New facets of matrix metalloproteinases MMP-2 and MMP-9 as cell surface transducers: Outside-in signaling and relationship to tumor progression

Abstract

This review focuses on matrix metalloproteinases (MMPs)-2 (gelatinase A) and-9 (gelatinase B), both of which are cancer-associated, secreted, zinc-dependent endopeptidases. Gelatinases cleave many different targets (extracellular matrix, cytokines, growth factors, chemokines and cytokine/growth factor receptors) that in turn regulate key signaling pathways in cell growth, migration, invasion, inflammation and angiogen-esis. Interactions with cell surface integral membrane proteins (CD44, αVβ/αβ1/αβ2 integrins and Ku protein) can occur through the gelatinases' active site or hemopexin-like C-terminal domain. This review evaluates the recent literature on the non-enzymatic, signal transduction roles of surface-bound gelatinases and their subsequent effects on cell survival, migration and angiogenesis. Gelatinases have long been drug targets. The current status of gelatinase inhibitors as anticancer agents and their failure in the clinic is discussed in light of these new data on the gelatinases' roles as cell surface transducers — data that may lead to the design and development of novel, gelatinase-targeting inhibitors.

Full text

Review
New facets of matrix metalloproteinases MMP-2 and MMP-9 as cell surface
transducers: Outside-in signaling and relationship to tumor progression
Brigitte Bauvois ⁎
INSERM U872, Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Université Paris Descartes, Paris, France
abstractarticle info
Article history:
Received 15 June 2011
Received in revised form 3 October 2011
Accepted 4 October 2011
Available online 12 October 2011
Keywords:
Gelatinase
Cell surface binding
Cancer
Inhibitor
Function
Outside-in signaling
This review focuses on matrix metalloproteinases (MMPs)-2 (gelatinase A) and -9 (gelatinase B), both of
which are cancer-associated, secreted, zinc-dependent endopeptidases. Gelatinases cleave many different
targets (extracellular matrix, cytokines, growth factors, chemokines and cytokine/growth factor receptors)
that in turn regulate key signaling pathways in cell growth, migration, invasion, inflammation and angiogen-
esis. Interactions with cell surface integral membrane proteins (CD44, αVβ/αβ1/αβ2 integrins and Ku pro-
tein) can occur through the gelatinases' active site or hemopexin-like C-terminal domain. This review
evaluates the recent literature on the non-enzymatic, signal transduction roles of surface-bound gelatinases
and their subsequent effects on cell survival, migration and angiogenesis. Gelatinases have long been drug
targets. The current status of gelatinase inhibitors as anticancer agents and their failure in the clinic is dis-
cussed in light of these new data on the gelatinases' roles as cell surface transducers —data that may lead
to the design and development of novel, gelatinase-targeting inhibitors.
© 2011 Elsevier B.V. All rights reserved.
Contents
1. Introducing MMPs and their roles in cancer ................................................ 29
2. Development of synthetic MMP inhibitors as cancer drugs ......................................... 31
3. Gelatinases (MMP-2 and MMP-9) as cancer biomarkers........................................... 31
4. Proteolysis-dependent functions of gelatinases ............................................... 31
5. Cell surface-associated gelatinases..................................................... 32
6. Outside-in signaling by cell surface-associated gelatinases ......................................... 33
7. Open questions and conclusions...................................................... 33
Acknowledgement .............................................................. 34
References .................................................................. 35
1. Introducing MMPs and their roles in cancer
The matrix metalloproteinase (MMP) family consists of at least 23
structurally related, zinc-dependent endopeptidases [1,2]. The family
shares specific functional and structural components, including a
hydrophobic signal peptide for secretion, a propeptide domain for
enzyme latency, a catalytic domain with a highly conserved zinc-
binding site and (for the majority of MMPs) a hemopexin-like
C-terminal domain (PEX) linked to the catalytic domain via aflexible
hinge region (Fig. 1A) [1,2]. The PEX domain binds endogenous tissue
