CD146, a novel target of CD44-signaling, suppresses breast tumor cell invasion
Ouhtit et al. Cell Communication and Signaling
CD146, a novel target of CD44-signaling, suppresses breast tumor cell invasion
Allal Ouhtit 0 1
Mohammed E. Abdraboh 0 3
Andrew D. Hollenbach 2
Hatem Zayed 5
Madhwa H. G. Raj 4
0 Equal contributors
1 Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University , Doha , Qatar
2 Department of Genetics, Louisiana State University, Health Sciences Center , New Orleans , USA
3 Department of Zoology, Faculty of Science, Mansoura University , Mansoura , Egypt
4 Department of Obstetrics and Gynecology, Louisiana State University, Health Sciences Center , New Orleans , USA
5 Department of Biomedical Sciences, College of Health and Sciences, Qatar University , Doha , Qatar
Background: We have previously validated three novel CD44-downstream positively regulated transcriptional targets, including Cortactin, Survivin and TGF-β2, and further characterized the players underlying their separate signaling pathways. In the present study, we identified CD146 as a potential novel target, negatively regulated by CD44. While the exact function of CD146 in breast cancer (BC) is not completely understood, substantial evidence from our work and others support the hypothesis that CD146 is a suppressor of breast tumor progression. Methods: Therefore, using molecular and pharmacological approaches both in vitro and in breast tissues of human samples, the present study validated CD146 as a novel target of CD44-signaling suppressed during BC progression. Results: Our results revealed that CD44 activation could cause a substantial decrease of CD146 expression with an equally notable converse effect upon CD44-siRNA inhibition. More interestingly, activation of CD44 decreased cellular CD146 and increased soluble CD146 through CD44-dependent activation of MMP. Conclusion: Here, we provide a possible mechanism by which CD146 suppresses BC progression as a target of CD44-downstream signaling, regulating neovascularization and cancer cell motility.
CD146; CD44; Breast cancer; Metastasis; MMPs
Cell adhesion molecules (CAMs) are glycoproteins
responsible for promoting cell-cell adhesion and
cellextracellular matrix interactions [
]. Most cancerous
cells exhibit loss of cell adhesion capability due to
alteration in CAMs, which mediate the movement of cancer
cells from their tissue of origin to the secondary sites of
]. Our research efforts have been focused
on understanding the mechanisms by which CD44
promotes breast tumor invasion [
]. CD44 is a member
of the immunoglobulin-like family, known to promote
BC metastasis through binding of CD44 to its main
ligand hyaluronan (HA), or to other ligands, such as
osteopontin, fibronectin, collagen IV, and laminin [
The binding of HA to CD44 initiates a series of cell
signaling events through the activation of CD44 C-terminal
bound proteins such as Ankyrin, merlin, and ERM (Ezrin/
Radixin/Moesin). The CD44 cytoplasmic tail is directly
associated with actin filaments in a process mediated by
ERM proteins [
]. In order to identify the transcriptional
targets of CD44-HA interactions mediating breast tumor
invasion, we initially employed two molecular
approaches: i) subtractive hybridization and microarray
analyses, using the two BC cell lines, MDA-MB-231
(highly metastatic cells expressing high levels of CD44)
and MCF-7 (weakly invasive BC cells without any
noticeable CD44 expression); and ii) The MCF-7 founder
cell lines were used to establish the CD44-tet Off
inducible system. RNA samples from both CD44-induced
and non-induced cells were compared using microarray
analysis. Microarray analysis revealed dozens of potential
CD44-downstreatm transcriptional targets. Among these
targets, we have already validated cortactin [
] and TGF-β2 [
] as CD44-positively regulated targets,
and further characterized their respective underlying
molecular players for each signaling pathway mediating
CD44-dependent BC cell survival and motility (CD44/
Cortactin, CD44/Survivin and CD44/ TGF-β2). However,
substractive hybridization experiments led to the
identification of the CAM, CD146 as a potential
downstream target, negatively regulated by
CD44signaling (unpublished data). Therefore, the present
study focused on the structural and functional
validation of CD146, and provided evidence that CD146 is a
downstream target of CD44, suppressed during BC cell
CD146, also known as MCAM or Mel-CAM and a
member of the immunoglobulin-like CAM family, is
activated through a dimerization of its ligand, leading to a
cascade of signal transduction events (reviewed in [
CD146, a known marker of endothelial cells [
commonly used marker for the prognosis of patients with
metastatic cancer [
]. Soluble CD146 (sCD146) is known
to play a key role in angiogenesis. In fact, Lin et al.
