ARTICLE
Auteur(s) : Federico Bussolino,
Francesca Caccavari, Donatella Valdembri, Guido Serini
Department of Oncological Sciences and Division
of Molecular Angiogenesis, IRCC, Institute for Cancer
Research and Treatment, University of Torino School
of Medicine, Candiolo, Italy
accepté le 12 Juin 2009
Aberrant vascular morphogenesis in cancer:
an adhesion issue?
Solid cancers initially arise as microscopic avascular lesions that
must elicit angiogenesis, i.e. the formation of new blood vessels
from pre-existing ones, to grow beyond a minimal size and
metastasize. Notably, the tumor vasculature displays multiple
morphological abnormalities that can be due to, what remain, poorly
characterized defects in the molecular mechanisms that orchestrate
the angiogenic remodeling [1]. As a direct outcome,
chemotherapeutic drugs and molecular oxygen, which is mandatory for
the formation of reactive oxygen species and hence successful
radiotherapy, are inefficiently delivered to cancer cells in many
solid tumors [2]. Therefore, as originally proposed by R. Jain [3],
normalization of tumor blood vessel architecture could result in a
sizeable increase in the effectiveness of standard anti-cancer
therapy. Thus, a deeper understanding of the molecular mechanisms
that support physiological vascular morphogenesis, and that are
likely perturbed in cancer, is required in order to attain the
capacity of converting aberrant tumor blood vessel in a
quasi-normal vascular network.
The final shape of the cardiovascular system results from the
balancing and combinatorial interaction of several factors: i)
local environmental factors, e.g. tissue oxygen and nutrient
demand; ii) the blood flow; iii) genetically programmed extrinsic
cues, e.g. VEGF-A, Ang-1, ephrins, and semaphorins [4]. During
angiogenic remodeling of pre-existing vessels, endothelial cells
(ECs) move and change their reciprocal positions and interactions
in response to the several guidance cues that control their
motility [5]. As a result of a balanced response to fluid shear
stress, chemoattractant and chemorepulsive agents, ECs dynamically
regulate their adhesiveness both in terms of cell-to-cell and
cell-to-extracellular matrix (ECM) contacts. In this regard, EC
interactions with the surrounding ECM are of particular relevance
since, in multicellular organisms, directed cell motility is a
coordinated process largely impinging on the regulation of cell
adhesion to ECM [6]. In particular, in vertebrate embryos the ECM
protein fibronectin represents the earliest and most abundant
vascular basement matrix molecule and is essential for embryonic
vascular development [7] and branching morphogenesis [8]. Hence, a
minimal hypothesis could be that defective vascular morphogenesis
results at least in part from perturbations of vascular EC
interaction with the surrounding ECM.
Regulation of integrin function by PTB
domain-containing proteins and small GTPases
Integrin αβ heterodimers are primary ECM receptors. In mammals,
18 α subunits and 8 β subunits of integrins assemble into
24 distinct receptors, and several integrin heterodimers have
been seen to be involved in angiogenesis [5]. Integrins exist in
diverse conformational states that are endowed with different
binding affinity for ECM proteins (figure 1), and the
modulation of integrin shape is central for the fulfillment of
their biological functions [9]. In the low affinity state, the
large extracellular domain of integrins is bent over the cell
surface, whereas α and β transmembrane and cytoplasmic domains
tightly interact and are probably stabilized through a
juxta-membrane salt bridge involving the arginine of the GFFKR
sequence in an α subunit cytodomain and an aspartic residue in the
β subunit tail [9]. Interaction of the phosphotyrosine-binding
(PTB) domain containing proteins (figure 1), such as talin
[10] and kindlin [11-14], with the two NPxY tandem repeats located
in the cytoplasmic tail of the integrin β subunit, causes its
separation from the α subunit cytodomain, the extension of the
extracellular domain, and the uncovering of the ECM binding site of
integrins [9]. The talin PTB domain is part of a larger trefoil
protein 4.1/ezrin/radixin/moesin (FERM) domain, and can be involved
in an intramolecular association with the rod domain that impairs
talin binding to the integrin β subunit [10]. Such an inhibition
can be relieved upon binding of plasma membrane
phosphatidylinositol-4,5 bisphosphate (PIP2) to the talin rod
domain (figure
1), thus allowing talin activation, head-to-tail
dimerization and integrin binding [10]. In addition, activated
talin can also bind the FERM domain of
phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ 661), increase
PIP2 levels at adhesion sites, and thus trigger a positive
feed-back loop (figure
1) supporting cell adhesion to the ECM [15].