inhibitors of MMPs (TIMPs) and certain MMP substrates and is in-
volved in MMP activation [1]. TIMPs include four members originally
described as inhibitors of MMP activities, which also have biological ac-
tivities that are independent of MMP inhibition and regulate cell growth,
migration, survival and angiogenesis [3–5]. MMPs include membrane-
Biochimica et Biophysica Acta 1825 (2012) 29–36
Abbreviations: MMP, matrix metalloproteinase; PEX, hemopexin-like C-terminal
domain; TIMP, tissue inhibitor of metalloproteinase; MT, membrane type; CBD,
collagen-binding domain; ECM, extracellular matrix; MMPI, matrix metalloproteinase
inhibitor; CLL, chronic lymphocytic leukemia; AML, acute myeloid leukemia; EGF,
epidermal growth factor; VEGF, vascular endothelial growth factor; TGF-β, transforming
growth factor-β;FGF,fibroblast growth factor; TNF, tumor necrosisfactor; IL, interleukin;
IFN, interferon; IGF-BP, insulin-like growth factor-binding protein; ICAM, intercellular
adhesion molecule; PF4, platelet factor-4; SDF-1, stromal-cell derived factor-1; IP-10,
IFN-γ-induced protein of 10 kDa; MCP, macrophage chemotactic protein; I-TAC, IFN-γ-
induced T cell-activated chemokine; MIG, monokine induced by interferon-γ;RECK,
reversion-inducing cysteine-rich protein with Kazal motif; LRP, low density lipoprotein-
related scavenger receptor; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated
protein kinase; ERK, extracellular signal-regulated protein kinase; FAK, focal adhesion
kinase; CAM, chick chorioallantoic membrane; HIF, hypoxia-induced transcription factor
⁎Centre de Recherche des Cordeliers, INSERM U872, 15 rue de l'Ecole de Médecine,
F-75270 Paris cedex 06, France. Tel.: +33 144 278 188; fax: + 33 144 278 161.
E-mail address: brigitte.bauvois@crc.jussieu.fr.
4
0304-419X/$ –see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbcan.2011.10.001
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anchored and secreted MMPs. The membrane-anchored MMPs (MT-
MMPs) are localized at the cell surface by a C-terminal (type I) trans-
membrane domain or a glycosylphosphatidylinositol anchor (Fig. 1B)
[4,6]. A type II transmembrane MMP (MMP-23) has also been described
[6]. TIMP-1 has a relatively low affinity for the MT-MMPs [5].MMPsse-
creted as latent pro-enzymes include collagenases, stromelysins, matrily-
sins and two gelatinases (A and B) (Fig. 1C) [4].Removalofthe
prodomain by the endopeptidase furin leads to MMP activation. TIMPs
inhibit most of the secreted MMPs [5]. The gelatinases differ from most
of the other MMPs in that they have a collagen-binding domain (CBD)
within the catalytic domain (Fig. 1C). The CBD is composed of three
fibronectin type II repeats and is involved in the binding of collagenous
substrates, elastin, fatty acids and thrombospondins [7].
Matrix metalloproteinases selectively degrade various compo-
nents of the extracellular matrix (ECM) and release growth factors
and cytokines that reside in the ECM [8,9]. The MMPs are also capa-
ble of activating various latent growth factors, cytokines and chemo-
kines and cleaving cell surface proteins (cytokine receptors, cell
adhesion molecules, the urokinase receptor, etc.) [1,2,10,11].
Through their proteolytic activity, MMPs play crucial roles in inva-
sion and metastasis and regulate signaling pathways that control
cell growth, survival, invasion, inflammation and angiogenesis
hinge
linker
Pre Pro catalytic Zn2+
SS
PEX
(A) Basic MMP structure
(C) Secreted matrilysins
collagenases,
stromelysins
gelatinases
A & B
SS
SS
CBD
(B) Membrane-anchored
SS
GPI
TM-I
cytoplasmic
tail
SS
Fig. 1. Structures of the MMPs. (A) The general domain structure of MMP family members. The signal peptide (Pre) guides the MMP into the rough endoplasmic reticulum during
synthesis. The propeptide domain (Pro) sustains the latency of MMPs. The catalytic domain houses a highly conserved Zn
2+
binding region. The hemopexin-like-C-terminal domain
(PEX) is linked to the catalytic domain by a short hinge region. (B) MT-MMPs include membrane-anchored MMPs localized at the cell surface through a C-terminal (type I) trans-
membrane domain (TM-I) or by a glycosylphosphatidylinositol anchor (GPI). (C) Secreted MMPs include stromelysins, matrilysins, collagenases and gelatinases. The gelatinases
(MMP-2 and MMP-9) contain repeats of fibronectin type II-like domains (the collagen binding domain, CBD) that interact with collagen and gelatin.
