNOWLEDGMENTS:
We thank Janet Delahanty for editing the manuscript.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
26
FOOTNOTES
The abbreviations used are: FAK, focal adhesion kinase; FBS, fetal bovine serum; RIPA,
radioimmune precipitation assay; PI 3-kinase, phosphatidylinositol 3-kinase; RAFTK,
Related adhesion focal tyrosine kinase; MAP, Mitogen-activated protein; SH2, Src
homology 2.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
27
REFERENCES
1. Hyder, S. M., Chiappetta, C., and Stancel, G. M. (2001) Int J Cancer 92, 469-73.
2. Kim, H., and Muller, W. J. (1999) Exp Cell Res 253, 78-87.
3. McEarchern, J. A., Kobie, J. J., Mack, V., Wu, R. S., Meade-Tollin, L., Arteaga,
C. L., Dumont, N., Besselsen, D., Seftor, E., Hendrix, M. J., Katsanis, E., and
Akporiaye, E. T. (2001) Int J Cancer 91, 76-82.
4. Verbeek, B. S., Adriaansen-Slot, S. S., Vroom, T. M., Beckers, T., and Rijksen,
G. (1998) FEBS Lett 425, 145-50.
5. Muller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M. E.,
McClanahan, T., Murphy, E., Yuan, W., Wagner, S. N., Barrera, J. L., Mohar, A.,
Verastegui, E., and Zlotnik, A. (2001) Nature 410, 50-6.
6. Youngs, S. J., Ali, S. A., Taub, D. D., and Rees, R. C. (1997) Int J Cancer 71,
257-66.
7. Hilakivi-Clarke, L. (2000) Cancer Res 60, 4993-5001.
8. Taichman, R. S., Cooper, C., Keller, E. T., Pienta, K. J., Taichman, N. S., and
McCauley, L. K. (2002) Cancer Res 62, 1832-7.
9. Scotton, C. J., Wilson, J. L., Scott, K., Stamp, G., Wilbanks, G. D., Fricker, S.,
Bridger, G., and Balkwill, F. R. (2002) Cancer Res 62, 5930-8.
10. Robledo, M. M., Bartolome, R. A., Longo, N., Rodriguez-Frade, J. M., Mellado,
M., Longo, I., van Muijen, G. N., Sanchez-Mateos, P., and Teixido, J. (2001) J
Biol Chem 276, 45098-105.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
28
11. Moore, B. B., Arenberg, D. A., Stoy, K., Morgan, T., Addison, C. L., Morris, S.
B., Glass, M., Wilke, C., Xue, Y. Y., Sitterding, S., Kunkel, S. L., Burdick, M. D.,
Strieter, R. M. (1999) Am J Pathol. 154, 1503-12.
12. Wu, J. Y., Feng, L., Park, H. T., Havlioglu, N., Wen, L., Tang, H., Bacon, K. B.,
Jiang, Z., Zhang, X., and Rao, Y. (2001) Nature 410, 948-52.
13. Wong, K., Park, H. T., Wu, J. Y., and Rao, Y. (2002) Curr Opin Genet Dev 12,
583-91.
14. Bashaw, G. J., and Goodman, C. S. (1999) Cell 97, 917-26.
15. Wu, W., Wong, K., Chen, J., Jiang, Z., Dupuis, S., Wu, J. Y., and Rao, Y. (1999)
Nature 400, 331-6.
16. Li, H. S., Chen, J. H., Wu, W., Fagaly, T., Zhou, L., Yuan, W., Dupuis, S., Jiang,
Z. H., Nash, W., Gick, C., Ornitz, D. M., Wu, J. Y., and Rao, Y. (1999) Cell 96,
807-18.
17. Fernandis, A. Z., and Ganju, R. K. (2001) Sci STKE 2001, E1.
18. Chen, J. H., Wen, L., Dupuis, S., Wu, J. Y., and Rao, Y. (2001) J Neurosci 21,
1548-56.
19. Ghose, A., and Van Vactor, D. (2002) Bioessays 24, 401-4.
20. Holmes, G. P., Negus, K., Burridge, L., Raman, S., Algar, E., Yamada, T., and
Little, M. H. (1998) Mech Dev 79, 57-72.
21. Itoh, A., Miyabayashi, T., Ohno, M., and Sakano, S. (1998) Brain Res Mol Brain
Res 62, 175-86.