(2007), showed that suppression of CD146 expression in
epithelial progenitor cells (EPC) led to a significant
reduction in recombinant CD146-stimulated angiogenesis,
probably due to decreased expression of pro-angiogenic
]. In another study, CD146 promoted
angiogenesis via induction of pro-angiogenic factors, such as vascular
endothelial growth factor (VEGF), urokinase plasminogen
activator (uPA), endothelial nitric oxide synthase (eNOS)
and matrix metalloproteinase 2 (MMP2) [
]. On the other
hand, Kebir et al. (2010) demonstrated that the short
isoform of CD146 promoted angiogenesis via stimulating
adhesion of circulating progenitor cells (CPC) to activated
human vascular endothelial cells [
CD146 was first identified as a viable biomarker for
], due to its high expression in 80% of
melanomas, particularly in advanced primary and
metastatic tumors [
]. More interestingly, while CD146
was detected in highly metastatic prostate cancer (PC)
cell lines such as PC3 and DU145, it was not detected in
non-metastatic PC cell line such as LNCaP and normal
prostate cells [
]. High levels of CD146 expression
were also associated with progression of prostate
adenocarcinoma in a mouse TRAMP model .
While CD146 is a promoter of melanoma and PC, its
role in BC remains nascent and controversial. In fact,
while some reports support the role of CD146 as a
promoter of BC cell growth and metastasis [
including our own work, have demonstrated the role of
CD146 as an inhibitor of BC cell growth and invasion
2, 22, 23
]. The rationale of this study is based on the
following observations: i) substantial evidence from the
literature indicate that CD146 can suppress BC growth
and invasion [
2, 22, 23
]; and ii) Our gene expression
profiling data revealed a six-fold down-regulation of
CD146 upon HA-mediated activation of CD44 (data not
shown). Therefore, based on these observations, we
hypothesized that CD44 down-regulates CD146 expression
during breast tumor progression. The aim of this study
was to validate CD146 as a negative transcriptional
target of CD44-HA downstream signaling mediating
breast tumor cell invasion. In addition to better
understand the relationship between CD44 and CD146, this
investigation has the potential to provide mechanistic
evidence of the role of CD146 as a tumor suppressor of
BC via CD44-HA interactions.
The previously described MCF7-B5 cell line, a primary
BC cell line stably transfected with a
tetracyclineregulated CD44 vector (tetracycline off ) [
], was stably
co-transfected with a vector that constitutively
expresses CD146 to obtain MCF7-B5-CD146 also called
B4 cells. The MCF7-B5 and MCF7-B5-CD146 cells
were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM) containing 10% (v/v) fetal bovine serum
(FBS), 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate
(Gibco, Gaithersburg, MD), 2.5 μg/ml doxycyline (a
tetracycline analog with greater chemical stability), 100 μg/ml
G418 (Roche Diagnostics Ltd. (GmBH), Lewes UK), and
1 μg/ml puromycin (Invivogene, CA). To induce the
expression of CD44, the cells were cultured in the same
media in the absence of doxycyline but with the addition
of 50 μg/ml Zeocin (Invivogene, CA), selecting agent of
Western blot analysis
Protein lystaes (40 μg) were prepared as previously
described (Ouhtit et al., 2007) and boiled for 5 min in an
equal volume of reducing buffer (5 mmol/L of Tris/HCl
(pH 7.4), 4% (w/v) sodium dodecyl sulfate, 20% (v/v)
glycerol, 10% (v/v) mercaptoethanol, 0.2% (w/v) bromophenol
blue), resolved on 12% polyacrylamide gels and
electroblotted onto nitrocellulose membranes. Membranes were
probed with mouse monoclonal anti-CD44 (1:1000
dilution; R&D Biosystems, MN), a mouse monoclonal
anti-CD146 (1:500 dilution; Novocastra HD, Leica
Biosystems, UK), and goat anti-actin antibodies (1:500
dilution; Santa Cruz Biotechnology, CA), followed by
incubation with donkey anti-mouse (1:2000 dilution;
Santa Cruz Biotechnology, CA) and donkey anti-goat
IgG-HRP (1:2000 dilution; Santa Cruz Biotechnology,
CA) secondary antibodies. The presence of the protein
was detected using the West Femto Supersignal
chemiluminescence kit (Thermo Scientific, IL).