MMPs
TIMPs
Proteolysis of ECM
Release of:
growth factors
angiogenic factors
Processing of:
cytokines
chemokines
growth factors
growth factor binding proteins
cell adhesion molecules
receptors
Growth Survival
Invasion Metastasis
Angiogenesis
Inflammation
endothelial,
inflammatory
and tumor cells
Fig. 2. A schematic overview of the roles of MMPs in cancer. MMPs degrade structural components within the ECM, facilitating tumor cell invasion and metastasis and thus releasing
growth factors, cytokines and angiogenic factors embedded in the ECM (VEGF, TGF-β, bFGF, IFN-γ, etc.). MMPs also generate angiogenesis inhibitors, such as angiostatin, endostatin
and tumstatin. MMPs process and activate or inactivate signaling molecules (cytokines, chemokines, growth factors) that target immune cells (inflammation), endothelial cells (an-
giogenesis) and tumor cells (cell growth, survival, migration, invasion and metastasis). MMP-mediated cleaving of adhesion molecules (E-cadherin, ICAM-1, integrins, etc.)
enhances tumor cell migration and invasion. ( negative regulation, ➞positive regulation).
30 B. Bauvois / Biochimica et Biophysica Acta 1825 (2012) 29–36

(Fig. 2). A number of excellent reviews have discussed the MMPs'
roles in cancer [1,2,8,12–14].
2. Development of synthetic MMP inhibitors as cancer drugs
Many different MMP inhibitors (MMPIs) have been designed to
target MMPs in cancer [13,15,16]. Although these compounds differ
in their inhibitory potencies towards MMPs, none of them are selec-
tive for a given MMP (including the gelatinases).
The first generation of MMPIs were peptidometics (such as bati-
mastat and marimastat) that mimic the structure of collagen. They
act as competitive inhibitors and chelate the zinc ion present at the
MMP's active site. To improve specificity and oral bioavailability,
non-peptidometics (such as tanomastat, prinomastat, BMS-275291,
CGS27123A, etc.) were synthesized on the basis of the active site's
three-dimensional conformation. Other MMPIs include tetracycline
derivatives (such as metastat/COL-3, minocycline and doxycycline)
that inhibit both the MMPs' enzymatic activity and their synthesis
(by blocking gene transcription) [17,18]. To date, all clinical trials of
these MMPIs in advanced cancer patients have failed, with the excep-
tion of metastat (which has entered Phase II trials for Kaposi's
sarcoma and brain tumors) [19,20]. The latest generation of MMPIs
includes biphosphonates [21,22] and S-3304, a D-tryptophan deriva-
tive that primarily inhibits gelatinases [23]. Novel biphosphonate de-
rivatives show benefits as a result of altering the expression pattern of
MMPs/TIMPs in breast cancer cells [13]. A Phase I clinical trial of
S-3304 in patients with advanced and refractory solid tumors found
that the compound was safe, well tolerated and achieved plasma con-
centrations above those required to inhibit gelatinases [24]. However,
it is not yet known whether S-3340 will be effective in Phase II/III
clinical trials.
There are several possible reasons for the failure of MMPIs in the
clinic. Firstly, most MMPIs have dose-limiting musculoskeletal toxici-
ty that limits efficacy. Secondly, the clinical trials were performed on
patients with terminal-phase cancer, where several overlapping
pathways come into play. Thirdly, the structural similarity of the var-
ious MMPs' catalytic domains makes it difficult to design MMPIs with
high selectivity [25]. Moreover, the role of MMPs in cancer progres-
sion appears not to be restricted to their ECM-degrading activity,
with involvement in many signaling pathways that influence tumor
cell behavior [26,27]. Lastly, recent evidence shows that MMPs may
have opposing functions in primary and metastatic cancer sites;
hence, MMPIs may produce protumorigenic effects in some situations
and may counterbalance the benefits of target inhibition [26,28,29].
3. Gelatinases (MMP-2 and MMP-9) as cancer biomarkers
Of the various MMPs thought to be involved in cancer, attention
has focused on the gelatinases because (i) they are overexpressed in
a variety of malignant tumors and (ii) their expression and activity
are often associated with tumor aggressiveness and a poor prognosis.