22. Brose, K., Bland, K. S., Wang, K. H., Arnott, D., Henzel, W., Goodman, C. S.,
Tessier-Lavigne, M., and Kidd, T. (1999) Cell 96, 795-806.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
29
23. Yuan, W., Zhou, L., Chen, J. H., Wu, J. Y., Rao, Y., and Ornitz, D. M. (1999)
Dev Biol 212, 290-306.
24. Kidd, T., Brose, K., Mitchell, K. J., Fetter, R. D., Tessier-Lavigne, M., Goodman,
C. S., and Tear, G. (1998) Cell 92, 205-15.
25. Stein, E., and Tessier-Lavigne, M. (2001) Science 291, 1928-38.
26. Bashaw, G. J., Kidd, T., Murray, D., Pawson, T., and Goodman, C. S. (2000) Cell
101, 703-15.
27. Seeger, M., Tear, G., Ferres-Marco, D., and Goodman, C. S. (1993) Neuron 10,
409-26.
28. Dallol, A., Da Silva, N. F., Viacava, P., Minna, J. D., Bieche, I., Maher, E. R., and
Latif, F. (2002) Cancer Res 62, 5874-80.
29. Cherla, R. P., and Ganju, R. K. (2001) J Immunol 166, 3067-74.
30. Fernandis, A. Z., Cherla, R. P., and Ganju, R. K. (2003) J Biol Chem 278, 9536-
43.
31. Fernandis, A. Z., Cherla, R. P., Chernock, R. D., and Ganju, R. K. (2002) J Biol
Chem 277, 18111-7.
32. Salcedo, R., Wasserman, K., Young, H. A., Grimm, M. C., Howard, O. M., Anver,
M. R., Kleinman, H. K., Murphy, W. J., and Oppenheim, J. J. (1999) Am J Pathol
154, 1125-35.
33. Ganju, R. K., Dutt, P., Wu, L., Newman, W., Avraham, H., Avraham, S., and
Groopman, J. E. (1998) Blood 91, 791-7.
34. Munshi, N., Ganju, R. K., Avraham, S., Mesri, E. A., and Groopman, J. E. (1999)
J Biol Chem 274, 31863-7.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
30
35. Barrett, J. M., Puglia, M. A., Singh, G., and Tozer, R. G. (2002) Breast Cancer
Res Treat 72, 227-32.
36. Huminiecki, L., Gorn, M., Suchting, S., Poulsom, R., and Bicknell, R. (2002)
Genomics 79, 547-52.
37. Chisholm, A., and Tessier-Lavigne, M. (1999) Curr Opin Neurobiol 9, 603-15.
38. Pratt, W. B., and Taylor, P. (1990) in Principles of Drug Action, 3rd Ed., pp 1-102
Churchill Livingstone Inc.
39. Park, S. Y., Avraham, H., and Avraham, S. (2000) J Biol Chem 275, 19768-77.
40. Avraham, H., Park, S. Y., Schinkmann, K., and Avraham, S. (2000) Cell Signal
12, 123-33.
41. Schlaepfer, D. D., Hauck, C. R., and Sieg, D. J. (1999) Prog Biophys Mol Biol 71,
435-78
42. Chernock, R. D., Cherla, R. P., and Ganju, R. K. (2001) Blood 97, 608-15.
43. Woods, M. L., and Shimizu, Y. (2001) J Leukoc Biol 69, 874-80.
44. Gold, M. R., Ingham, R. J., McLeod, S. J., Christian, S. L., Scheid, M. P.,
Duronio, V., Santos, L., and Matsuuchi, L. (2000) Immunol Rev 176, 47-68.
45. Sulis, M. L., and Parsons, R. (2003) Trends Cell Biol 13, 478-83.
46. Choi, Y., Zhang, J., Murga, C., Yu, H., Koller, E., Monia, B. P., Gutkind, J. S.,
and Li, W. (2002) Oncogene 21, 5289-300.
47. Kampen, G. T., Stafford, S., Adachi, T., Jinquan, T., Quan, S., Grant, J. A., Skov,
P. S., Poulsen, L. K., and Alam, R. (2000) Blood 95, 1911-7.
48. Zhan, Y., Kim, S., Izumi, Y., Izumiya, Y., Nakao, T., Miyazaki, H., and Iwao, H.
(2003) Arterioscler Thromb Vasc Biol 13, 13
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
31
49. Nabeshima, K., Inoue, T., Shimao, Y., and Sameshima, T. (2002) Pathol Int 52,
255-64.