RNAi-mediated depletion of CD44 and CD146
Oligonucleotides specific for human CD44, human CD146,
along with a Silencer® Negative Control #1 siRNA were
synthesized commercially (Ambion, TX) for the siRNA
knockdown of CD44. The sequences used to inhibit
CD44 are, 5′-GGAAAUGGUGCAUUUGGUGTT-3′ (sense)
and 5′-TTCCUUUACCACGUAAACCAC-3′ (antisense),
and for CD146 are, 5′-GGCACAGCUGGUUAAAGAA
TT -3′ (sense) and 5′-UUCUUUAACCAGCUGUGCC
Cells were seeded in 6-well dishes and grown to
approximately 50% confluency, washed twice with sterile
PBS and then incubated with a transfection cocktail
containing OPTI-MEM, Lipofectamin-2000 (Invitrogen,
CA) and the siRNA or scrambled oligonucleotides at a
final concentration of 50 nM for CD44 and CD146 at
37 °C for 6–7 h in DMEM culture media without
doxycyline supplemented with 20% bovine serum albumin
(BSA). After incubation, the media was replaced with
10% (v/v) FBS-enriched growth media and subsequently
incubated overnight at 37 °C. Transfected cells were
retransfected (24 h after the first transfection), as
described to ensure CD44 or CD146 inhibition. Total cell
extracts were prepared within 48 h after the second
transfection and protein expression was confirmed by
Western blot analysis, as described above.
The cells were treated with CD44 siRNA as described
above and fixed with 2% paraformaldehyde in PBS. Cells
were washed twice with PBS, blocked with 1% BSA/PBS
and incubated overnight at 4 °C with rabbit anti-CD44
(1:30 dilution; Santa Cruz Biotechnology, CA) and mouse
anti-CD146 (1:30 dilution; Novocastra HD, Leica
Biosystems, UK) primary antibodies, prepared in 1% BSA/PBS.
Cells were washed and incubated with Alexa Fluor 488 or
546 (Invitrogen, CA) for 1 h at room temperature. Slides
were mounted in Vectashield (Vector Laboratories, CA)
and the images were captured using a Laser Scanning
Confocal Microscope (Bio-Rad).
Cell invasion assay
MCF7-B5-CD146 cells were cultured in the presence of
dox and Zeocin in the absence of HA (100 μg/ml) for a
period of 24 h in order to inhibit CD44 expression
(CD44-,CD146+). Cells were then transfected with
50 nM of human CD146 specific siRNA as described
above to inhibit CD146 expression and 24 h after siRNA
treatment; the cells were washed twice with sterile PBS
and harvested by trypsinization. The harvested cells
(5.0 × 104) were then re-suspended in DMEM
supplemented with 0.5% BSA and plated in Millicell culture inserts
(8 μm pore size, Millipore, MA), which were previously
coated with a thin layer of 200 μg/ml of Matrigel™ (BD
Biosciences, MA). The inserts containing the cells were
placed into a tissue culture dish (lower chamber)
containing the “attracting medium”, consisting of
MCF7B5-CD146 special medium as stated above. Cells were
incubated for 22 h at 37 °C and the Millicell culture
insert was removed and the upper surface of the insert
was wiped immediately with a cotton swab in order to
remove non-invasive cells. The cell culture inserts were
dried under laminar flow hood for 4 h, and the cells
that had invaded and were present at the bottom of the
filter were stained using the Diff-Quick staining kit,
according to the manufacturers’ protocol (Dade Behring Inc.). The
stained cells were counted under a phase-contrast
microscope, equipped with ocular grids. Data was presented as
Mean ± Standard deviation of at least triplicates from three
experiments and statistically analyzed by SPSS program
using Student’s t-test. The difference is considered
statistically significant when p < 0.05.