The small GTPase R-Ras (figure 1), which, in vivo,
is largely expressed by vascular ECs and smooth muscle cells [16],
has been found to promote integrin-mediated cell adhesion to
different ECM proteins [17]. However, the molecular details of the
effectors that directly mediate R-Ras activity on integrin function
are still poorly defined (figure 1). R-Ras (also
called R-Ras1), together with TC21 (also known as R-Ras2) and
M-Ras (also named R-Ras3), constitute the R-Ras branch of the wide
Ras superfamily of small GTPases [18]. Remarkably, integrin
function is activated by R-Ras and TC21/R-Ras2 and inhibited
by H-Ras [17]. R-Ras is 55% identical to H-Ras, displays a
26 amino acid N-terminal extension, and its effector region is
identical to H-Ras [18]. Intriguingly, H-Ras and R-Ras display a
significantly different plasma membrane compartmentalization [19,
20]. Active R-Ras-GTP is targeted to FAs via a specific sequence
(aa 175-218) within the C-terminal region [19] that is known to be
highly variable among different GTPases [18]. Moreover, in R-Ras, a
stretch of prolines at the C-terminus bind the SH3 domain of
the adaptor protein Nck; changing the prolines at positions
202 and 203 disrupted R-Ras-induced adhesion to the ECM
[21]. In addition, while active R-Ras localizes into lipid rafts
[20], H-Ras translocates from lipid rafts into non-raft
microdomains upon activation [22]. Swapping experiments with H- and
R-Ras showed that the R-Ras hypervariable region (aa 193-218)
contains a transferable, molecular determinant endowed with the
ability of activating integrins and re-localizing the GTPase to
lipid rafts [20]. However, changing R-Ras aa 208-218 into
H-Ras allows its subcellular compartmentalization in lipid rafts,
but does not confer it with the ability of activating integrins
[20]. The fact that R-Ras localizes both in FAs and lipid rafts is
compatible with the recent observation that integrin signaling
regulates lipid raft distribution [23-25]. Together, these data
indicate that both targeting to FAs and interaction with specific
downstream effectors must be involved in defining the unique
capability of R-Ras-GTP to promote integrin function. This is in
agreement with the concept that a bipartite recognition process
generates specificity within the Ras superfamily. One part of the
effector binds to the switch regions and senses the active state of
the GTPase, whereas the interaction of another region confers the
specificity of the effector [26]. Subcellular localization of
active GTPases and specific effectors would further contribute to
specificity. The 100% homology of the amino acid sequence of the H-
and R-Ras effector loop region is probably why all R-Ras
interactors isolated up to now (e.g. Raf, RalGDS, Nore1, and PI3K)
bind H-Ras as well [17]. These shared interactors cannot provide a
reasonable explanation for the opposing behavior that these two
GTPases exert on integrin function. Accordingly, the fact that the
effector loop mutant D64A of the constitutively active R-Ras
V38 still activates integrins, but completely loses any
interaction with shared effectors, clearly indicates that these
proteins are not needed for integrin activation [27]. Activated
R-Ras-GTP is thought to promote cell adhesion by favoring the
activation of other small GTPases such as Rap1 [28] and Rac1 [19].
In this respect, it is interesting to note that binding of
activated R-Ras to RLIP (Ral interacting protein) 76 leads to
Arf (ADP-ribosylation factor) 6 activation, which promotes
adhesion-induced GTP loading of Rac1 [29]. However, further work is
required to thoroughly dissect the molecular mechanisms by which
R-Ras activates integrins.
A balancing act between integrin activation
and inhibition
High affinity integrins are highly concentrated at adhesion sites
[30] and are at the leading edge of migrating ECs [31] where they
promote new adhesions to support directed cell motility. Major
determinants of vascular remodeling, such as fluid shear stress and
angiogenic growth factors, activate integrin adhesive function [5].
Moreover, during vascular development and experimental
angiogenesis, ECs generate autocrine chemorepulsive signals of
class 3 semaphorins (Sema3) that endow the vascular system
with the plasticity required for reshaping by inhibiting integrins
(figure 1) [5,
32]: the inhibitory autocrine loops of Sema3 can hence assure
a continuous and subtle modulation of integrin function versus an
all-or-none activation. Such a fine-tuning of integrin-mediated
adhesion to the ECM allows a graded control of EC migration and
redirectioning during physiological vascular remodeling.
Accordingly, after few minutes of stimulation with either Sema3A or
Sema3F, adherent ECs lose their focal adhesions [33] and Sema3A
inhibits integrin activation elicited by several pro-angiogenic
factors such as VEGF-A165, bFGF, and PlGF2 [32]. Notably, vascular
abnormalities usually observed in solid tumors appear to be due to
an inbalance between integrin activators and inhibitors in favor of
the former (figure
2), e.g. VEGF-A [2, 34] and bFGF [35]. Correspondingly,
treatment of solid tumors with VEGF-A inhibitors transiently
reverts the structural and functional abnormalities of tumor blood
vessels (figure
2), resulting in an improved capacity for drug and oxygen
delivery [2-4, 34]. Since mutually antagonistic autocrine loops of
VEGF-A [36-38] and Sema3 [32, 39-41] are present in ECs, both in
vitro and during normal angiogenesis in vivo, an imbalance in the
ratio of autocrine VEGF-A/Sema3 in ECs might happen during
tumor progression (figure 2) and contribute
to the structural and functional defects of the tumor blood vessels
[2, 3, 34]. In support of this hypothesis, bone marrow ECs of
patients with malignant multiple myeloma lose autocrine loops of
Sema3A in favor of endogenous VEGF-A [41]. Therefore, it is
tempting to speculate that Sema3A is part of a negative autocrine
loop that brings under control VEGF-A signaling in order to
self-limit angiogenesis and encourage the development of a
functional vasculature (figure 2). Seven, class
3 Sema exists (Sema3A to Sema3G), and it is thus likely that
other Sema3 could function as angiogenesis regulators [42].