50. Itoh, Y., and Nagase, H. (2002) Essays Biochem 38, 21-36
51. Latil, A., Chene, L., Cochant-Priollet, B., Mangin, P., Fournier, G., Berthon, P.,
and Cussenot, O. (2003) Int J Cancer 103, 306-15.
52. Xian, J., Clark, K. J., Fordham, R., Pannell, R., Rabbitts, T. H., and Rabbitts, P.
H. (2001) Proc Natl Acad Sci U S A 98, 15062-6.
53. van Golen, K. L. (2003) Breast Cancer Res 5, 174-9.
54. Guan, J. L. (1997) Int J Biochem Cell Biol 29, 1085-96.
55. Verbeek, B. S., Vroom, T. M., and Rijksen, G. (1999) Exp Cell Res 248, 531-7.
56. Soltoff, S. P., Avraham, H., Avraham, S., and Cantley, L. C. (1998) J Biol Chem
273, 2653-60.
57. Wong, K., Ren, X. R., Huang, Y. Z., Xie, Y., Liu, G., Saito, H., Tang, H., Wen,
L., Brady-Kalnay, S. M., Mei, L., Wu, J. Y., Xiong, W. C., and Rao, Y. (2001)
Cell 107, 209-21.
58. Rhee, J., Mahfooz, N. S., Arregui, C., Lilien, J., Balsamo, J., and VanBerkum, M.
F. (2002) Nat Cell Biol 4, 798-805.
59. Hirsch, E., Katanaev, V. L., Garlanda, C., Azzolino, O., Pirola, L., Silengo, L.,
Sozzani, S., Mantovani, A., Altruda, F., and Wymann, M. P. (2000) Science 287,
1049-53.
60. Saxton, T. M., and Pawson, T. (1999) Proc Natl Acad Sci U S A 96, 3790-5.
61. Manes, S., Mira, E., Gomez-Mouton, C., Zhao, Z. J., Lacalle, R. A., and
Martinez, A. C. (1999) Mol Cell Biol 19, 3125-35.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
32
62. Acosta, J. J., Munoz, R. M., Gonzalez, L., Subtil-Rodriguez, A., Dominguez-
Caceres, M. A., Garcia-Martinez, J. M., Calcabrini, A., Lazaro-Trueba, I., and
Martin-Perez, J. (2003) Mol Endocrinol 17, 2268-82.
63. Ganju, R. K., Brubaker, S. A., Meyer, J., Dutt, P., Yang, Y., Qin, S., Newman,
W., and Groopman, J. E. (1998) J Biol Chem 273, 23169-75.
64. Fincham, V. J., James, M., Frame, M. C., and Winder, S. J. (2000) Embo J 19,
2911-23.
65. Janowska-Wieczorek, A., Marquez, L. A., Dobrowsky, A., Ratajczak, M. Z., and
Cabuhat, M. L. (2000) Exp Hematol 28, 1274-85.
66. Libura, J., Drukala, J., Majka, M., Tomescu, O., Navenot, J. M., Kucia, M.,
Marquez, L., Peiper, S. C., Barr, F. G., Janowska-Wieczorek, A., and Ratajczak,
M. Z. (2002) Blood 100, 2597-606.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
33
FIGURE LEGENDS
FIG. 1: Expression of Robo mRNA in DU4475 and MDA-MB-231 cells. A) RNA
isolated from breast cancer cells was subjected to RT-PCR analysis with primers
amplifying human Robo 1 (R1) and Robo 2 (R2) using the TITANIUMΤΜ one step RT-
PCR kit as described in Methods. The PCR was carried out with an annealing temp of
60οC. Mouse liver total RNA and control mouse β-Actin Primer mix were used for the
positive control, and PCR enzyme mix without Reverse Transcriptase was taken as the
negative control. M = marker
B) DU4475 or MDA-MB-231 cells (1 x 106) were treated with Robo 1, Robo 2 (solid
lines) or normal goat IgG antibodies as a control (dotted lines), and stained with anti-goat
antibody conjugated with FITC. Cells were analyzed by flow cytometry.
FIG. 2: Expression profile of Robo in breast cancer tissues. Paraffin-mounted slides
of patient-derived breast cancer tissues were processed for immunohistochemistry
analysis as described in Experimental Procedures using the DAB staining kit. The slides
were stained for Robo 1, Robo 2, or control IgG (AbC) and then developed with 3,3’-
diaminobenzidine (DAB) as substrate, mounted with aqua mount and photographed.