Paraffin blocks including normal and tumor breast
tissue were obtained from the archives of the Department
of Pathology at Louisiana State University Health
Sciences Center, New Orleans. Immunohistochemical
assays were performed by examining adjacent sections
for both CD44 and CD146 expression using mouse
anti-human CD44 antibody (1:100 dilution, R&D, CA)
and polyclonal mouse anti-CD146 antibody (1:25 dilution,
Novocastra) after antigen retrieval carried out by boiling
the samples in 500 ml of 9 mM citrate buffer (pH 6)
(Invitrogen, Carlsbad, CA) for 25 min. Primary
antibodies were applied overnight at 4°C. Biotinylated
secondary antibody of vector labs VECTASTAIN ABC
systems universal kit and DAB substrate were applied
according to the manufacturers specifications (Vector labs,
CA). For the intensity of immunostaining, we adopted a
simple comparison of the intensity of immunostaining
using 1+ for low expression, 2+ for intermediate
expression and 3+ for high expression.
MCF7-B5-CD146 cells were cultured for 24 h in
(HA-, Tet+) supplemented media to induce CD146
expression at the cell surface. The culture media was
then replaced with a (HA+, Tet-) supplemented
media in order to induce CD44 expression.
Following culturing, the culture media was collected at 0, 24
and 48 h concentrated using Millipore minicon
concentrator columns (Millipore, MA) and was frozen at -80 °C till
use. Sample aliquots of 30 μg were separated using casein
zymography pre-casted gels according to the
manufacturer’s protocol (Invitrogen, CA).
Differences were assessed for statistical significance by
SPSS program using Student’s t-test two-tailed. Data was
presented as Mean ± Standard deviation of at least
triplicates from three experiments. The difference is
considered statistically significant when p < 0.001 (*).
The inverse relationship between cellular CD44 and CD146
in MCF7-B5 cells
The majority of previous studies related to CD146 in BC
focused generally on the detection of its expression in
circulating endothelial cells, present in the blood of BC
]. In contrast, only few reports examined
the expression of CD146 in BC [
], and yet
however, significant discrepancies exist. In fact, while some
reports concluded that CD146 promotes BC cell growth
and metastasis [
], other studies showed that
CD146 inhibits BC progression [
2, 22, 23
]. To address
this discrepancy, we have initially employed two
molecular approaches: subtractive hybridization and microarray
analyses using the two BC cell lines, MDA-MB-231
(high CD44 expression) and MCF-7 (no CD44
expression) tetracycline (Tet)-off CD44-inducible cells. Our
results revealed a six-fold down-regulation of CD146
when CD44 was activated by HA (data not shown).
Molecular and functional approaches were employed in the
present study to validate CD146 as a negative
transcriptional target of CD44-HA signaling bot at structural and
functional levels. First, we examined the expression of
CD44 and its potential target CD146 in BC cells. In
order to investigate the relationship between CD44 and
CD146, MCF7-B5 cells, which allow the inducible
expression of CD44, were stably co-transfected with a
construct allowing the constitutive expression of CD146
], also named B4 clone). The
expression of CD44 was induced by the removal of
doxycycline (dox) and activated by the addition of HA. The
expression levels of CD44 and CD146 were then
determined by Western blot analysis (Fig. 1a). A significant
induction of CD44 expression was observed upon the
removal of dox (Fig. 1a). Interestingly, a distinct inverse
relationship between CD44 and CD146 expression was
observed, with a significant reduction in CD146 levels
upon induction and activation of CD44. In addition,
induction of CD44 decreased the levels of CD146
regardless of the presence or absence of HA (data not shown).