Elevated levels of MMP-2 and/or MMP-9 are found in breast, brain,
ovarian, pancreas, colorectal, bladder, prostate and lung cancers and
melanoma [2,8,30–32]. Dysregulated MMP expression is also ob-
served in hematological malignancies such as acute lymphoblastic
leukemia, adult T-cell leukemia, chronic B lymphocytic leukemia
(CLL), acute myeloid leukemia (AML), chronic myeloid leukemia,
myelodysplastic syndromes and Hodgkin's and non-Hodgkin's lym-
phoma [31,33].
4. Proteolysis-dependent functions of gelatinases
Gelatinases are secreted as inactive zymogens (proMMP-2:
72 kDa, proMMP-9: 92 kDa), with cleavage of a prodomain yielding
the active form (MMP-2: 65 kDa; MMP-9: 82 kDa). An 85 kDa pro-
form of MMP-9 lacking complex carbohydrates has been reported in
breast tumors and AML cell lines [34,35]. Several mechanisms can
stimulate the activation process. The main route for activation of
proMMP-2 on the cell surface occurs through the formation of a mo-
lecular complex containing proMMP-2 (via its PEX domain), MT1-
MMP (via its catalytic domain) and TIMP-2 (reviewed in [2]). This
cell surface interaction leads to clustering of proMMP-2 near a
TIMP-free, active MT1-MMP which initiates activation of proMMP-2.
MMP-2 can also be activated by MMP-1, MMP-7, thrombin and acti-
vated protein C [7,36]. MMP-9 can be activated by plasmin, trypsin-
2, MMP-2, MMP-13 (activated by MMP-2) and MMP-3 (activated by
plasmin) [7,36]. Other activation mechanisms have been suggested
in order to explain proMMP-2 and proMMP-9's catalytic activity in
the presence of the propeptide. For example, binding of proMMP-9
to a gelatin- or type IV collagen-coated surface could lead to revers-
ible activation of MMP-9 via disengagement of the propeptide from
the active site [37]. Interaction of hemin or β-hematin with the
proMMP-9 PEX domain primes MMP-9 activation via an autocatalytic
process [38]. Interaction of proMMP-2 with low concentrations of col-
lagen α2 VI chain induces its auto-activation [39]. Lastly, the
reversion-inducing cysteine-rich protein with Kazal motifs (RECK,
an integral membrane protein that forms a complex with MT1-
MMP) has been found to inhibit gelatinase secretion and activity
[40,41]. Whether these mechanisms occur in vivo remains to be
established.
Activated gelatinases are able to degrade various components of
the ECM and non-matrix proteins (Table 1)[1,2,8,11,12]. Cell migra-
tion and invasion are complex processes that involve the ECM, pro-
teinases, chemokines, adhesion receptors and (for invasion)
basement membrane. Angiogenesis (defined as the generation of
new blood vessels from preexisting ones) is critically important for
tumor growth and metastatic spreading. It was initially suggested
that gelatinases played a dominant role in basement membrane-
invasive events because of their ability to degrade collagen IV [42].
However, studies in MMP-9
(−/−)
and MMP-2
(−/−)
/MMP-9
(−/−)
mu-
rine models of inflammation [43], cancer cells engineered to express
active MMP-2 and MMP-9 [44] and fibroblasts isolated from MMP-
2
−/−
and MMP-9
−/−
mice [45] strongly suggest that gelatinases do
not promote basement membrane invasion. In fact, recent evidence
shows that gelatinases play major but indirect roles in cell signaling
by controlling the bioavailability and bioactivity of molecules that tar-
get specific receptors regulating cell growth, migration, inflammation
and angiogenesis (Table 1).
Table 1
Gelatinase substrates.