Breast cancer tissue slides were also stained for hematoxylin and Eosin (H&E) to check
the pathology of the tissue section.
FIG. 3: Slit-3 blocks CXCL12-induced chemotaxis, chemoinvasion and cell
adhesion. DU4475 (A) or MDA-MB-231 (B, C and D) breast cancer cells were
pretreated with varying concentrations of Slit-3 (µg/ml) or control (µg/ml), treated with
CXCL12 (10 nM), then subjected to chemotactic (A and B) or chemoinvasion (C) assays
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
34
as described under Methods. (D) MDA-MB-231 cells were grown on plastic or on plates
precoated with either fibronectin (FN, 25 µg/ml) or Collagen IV (Col IV, precast). The
cells were then untreated or treated with CXCL12 alone or together with partially
purified Slit-3 or control preparation. Adherent cells were analyzed by using the Roche
MTT Cell Proliferation Assay Kit. The experiments were done in triplicates and are
represented as the mean ± s.e.m. The data are representative of 4 different experiments.
The % chemoinvasion was calculated by using the following equation: [(Mean number of
cells invading through the membrane / Mean number of cells migrating through the
control membrane)x 100]. The % cell adhesion was calculated by considering the Optical
Density (O.D.) of the untreated control as 100%. * P < 0.05 for all experiments.
FIG. 4: Effect of Slit-3 immunodepleted supernatant and purified Slit-3 on
chemotaxis. (A) The Slit-3 or control supernatant concentrates were incubated with anti-
myc antibodies for 1 h at 4οC, followed by overnight incubation with Protein A
Sepharose. The beads were removed by centrifugation and the supernatant was used as
the immunodepleted sample (I.D.). DU4475 breast cancer cells were pretreated with the
immunodepleted control (Control I.D., 100 µg/ml), immunodepleted Slit-3 (Slit-3 I.D.,
100 µg/ml) or the undepleted concentrates (either Slit-3 or control; 100 µg/ml). The cells
were untreated (UN) or treated with CXCL12 as indicated and then subjected to
chemotactic assays. (B) The DU4475 breast cancer cells were pretreated with FPLC
purified Slit at varying concentrations as indicated and then subjected to chemotactic
assays using CXCL12 (10 nM). The IC50 value (111 ng/ml) was determined by using
linear interpolation. * P < 0.05. (C) shows a chemotactic assay in the presence of
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
35
purified Slit-3 using different concentrations of CXCL12. Experiments were repeated
three times, and a representative one is shown.
FIG. 5: Role of Slit-3 in CXCL12-mediated CXCR4 down-modulation and CXCL12
binding to CXCR4. (A) DU4475 cells preincubated with Slit-3 (100 µg/ml) were
stimulated for different time points with or without CXCL12 (10 nM) or with CXCL12
alone, as indicated. The cells were collected and analyzed for CXCR4 surface expression
by FACS, as described in Experimental Procedures. The figure is representative of 3
different experiments.
(B) Effect of Slit-3 on CXCL12 binding to CXCR4. Binding of 125I-labelled CXCL12 in
the presence of various concentrations of Slit-3 or unlabelled CXCL12 (100 ng/ml) was
performed in DU4475 cells as described in Experimental Procedures. The data are shown
as the mean ± s.e.m. of three independent experiments.
FIG. 6: Effect of Slit-3 on the CXCL12-induced tyrosine phosphorylation of focal
adhesion molecules. Slit-3 (100 µg/ml) or control (100 µg/ml) pretreated DU4475 cells
were unstimulated (0) or stimulated with CXCL12 (10 nM) for the indicated time points.
The cells were lysed and the lysates analyzed by serial immunoblotting with antibodies
recognizing the site-specific tyrosine-phosphorylated residues of RAFTK (A) or FAK
(B). The blots were stripped and probed with RAFTK (A, bottom panel) or FAK (B,
bottom panel) antibodies. (C) The stimulated cell lysates were immunoprecipitated (I.P.)
with anti-paxillin antibodies. The immune complexes were resolved on SDS-PAGE and
Western blotted (W.B.) with anti-phosphotyrosine antibodies (pY99) (C, upper panel).
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
36
The same blots were stripped and reprobed with anti-paxillin (C, bottom panel) antibody.
Ab= antibody control, TCL= total cell lysates.