This results suggest that CD44 downregulates CD146
through other known CD44-ligands or via other unknown
mechanism. Similarly, immuno-fluorescence staining of
CD146 in MCF7-B5-CD146 cells, showed an upregulation
of CD146 expression at the cell surface in the absence of
CD44 (Fig. 1b).
To further validate these results, an alternative
approach in which CD44 expression was inhibited by
human specific CD44 siRNA was employed. Treatment of
the cells with CD44-specific siRNA effectively inhibited
the expression of CD44 (Fig. 2). Consistent with the
results shown in Fig. 1b, we observed a significant increase
in the expression of cellular CD146 upon siRNA specific
inhibition of CD44 (Fig. 2).
Expression of CD146 and CD44 in breast tumor tissues
Next, we examined both normal and tumor breast tissue
samples from four patients with highly aggressive grade
3 breast adenocarcinoma, for the expression levels of
CD44 and CD146, by immunohistochemical analysis.
The neighboring normal human breast tissue from the
same patient was used as a control. We observed low
CD44 staining (1+) in the normal human breast tissue
with a strong staining (3+) for CD146 (Fig. 3a, c, and e,
and Table 1). In contrast, the breast tumor sample
contained high levels (3+) of CD44 expression with the
concomitant low (1+) CD146 expression levels (Fig. 3b,
d, and f, and Table 1), and these findings were consistent
with our Western blotting results (Fig. 1). Interestingly,
the low (1+) CD146 staining present in the breast tumor
tissue was specifically localized in blood vessel
endothelial cells, consistent with its known role as a marker for
endothelial cells [
Cellular localization of CD44 and CD146
To further examine the effect of CD44 on CD146
expression, we examined the cellular localization of both
these proteins by immunofluorescence. We observed a
perceptible reduction of CD44 expression on the cell
surface of the MCF7-B5-CD146 cells upon treatment
with CD44-specific siRNA; a phenomenon not observed
using the non-specific control siRNA construct (Fig. 4).
Consistent with our Western blot results,
downregulation of CD44 expression was accompanied by visible
increase in the cell surface expression of CD146 (Fig. 4).
Interestingly, low expression (1+) of the cell surface
CD146 was observed in the presence of CD44. However,
this CD146 expression was restricted to cellular surfaces
adjacent to “empty spaces” and was not present in
regions of cell-cell contact (Fig. 4, control). Further, upon
the decrease of CD44 expression, CD146 was
subsequently expressed on all cellular surfaces, including those
mediating cell-cell interactions (Fig. 4, siRNA CD44).
Taken together, these results demonstrate an inverse
relationship between the expression of CD44 and CD146 in
MCF7 BC cells.
Cell surface vs soluble CD146
The inverse relationship between CD44 and CD146
cellular levels, illustrated in the data described above prompted
us to further examine the mechanism by which CD44
controls CD146 at cellular levels. In order to test this
mechanism we collected culture media of
MCF7-B5CD146 cells after induction of CD44 expression (−Dox,
+HA). One big band correspondingThe protein levels of
both CD44 and CD146 were determined using Western
blot analysis. Our data showed significant increase of
sCD146 levels in the culture media after 48 h of CD44
induction (Fig. 5). These results suggest that CD44 might
down-regulate CD146 at cellular levels, perhaps through
CD44-dependant detachment of CD146 from the cell
surface into the culture medium.
Expression of CD44 promotes the MMP-dependent
release of CD146 from the cell surface
Our results showing that increased expression of CD44
resulted in decreased expression of CD146 on the cell
surface of BC cells, suggest that CD44 signaling may induce
the release of CD146 from the cell surface. Recent studies
have discussed the relationship between CD44 expression
on cancer cell surface and activation of Matrix
Metalloproteinases (MMPs) [
]. Based on these studies, we
hypothesized that CD44-dependent release of CD146 may
occur following an increase of MMPs activity. To test this
hypothesis, we used casein zymography approach to
evaluate MMPs activity in culture medium upon
induction of CD44. Our results showed a 40% increase in the
activity of MMP2 (Fig. 6a) 24 and 48 h post-CD44
induction, and 20% increase in the activity of MMP9 (Fig. 6b)
after 24 h of CD44 induction.