Substrates MMP-2/gelatinase A MMP-9/gelatinase B
ECM
substrates
Collagens I, IV, V, VII, X and XI Collagens III, IV and V
Gelatin Gelatin
Tenascin Elastin
Elastin Vitronectin
Fibronectin Entactin
Laminin-5
Other
substrates
proTGF-βproTGF-β
proIL-1βproTNF-α
proTNF-αIL-2Rα
proHB-EGF ICAM-1
FGFR-I EGFR-1
IGFBP-3, -5, -6 Kit ligand
CXCL12/SDF-1 CXCL1/GRO-α
CCL7/MCP-3 CXCL4/PF4
CX3CL1/fractalkine CXCL8/IL-8
KISS-1 CXCL9/MIG
CXCL11/ITAC
CXCL12/SDF-1
α1 proteinase inhibitor
Plasminogen
KISS-1
IFN-β
31B. Bauvois / Biochimica et Biophysica Acta 1825 (2012) 29–36

By degrading the ECM, gelatinases generate or release bioactive
molecules that influence tumor progression. Gelatinase activity can
cause the release of cryptic information from the ECM, leading to
cell migration and angiogenesis. For example, the proteolytic cleav-
age of collagen IV by MMP-9 unmasks cryptic sites that are critical
for angiogenesis [46,47]. Similarly, cleavage of laminin-5 by MMP-2
results in the exposure of a cryptic epitope that enhances endothelial
cell migration [48]. MMP-9 can release ECM-sequestered factors
VEGF, TGF-βand FGF-2, which stimulate proliferation and migration
of endothelial cells and thus promote angiogenesis and tumor growth
[49–53]. In contrast, tumstatin and endostatin (generated by the
MMP-9-mediated proteolysis of type IV collagen and type XVIII colla-
gen, respectively) are active inhibitors of angiogenesis [54,55].
Gelatinases target immunomodulating cytokines and growth fac-
tors and cytokine/growth factor receptors. For example, gelatinases
shed and activate TNF-α, TGF-βand IL-1β, which are intimately in-
volved in the regulation of growth, angiogenesis and inflammation
[56,57]. FGF-R1 may be a specific cell-surface target for MMP-2, yield-
ing a soluble FGF receptor that modulates the mitogenic and angio-
genic activities of FGF-2 [58]. MMP-9 cleaves IFN-βand thus kills
the cytokine's antiviral and immunotherapeutic activity [59]. MMP-
2 is able to cleave certain insulin-like growth factor-binding proteins
(IGFBPs) and thus release active insulin-like growth factors (IGFs) in-
volved in tumor cell growth [11,60]. Both gelatinases process the
tumor suppressor protein KISS-1 to generate metastin, which en-
hances cell invasion [61,62]. MMP-9 suppresses the proliferation of
T lymphocytes through disruption of the IL-2Rαsignaling that may
constitute a mechanism of cancer-mediated immunosuppression
[63]. Moreover, MMP-9 releases Kit-ligand, which plays a crucial
role in tumor growth and angiogenesis [64,65]. MMP-9-dependent
shedding of ICAM-1 augments tumor cell resistance to natural-
killer-cell-mediated cytotoxicity [66].
Chemokines play an essential role in modulating tumor growth via
regulation of tumor-associated angiogenesis, activation of host immu-
nological responses and direct inhibition of tumor cell proliferation.
Gelatinases generate either inactivated chemokine fragments (e.g.
GRO-α, PF4, SDF-1, MCP-3, IP-10, MIG) or truncated chemokines
with enhanced activity (IL-8, I-TAC) [9]. The gelatinase-mediated pro-
teolysis of chemokines might have direct consequences on tumor
growth (e.g. I-TAC), migration (e.g. SDF-1 and MCP-3) and angiogene-
sis (e.g. IL-8, PF4, MIG, IP-10 and SDF-1) [7,9,11]). For example, the
MCP-3 generated by MMP-2 can bind to CC chemokine receptors
and inhibit migration, and suppresses inflammation [67]. In contrast,
processing of IL-8 by MMP-9 increases its chemotactic activity in neu-
trophils [68].
5. Cell surface-associated gelatinases
Gelatinases have been shown to interact with the cell surfaces of
leukocytes and epithelial and endothelial cells [7,36,69,70]. As men-
tioned above, the activation of proMMP-2 requires interaction with
MT1-MMP and TIMP-2 [7]. Furthermore, gelatinases bind to collagens
and fibronectin at the surface of cancer cells through their CBD do-
main [7]. Gelatinases also bind to the low-density lipoprotein-
related scavenger receptor (LRP), which is responsible for the inter-
nalization of various ligands including these enzymes [71–73].