FIG. 7: Slit-3 blocks CXCL12-induced Src kinase and PI 3-kinase activation.
DU4475 cells were preincubated with Slit-3 (100 µg/ml) or control (100 µg/ml) and
stimulated with CXCL12 (10 nM) for the indicated time points. The stimulated cells were
lysed and equal amounts of protein lysates were immunoprecipitated with either Src
kinase antibodies (A) or p85 antibodies (B). The immune complexes were subjected to in
vitro kinase assays as described in Methods. For the Src kinase activity, enolase was used
as a substrate (A). The p85 immunoprecipitates were assayed for PI 3-kinase activity with
phosphatidylinositol and radiolabeled ATP as substrates. The radiolabeled lipids were
then extracted and chromatographed on silica gel TLC plates. The labeled lipids were
then visualized by autoradiography. The experiment was done three times, and a
representative one is shown. (C) The cell lysates were analyzed for PTEN activity by
using the Malachite Green Assay Kit with Ptdlns(3,4,5) P3 as substrate. The inorganic
phosphate liberated was detected by following the procedures of the assay kit.
Absorbance was measured at 650 nm. The activity of PTEN is represented as pmoles of
phosphate/µg of protein. The values are shown as the mean ± s.e.m. of three independent
experiments.
FIG. 8: Slit-3 specifically inhibits CXCL12-induced p44/42 MAP kinase activity.
Cell lysates were obtained from Slit-3 (100 µg/ml) or control (100 µg/ml) pretreated
DU4475 cells after stimulation with CXCL12 (10 nM). The lysates were
immunoprecipitated with p44/42 (A), p38 MAP kinase (B) or JNK kinase (C) antibodies.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
37
The immune complex for p44/42 was run on SDS-PAGE and immunoblotted with
phospho-p44/42 antibody (p-p44/42) (A, upper panel). The protein levels were monitored
by stripping the blot and reprobing with anti-p44/42 antibodies (A, lower panel). The
p38 MAP kinase and JNK immunoprecipitates were subjected to in vitro kinase assay
using myelin basic protein (MBP) and c-Jun, respectively, along with labeled ATP as
substrates.
FIG. 9: Slit-3 blocks CXCL12-induced MMP activity. Conditioned media were
concentrated from MDA-MB-231 cells pretreated with partially purified Slit-3 (100
µg/ml) or control preparation (100 µg/ml) in the presence of CXCL12 (10 nM). Equal
volumes of the concentrates were analyzed for the level of MMP-2 using immunoblot
analysis with anti-MMP-2 antibodies (A). For the gelatin zymography, similar volumes
of concentrates were loaded with Laemmli buffer without boiling and run on 10% SDS-
polyacrylamide gels containing 0.3% gelatin. The gel was then stained with Coomassie
Blue (B). The activity of MMP was quantified by inverting the image and scanning for
band intensity (C). The gelatin zymography was done 4 times and the experiment is a
representative one. The graph depicts the average negative band intensity based on 4
different experiments. Data are represented as the mean ± s.e.m. MDA-MB-231 cells
were pretreated with partially purified Slit-3 or control preparation as indicated, followed
by stimulation with or without CXCL12 (100 ng/ml). The supernatant concentrates from
these samples were then assayed for MMP-2 (D) and MMP-9 (E) expression using the
quantikine ELISA assay. * P < 0.05 for all experiments.
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Figure 1
R1 R1R2 R2
DU
4475
MD
A-M
B-2
31
Posi
tive
Con
trol
Neg
ativ
e C
ontr
ol
2.0 Kbp1.2 Kbp0.8 Kbp
21%
35%20%
45%
A.
B.
(i) (ii)
M1
M1
Robo
DU4475 MDA-MB-231
Robo 1Robo 1
Robo 2 Robo 2
M
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Figure 2
H&E AbC
Robo 1 Robo 2
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Figure 3
*
A.
0
25
50
75
100
125
150
Cel
ls m
igra
ted
(x10
3 )CXCL12 - + + + + + - -
Control - - 10 - 100 - 100 Slit-3 - - - 10 - 100 - 100
0
20
40
60
80
100
Cel
ls m
igra
ted
(x10
3 )
CXCL12 - + + + + + - -
Control - - 10 - 100 - 100 -Slit-3 - - - 10 - 100 - 100
0
20
40
60
80
% C
ell i
nvas
ion
CXCL12 - + + + +
Control - - 100 - -Slit-3 - - - 10 100
*
0
50
100
150
200
250
300
FN Col IV
% C
ell a
dhes
ion
Untreated
CXCL12
Control + CXCL12
Slit-3 + CXCL12
Plastic
D.