Determining the role of CD146 in breast cancer cell
We have previously reported that CD44 induction
enhances the invasiveness of BC cell lines in vitro, and
induced breast tumor metastasis to the liver [
data prompted us to examine whether increased
invasiveness may result from a decrease in CD146
expression. To test this hypothesis, we transiently transfected
MCF7-B5-CD146 cells with a CD146-specific siRNA or
a non-specific siRNA control oligonucleotide, and then
determined the invasive ability of these cells using the
Boyden chamber invasion assay. We observed greater
than two-fold increase in the invasive capacity of the
cells transfected with CD146-specific siRNA relative to
the negative control (Fig. 7). This result suggests that
loss of CD146 increased the invasive ability of
MCF7B5-CD146 cells and provides a link between our
previous data, in which CD44 expression increased cellular
invasiveness, and our present results, in which CD44
expression decreased the levels of cellular CD146.
The role of CD146 in promoting progression and
metastasis of melanoma and PC has been widely
]. The upregulation of CD146
expression contributes to increased tumorigenicity,
poor prognosis in metastatic melanoma [
] and is
believed to contribute to melanoma metastasis [
Similarly, CD146 overexpression has been reported in
metastatic PC cell lines but not in normal and
nonA simple comparison of the intensity of immunostaining was adopted using 1+ for low expression, 2+ for intermediate expression and 3+ for high expression,
and the data was summarized in a table
metastatic cells [
]. However, although the role of
CD146 in these cancers was confirmed, the very little
known about its role in BC metastasis is controversial
2, 20, 21, 23
]. One group observed the expression of
CD146 in normal breast tissue but only in 17% of the
metastatic tumors, suggesting an inhibitory effect on
BC metastasis [
]. In contrast, another group
demonstrated high levels of CD146 expression in metastatic
BC, restricted to the vascular endothelium, which is
obvious because CD146 is known as a marker of
endothelial cells [
Several lines of evidence suggest a link between CD44
signaling and CD146 expression. Our previous
microarray data indicated that the upregulation and activation
of CD44 resulted in the alteration of CD146 expression
in MCF7-B5 cells (data not shown). Further, we have
previously reported that the cytoskeletal actin filament
associated protein, cortactin is a downstream target of
CD44 signaling, a mechanism by which CD44 induces
actin filament polymerization [
] and may potentiate BC
motility and in-vivo cell invasion [
]. Further, CD146 is
also associated with the cytoskeletal actin filament,
suggesting that, like cortactin, CD146 may also
contribute to CD44-dependent BC development [
In the present study, we provide evidence to support
our hypothesis that CD44 signaling contributes to the
inhibition of cellular expression of CD146, which
subsequently contributes to BC invasiveness. As matter of
fact, clinical studies have reported a loss of CD146 in
metastatic breast tumors supporting our current
findings. First, we showed that the inducible expression and
activation of CD44 resulted in a substantial decrease in
the expression of cellular CD146 (Fig. 1) with an equally
notable converse effect upon siRNA inhibition of CD44
expression (Fig. 2). Second, we observed an inverse
relationship between CD44 and CD146 in late stage breast
adenocarcinoma tissue samples relative to the adjacent
normal tissue (Fig. 3; Table 1), thus supporting our in
vitro results (Fig. 1). Furthermore, although there is a
significant decrease in CD146 expression in late stage
breast tumors, it is restricted to regions of
neovascularization. Third, our immunofluorescence results confirm
the Western blot data, demonstrating that although
there was a significant increase in CD146 expression
upon siRNA inhibition of CD44, there was alteration in
the localization of CD146; It was absent from the surface
of cells involved in making cell-cell contacts to being
present on the surface of all cells (Fig. 4). Fourth,
consistent with our immunofluorescence data, we observed
that an increase in CD44 expression resulted in a
decrease in cellular CD146 and an increase in soluble
CD146 (Fig. 5). This result is further confirmed by the
CD44-dependent activation of MMP (Fig. 6); these data
suggest that MMP activation by CD44 resulted in the
cleavage of CD146 from the surface of the cell. Finally,
the decrease in CD146 subsequently promotes BC cell
invasiveness (Fig. 7), suggesting that loss of cell surface
CD146 is required for the promotion of BC invasiveness.