In addition to these cell surface associations, gelatinases reported-
ly bind to other integral membrane proteins, such as the DNA repair
protein Ku (via its integrin I-like domain) [74], CD44 [50,75–80] and
the integrins (αVβ3, αVβ1, αβ2, αVβ5, α4β1 and α5β1) [70,80–93]
(Table 2). The gelatinases' catalytic and PEX domains are variously in-
volved in these interactions (Table 2). For example, the integrin α
M
,
α
L
and β2 subunits can bind to MMP-9's catalytic domain, whereas
CD44 and the β5, α4 and β1 subunits interact with the PEX domain
(Table 2). However, doubt has been cast on the reported molecular
interaction between MMP-2 and integrin αVβ3via the PEX domain
in endothelial cells [82], with a suggestion that the recombinant
PEX polypeptide was possibly contaminated by lipopolysaccharide
[94]. On mesenchymal cells, active MMP-2 can bind to αVβ3via
Table 2
Examples of binding between gelatinases and integral membrane proteins.
Cell type Gelatinase (domain involved in the binding) Integral membrane protein Refs Positive effect on cell process(es)
Melanoma
Primary and metastatic melanomas active MMP-2 αVβ3[81,85] Growth collagen IV degradation
MC cells pro/active MMP-9 CD44 [76] Growth migration
1
Squamous cell carcinoma
SCC12F2 cells pro/active MMP-9 α5β1[88] Migration
2
Breast cancer
MCF7, MCF10A cells pro/active MMP-2 αVβ3[82,83 Migration angiogenesis
3
Met-1, MDA-MB435 cells active MMP-9 αVβ3[75,87 Migration
4, 5
MMP-9 transfected MCF-7 cells proMMP-9 (PEX) CD44 [78,79 Migration
5
Lung cancer
A549 cells MMP-2 (catalytic) αVβ3[93] VEGF release and angiogenesis
6
Fibrosarcoma
HT1080 cells proMMP-9 (PEX) αVβ5[89] Migration
5,7
AML
THP-1, OCI-AML3 cells pro MMP-2 (catalytic) α
M
β2, α
L
β2[90] migration
5
THP-1 cells proMMP-9 (catalytic) α
M
β2, α
L
β2[90,91] Growth migration
5
transendothelial migration
8
U937, HL-60, THP-1, primary AML blasts proMMP-9 (PEX) Ku protein [35,74] Migration
9
CLL
Primary CLL cells pro/active MMP-9 (PEX) α4β1 and CD44v [80,96] Survival
Leukocytes Migration
5
Neutrophils, monocytes proMMP2 and proMMP-9 (catalytic) α
L
β2, α
M
β2[91] Transendothelial migration
8
Monocyte-derived dendritic cells active MMP-9 (catalytic) α
M
β2 and CD44 [77] Migration
10
Endothelial cells
ECV304 cells proMMP-2 αVβ3[82,85] Growth angiogenesis
3
HUVEC cells active MMP-2 αVβ1[86] Apoptosis
1
In vitro culture of TA3 cells on G8 myoblast monolayers; in vitro endothelial tube formation;
2
cell migration in fibronectin-coated Transwell
®
chambers;
3
CAM angiogenesis assay;
4
cell migration in purified ECM proteins-coated Transwell
®
chambers;
5
cell migration in Transwell
®
chambers with 10% fetal calf serum as chemoattractant in the lower chamber;
6
in vitro culture of HMEC-1 cells on Matrigel
®
and formation of capillary-like structures; xenograft mice;
7
cell migration in Matrigel
®
-coated Transwell
®
chambers;
8
transendothe-
lial cell migration in HMECs-coated Transwell
®
chambers;
9
cell migration in collagen IV-coated Transwell
®
chambers;
10
cell migration in Matrigel
®
-coated Transwell
®
chambers
with chemokine in the lower chamber.
32 B. Bauvois / Biochimica et Biophysica Acta 1825 (2012) 29–36

PEX [92]. It therefore remains to be definitively established whether
αVβ3 can bind to the PEX domain of MMP-2.