*
*
*
B.
C.
*
*
-
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Figure 4
0
40
80
120
UN
CXCL12 (10 nM)
Slit-3
Slit-3
I.D.
Contro
l
Contro
l I.D
.
Cel
ls m
igra
ted
(x10
3 )
A.
Cel
ls m
igra
ted
(x10
3 )
B.
Slit-3 (ng/ml) - - 1 10 100 250 500 1000 CXCL12 (10 nM) - + + + + + + +
C.
Cel
ls m
igra
ted
(x10
3 )
CXCL12 (ng/ml) 0 10 50 100 250 500 0
20
40
60
80
100
120
140ControlSlit-3
0
40
80
120
160
*
(+)
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Figure 5
0
20
40
60
80
100
120
0 1 2 3 4 5Time (hrs)
% C
XC
R4
surf
ace
expr
essi
on
CXCL12 Slit-3 Slit-3 + CXCL12A.
B.
0
10000
2000
4000
6000
8000
Bin
ding
of
125 I
-CX
CL
12 (
cpm
)
Slit-3 (ng/ml) 0 100 250 500 -CXCL12 (ng/ml) - - - - 100
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Time (min) 0 0 2.5 2.5
Slit-3 - + - +Control + - + -
RAFTK (pY580)
RAFTK (pY881)
RAFTK
A.
Time (min) 0 0 2.5 2.5
Slit-3 - + - + Control + - + -
FAK 576
FAK
B.
Figure 6
Time (min) Ab 0 0 2.5 2.5 5 5 TCL
Slit-3 - + - + -
IP: Paxillin; WB: pY99
IP: Paxillin; WB: Paxillin
CXCL12 (10 nM)
Control + - + -
C.
CXCL12 (10 nM)
CXCL12 (10 nM)
+ -
+
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Figure 7
PI 3-kinase activity
Time (min) 0 0.5 2 5 20 0 0.5 2 5 20
CXCL12 (10 nM) CXCL12 (10 nM)
Control Slit-3B.
CXCL12 stimulation (min)
pmol
es o
f ph
osph
ate/
µg
o
f pr
otei
n
0
20
40
60
80
100
120
140
0 0.5 2.5 5 20 60
C.
Time (min) 0 0 2.5 2.5
Slit-3 - + - +Control + - + -
Src kinase assay
CXCL12 (10 nM)A.
Enolase
ControlSlit-3
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Time (min) 0 0 2.5 2.5
Figure 8
Slit-3 - + - + Control + - + -
p-p44/42
p44/42
p38 MAP kinase
JNK kinase
CXCL12 (10 nM)A.
B.
C.
MBP
c-Jun
Slit-3 - + - + Control + - + -
CXCL12 (10 nM)
Slit-3 - + - + Control + - + -
CXCL12 (10 nM)
Time (min) 0 0 2.5 2.5
Time (min) 0 0 2.5 2.5
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Figure 9
0
10
20
30
40
MM
P-2
(ng/
ml)
Slit (µg/ml) 50 - 100 - 100 -
Control (µg/ml) - 50 - 100 - 100
CXCL12 (ng/ml) 100 100 100 100 - -
Slit (µg/ml) 50 - 100 - 100 -
0
20
40
60
80
100
MM
P-9
(ng/
nl)
Control (µg/ml) - 50 - 100 - 100
CXCL12 (ng/ml) 100 100 100 100 - -
E.D.
*
Control + -
MMP-2
MMP-9
Slit-3 - +Control + -
CXCL12 (10 nM)
0
50
100
150
200
250
% B
and
Inte
nsity
Control + CXCL12Slit-3 + CXCL12
MMP-9 MMP-2
B.
Slit-3 - +
CXCL12 (10 nM)A. C.
MMP-2* *
*
* *
*
*
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Anil Prasad, Aaron Z. Fernandis, Yi Rao and Ramesh K. Ganjusignaling pathways in breast cancer cells
Slit-3 protein-mediated inhibition of CXCR4-induced chemotactic and chemoinvasive
published online November 26, 2003J. Biol. Chem.
10.1074/jbc.M308083200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on April 30, 2020
http://ww
w.jbc.org/
Dow
nloaded from