Two apparently contradicting reports studied the
relevance of CD146 in BC cell motility and invasion.
While, Zabouo et al., (2009) demonstrated that
siRNAdependent decrease in CD146 reduced the motility of
BC cells; preliminary data from our group demonstrated
that increase in CD146 expression significantly reduced
cell invasion [
]. In this study, we confirmed the latter
observation by providing substantial data demonstrating that
the inhibition of CD146 expression significantly induced
cell invasiveness. We have extended these observations
and demonstrated an inverse relationship between the cell
adhesion molecules, CD44 and CD146 in BC cell lines
where there was an increase in CD44 expression resulting
in the decrease in cellular expression of CD146. These
data put together, suggest a model in which the expression
and activation of CD44 results in the MMP-dependent
cleavage of CD146 from the surface of the cell. The loss of
CD146 from the cell surface further alters the intercellular
interactions of cells within the microenvironment to
subsequently induce the motility and invasiveness of BC cells,
thereby resulting in tumor cell metastasis.
In this model, there are two main functions of CD146
within the tumor microenvironment. First, CD146 is
greatly expressed by circulating endothelial and
progenitor cells, which are responsible for the
neovascularization process essential for tumor growth [
], and as
such plays a fundamental role in promoting angiogenesis
and neovascularization . Second, CD146 is known as
an endothelial cell marker responsible for the tight
adhesion between endothelial cells [
]. Therefore, it is
conceivable that CD44-dependent expression of cell surface
CD146 may have dual functions at different stages of BC
tumor development that are most likely dependent on
the tumor microenvironment. In early stages of BC,
CD44 expression is low, resulting in higher levels of
CD146 expression therefore promoting the tight
adhesion between cells. During early stages of metastasis, the
BC cell expression of CD44 is increased resulting in the
elevation of MMP2 and MMP9 activation [
CD44 acts as a docking site for MMP9, thus inducing its
activation and redistribution to the cell surface, thereby
bringing MMP9 to a direct proximity to cell surface, thus
allowing MMP9 to maintain its proteolytic activity on the
cell surface [
]. The proximity of MMPs to cell surface
CAMs, such as CD146, would subsequently release
proteins such as CD146 into the supernatant. The absence of
CD146 on the cell surface would then decrease cell-cell
adhesions, thereby promoting cell metastasis by degrading
the tumor tissue basement membrane and extracellular
]. Soluble CD146 then acts as a chemotactic
factor for the migration of circulating progenitor cells,
which is a crucial step for neovascularization and
]. Once vascularization has been
initiated, the upregulation of soluble CD146 intracellular
levels enhances VEGFR2 expression and VEGF
secretion, which in turn signals for angiogenesis [
These results put together suggest that CD44 activates
MMP resulting in the cleavage of CD146 from the cell
surface, thereby promoting BC cell invasion. Here, we
provide a possible mechanism by which CD146 suppresses
BC progression as a target of CD44-downstream signaling,
regulating neovascularization and cancer cell motility.
CD146 may in fact exert two distinct functions in BC
progression: i) the loss of cell surface CD146 promotes the
motility and invasion of tumor cells; and ii) the released
soluble form then initiates the neovascularization process
at secondary sites of metastasis.
AO designed the experiments; ADH helped in designing few experiments;
MEA carried out the experiments; HZ carried out statistical analyzes, edited
figures and manuscript; AO, ADH and MHGR supervised MEA; All the authors
reviewed the paper. “All authors read and approved the final manuscript.”
Ethical approval and consent to participate
Funded by Intramural Grant from Qatar University (A.O.), and by The Egyptian
Government scholarship (M.E.A.).
Availability of data and materials
All data generated during this study are included in this published article
and its Additional files.
All authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
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