Consequently, cell growth, migration and angiogenesis appear to
depend on cell surface associations between gelatinases and these in-
tegral membrane proteins (Table 2). For example, disrupting MMP-2/
αvβ3 binding on the surface of melanoma cells is associated with the
inhibition of tumor growth and migration [84,95]. Similarly, the com-
plex formed by Ku protein and proMMP-9 (via the latter's PEX do-
main) is involved in the migration of AML cells [35]. Both pro-
MMP-9 and active MMP-9 bound to the membrane via α4β1 and
CD44 are involved in the survival of CLL cells [80,96]. ProMMP-9 en-
hances epithelial cell migration via a non-proteolytic mechanism
that involves its PEX domain and CD44 [78]. Mesenchymal invasive
behavior might be dependent on MMP-2/αVβ3 binding [92]. In neu-
trophils, both the active site and PEX domain of MMP-9 are involved
in the induction of FGF-2-mediated angiogenesis [53].
6. Outside-in signaling by cell surface-associated gelatinases
Observation of these binding associations between surface recep-
tors and gelatinases raised the possibility that the latter have the po-
tential to directly influence cell behavior and to activate the classical
signaling pathways involved in major biological events (cell growth,
migration, survival, etc.).
Experiments with inhibitors strongly suggest the involvement of
cell signaling pathways in MMP-9-mediated cell migration. For exam-
ple, the JNK inhibitor SP600125 blocked MMP-9-mediated dendritic
cell migration [97], whereas the MAPK inhibitor PD98059 and the
PI3K inhibitor LY-294002 inhibited MMP-9-induced epithelial cell
migration [78]. By studying the adenovirus-mediated delivery of
MMP small interfering RNA, Rao, Bhoopathi and colleagues showed
a clear relationship between the loss of MMP-9 expression and apo-
ptosis induction in medulloblastoma cells (associated with the activa-
tion of β1 integrin, ERK and NF-κB) [98,99].
By using a combination of strategies to respectively target MMPs
(with siRNA, recombinant MMPs and enzyme inhibitors),
gelatinase-integral protein interactions (with antibodies) and signal
transduction pathways (with signaling inhibitors and siRNA), three
recent studies have described the signaling properties of MMP-2
and MMP-9. Redondo-Munoz and colleagues showed that the bind-
ing of proMMP-9's PEX domain to its docking receptors α4β1 integrin
and CD44 induces an intracellular signaling pathway that favors the
survival of CLL cells [96]. This pathway (Fig. 3A) consists of Lyn kinase
activation, STAT3 phosphorylation and up-regulated expression of
the pro-survival protein Mcl-1 (a member of the Bcl-2 family). Ac-
cordingly, high levels of proMMP-9 and Mcl-1 are found in CLL cells
from blood [100,101] and lymphoid organs [96]. The data presented
by Dufour et al. [79] indicate that MMP-9-dependent epithelial cell
migration involves the heterodimerization of the PEX domain of
proMMP-9 with CD44, leading to activation of the tyrosine kinase epi-
dermal growth factor receptor (EGFR) and subsequent phosphoryla-
tion of its downstream kinase effectors ERK, AKT and FAK (focal
adhesion kinase) (Fig. 3B). Indeed, EGFR can stimulate various down-
stream cell signaling cascades, including the PI3K/AKT pathway that
favors cell migration and cancer cell invasion [102]. Moreover, FAK
reportedly coordinates cell adhesion, polarization, migration, survival
and death [103]. Chetty and colleagues suggested a role for MMP-2 in
VEGF-induced lung tumor angiogenesis [93]. The interaction of
proMMP-2 with integrin αvβ3 on A549 epithelial cells induces
PI3K/AKT-mediated VEGF expression and related angiogenesis in
vitro (Fig. 3C). The part of MMP-2 that binds to αvβ3 remains to be
determined. Importantly, these results have been validated in vivo
in a spontaneous lung metastasis mouse model [93].