For example, Sema3F, which signals through Nrp-2, is known to
inhibit integrins [32], induce the disassembly of ECM adhesions
[33], and inhibit both VEGF-A and bFGF activity on cultured EC
[32]. When overexpressed, Sema3F lessens the angiogenic and
metastatic phenotype of melanomas and transforms these lesions into
benign and encapsulated tumors [43]. Recombinant Sema3A and/or
Sema3F could therefore be employed as pharmacological agents in
order to restrain excessive VEGF-A- or bFGF-elicited tumor
angiogenesis, inhibit tumor progression, and perhaps normalize
tumor blood vessels thus favoring the activity of chemotherapeutic
drugs and radiotherapy (figure 2). To this end, it
is therefore crucial to characterize the signaling pathways that
regulate the function of endothelial integrins.
In different experimental systems, growth factors [5] and
semaphorins [44-46] regulate integrin function by exerting opposite
effects on the activation of the small GTPase R-Ras (figure 1). Neuropilins
(Nrp) and type A or D plexins are the ligand binding and the
signal transducing subunits of SEMA3 receptors respectively.
The cytoplasmic domain of plexins is endowed with an R-Ras
GTPase-activating protein (GAP) activity (figure 1). Specifically,
the juxtamembrane basic sequence of class A plexins directly
interacts with FARP2 (figure 1), a Rac guanosine
exchange factor (GEF). Sema3A binding to the
Nrp-1/plexinA1 complex induces the dissociation of
FARP2 from plexinA1 [47]; next, FARP2 GEF activity
elicits a rapid increase of active Rac1-GTP that in turn
facilitates the binding of the small GTPase Rnd1 to the linker
region of plexinA1 cytodomain (figure 1) [48]. This event
finally activates the R-Ras GAP activity of plexin A1 that
that impairs the function of the small GTPase R-Ras and is required
for Sema3A inhibition of integrins. In addition, FARP2 holds a
FERM domain that mediates its binding to plexin A1 [47]. Upon
dissociation from plexinA1, the FERM domain of FARP2 competes
with talin for binding to PIPKIγ661 and hence impairs the
talin/PIPKIγ661/PIP2/talin positive feedback that supports the
formation of ECM adhesion sites (figure 1).
Conclusion
It is well accepted that that tissue neo-vascularization is crucial
for cancer growth and therapy [49]. However, the architecture and
function of tumor vasculature are abnormal, and are ineffective for
successful chemotherapy and radiotherapy [2, 3, 34]. The
development of a properly patterned vascular tree depends also on
the modulation of integrin function by chemoattractant and
chemorepulsive molecules, e.g. angiogenic growth factors and
semaphorins [5]. Such a fine-tuning of endothelial integrin
function is likely to be disrupted in solid tumors. Indeed,
opposing autocrine loops of VEGF-A [36-38] and Sema3A [32, 39-41]
are present in angiogenic ECs, and an imbalance in the ratio of
autocrine VEGF-A/Sema3A in ECs could support cancer progression and
contribute to the defects in tumor blood vessels (figure 2). Notably,
resistance to VEGF-A-targeted therapies, due to loss of
responsivity to VEGF-A inhibitors, has been reported [34].
Recently, Vacca and colleagues showed that tumor ECs can lose the
autocrine loops of Sema3A in favor of endogenous VEGF-A [41]. Thus,
restoring Sema3A in tumors could synergize with VEGF-A blockers and
help to improve the efficacy of current anti-angiogenic therapies
(figure 2). In
addition, it will be important to characterize the molecular
mechanisms that regulate integrin function in normal ECs and that
could be disrupted in cancer ECs.
Acknowledgments
This research is supported by Telethon Italy (GGP04127 to GS),
Fondazione Guido Berlucchi (to GS); Associazione Augusto per la
Vita (to GS); Associazione Italiana per la Ricerca sul Cancro (to
GS and FB); Ministero della Salute - Programma Ricerca Oncologica
2006 and Ricerca Finalizzata 2006 (to GS and FB); Regione Piemonte
- Ricerca Sanitaria Finalizzata 2006 and 2008, Ricerca Scientifica
Applicata 2004: grants D10 and A150, Ricerca industriale e sviluppo
precompetitivo 2006: grants PRESTO, SPLASERBA(to GS and FB),
Convergine technologies 2007: grant PHOENICS (to FB); Sixth
Framework Programme of European Union Contract LSHM-CT-2003-503254
(to FB); Fondazione Cassa di Risparmio di Torino (to FB); Ministero
dell’Istruzione dell’Università e della Ricerca (PRIN 2007BMZ8WA);
Compagnia di San Paolo - Programma Neuroscienze 2008-2009 (to GS);
Regione Piemonte: "Piattaforme innovative", grant DRUIDI (to FB)
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