7. Open questions and conclusions
The gelatinases' established functions depend on their proteolytic
activity. By cleaving ECM components, releasing ECM-associated
growth factors, shedding membrane-anchored cytokines and recep-
tors and regulating chemokine activity, gelatinases process signaling
molecules that in turn influence tumor cell growth, migration, inva-
sion and angiogenesis. The publications detailed in this review high-
light the ability of cell-surface-associated gelatinases to directly
trigger intracellular signaling pathways that control the afore-
mentioned critical cellular processes and behavior. The observed
cell surface association of gelatinases with integrins or other integral
(A) (B) (C)
Fig. 3. Schematic representations of the signaling pathways induced by gelatinases and integral receptors. (A) ProMMP-9 interacts (via its PEX domain) with α4β1 and CD44 on B-
CLL cells, leading to Lyn activation, STAT3 phosphorylation and MCL-1 up-regulation. Mcl-1 (a member of the Bcl-2 family) is essential for lymphocyte survival. (B) The PEX domain
of proMMP-9 interacts with CD44 on tumor epithelial cells (COS-1/kidney, HT1080/fibrosarcoma, MDA-MB435/breast cancer cells) leading to the activation of EGFR and stimula-
tion of various downstream cell signaling cascades, such as PI3K/AKT, ERK and FAK signals that coordinate cell migration. (C) The interaction of proMMP-2 with integrin αvβ3on
lung cancer cells (A549 cells) activates PI3/AKT signaling, leading to the activation of hypoxia-induced transcription factor-1α(HIF-1α). The latter regulates the expression of the
primarily pro-angiogenic vascular endothelial growth factor VEGF-A. VEGF/VEGFR activation drives vascular sprouting, endothelial cell differentiation and then microtubule
formation.
33B. Bauvois / Biochimica et Biophysica Acta 1825 (2012) 29–36

membrane proteins suggests that the signals triggered by these en-
zymes are intertwined with those triggered by integral proteins;
hence, gelatinases may be involved in regulating other aspects of
cell behavior, such as proliferation, differentiation, adhesion and apo-
ptosis. Furthermore, the sheer variety of well-known and newly dis-
covered functions for gelatinases (i.e. secreted forms versus
membrane-bound forms, proforms versus active forms, etc.) begs sev-
eral questions. To what extent are catalytic and non-catalytic activities
(via CBD and PEX) related or interdependent? Are all cell types able to
activate signaling cascades in response to membrane-bound gelati-
nase? Is MMP-2- and MMP-9-mediated outside-in signaling relevant
in other disease states (e.g. inflammation and cardiovascular disease)?
Does gelatinase-mediated outside-in signaling extend to other secret-
ed MMPs that might colocalize with integral proteins? At present, it is
thought that proMMP-1 interacts with α2β1 integrin on epithelial
cells [104] and on keratinocytes via its PEX domain [105], whereas
MMP-19 binds to myeloid cells via its PEX domain [106].
Antiproteolytic therapies have sought to target the MMPs' catalyt-
ic activity and thus inhibit tumor progression [25]. The failure of
MMPIs as cancer drugs in the clinic may be explained by their lack
of selectivity towards MMPs (including gelatinases). In light of our
current knowledge of the gelatinases' proteolytic and non-
proteolytic (i.e. outside-in signaling) roles, the enzyme inhibitor ap-
proach may no longer be sufficient because it does not affect the gela-
tinases' interactions with cell surface proteins and consequent
signaling. New therapeutic strategies are focusing on more selective
MMPIs and targeting motifs outside the active site (the “exosite”)of
individual MMPs; newly designed inhibitors include peptides that
block exosite-mediated cell surface interactions and function-
blocking anti-MMP antibodies [28,107,108]. These approaches have
already been used to target MMP-9 [79,89,109]. A neutralizing anti-
body targeting the PEX domain of MMP-9 bound to LRP-1 at the sur-
face of Schwann cells, blocks cell migration in vitro [109].An
inhibitory peptide that binds selectively to the MMP-9 PEX domain
has already been developed [89]. This small inhibitor prevents PEX
from binding to α1β5 integrin and blocks cell migration in vitro and
tumor xenograft growth in vivo [89]. Synthetic peptides targeting
specific sites of the PEX domain of MMP-9 inhibit the motility of
HT-1080 and MDA-MB-435 tumor cells [79]. These studies indicate
that targeting the PEX domain of MMP-9 by antibodies or peptides
may be a viable approach to abrogate MMP-9-mediated cell function.
In conclusion, recent insights into the potential role of gelatinases as
outside-in signalingmolecules may provide opportunities for the devel-
opment of new gelatinase inhibitors (such as antibodies and peptides)
and validation in relevant animal models, before use as independent
agents or in combination with other cancer treatment strategies.
Acknowledgement
Funding for this work was from the Institut National de la Santé et
de la Recherche Médicale (INSERM).
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