Regulation of Angiogenesis
D. Cheresh, R.
Klemke, C.A. Andrews, L.M. Collins,
B. Eliceiri, A. Le, J. Leng, R.
Molander, A.C. Reddy, M. Rosenfeld,
S. Silletti, K. Spencer, C.M.A.
Storgard, M. Storgard, D. Stupack,
T.L. von Schalscha, L.M. Yeh, W. Laug*
* University
of Southern California, Los Angeles,
CA
CLINICAL TRIALS IN LATE-STAGE CANCER PATIENTS
Previous studies
from our laboratory implicated
integrins
 vß3
and vß5
in angiogenesis, the growth of new
blood vessels. We found that
antagonists of these integrins
disrupt angiogenesis by causing
unscheduled apoptosis in angiogenic
blood vessels and that use of the
antagonists can cause regression of
preestablished tumors in animals.
These studies led to the clinical
testing of both antibody and peptide
antagonists of these integrins.
Specifically, a phase 1 clinical
trial of Vitaxin, the humanized
version of a mouse monoclonal
antibody to
vß3,
in patients with late-stage cancer
has been completed. The trial was
conducted by J. Gutheil at the Sidney
Kimmel Cancer Center, San Diego.
By definition, a phase 1 clinical
trial is designed to simply examine
the toxicity of a given drug.
Beginning in February 1997, Vitaxin
was given once a week for 6 weeks to
15 patients with late-stage cancer in
a dose-escalation study. No side
effects or toxic reactions were
observed. However, surprisingly, 9 of
the patients had either disease
stabilization or clinical benefit.
One patient who responded early to
the treatment was allowed to continue
treatment and has been taking the
drug for more than 18 months. This
patient has experienced steady
shrinkage of tumor and no side
effects. Although these results are
preliminary and anecdotal, a phase 2
trial will begin soon to actually
measure the potential clinical
benefits of this drug.
A second phase 1
clinical trial has now been started
in Europe. This trial involves the
administration of a chemically
modified cyclic peptide antagonist of
integrins
vß3
and vß5.
This peptide is also being given to
patients with late-stage cancer in a
dose-escalation study to determine a
possible maximum tolerable dose. This
trial should be completed by the end
of 1998.
MOLECULAR BASIS OF ANGIOGENESIS
Our goals in
current studies are to gain a
molecular understanding of
angiogenesis and to determine the
mechanism by which antagonists of
integrins
vß3
and vß5
disrupt this growth of new vessels.
It is clear that angiogenesis depends
on growth factors such as basic
fibroblast growth factor and vascular
endothelial cell growth factor and
the ligation of integrin
vß3
or vß5.
These cell-surface receptors
cooperate to promote intracellular
signaling that potentiates survival,
migration, proliferation, and
differentiation of endothelial cells
during the formation of new blood
vessels.
We have taken a
gene-delivery approach to determine
some of the key signaling molecules
regulated by the ligation of growth
factor receptors and integrins that
ultimately lead to angiogenesis. For
example, retroviral delivery of a
kinase inactive mutant of Src
selectively disrupts angiogenesis
induced by vascular endothelial cell
growth factor on chick
chorioallantoic membranes. In
contrast, an active form of Src
actually promotes angiogenesis on
such membranes, which can be blocked
by a selective antagonist of
vß5
but not by one of
vß3.
Therefore, Src appears to play a
critical role in a pathway of
angiogenesis that depends on vascular
endothelial cell growth factor and
vß5
A second genetic
approach for regulating angiogenesis
is based on use of adenoviral vectors
containing activated or inactivated
forms of the small GTPase Ras. Gene
delivery of a mutationally active
form of Ras promotes growth of blood
vessels on chick chorioallantoic
membranes, and this growth can be
blocked by the mouse monoclonal
antibody to
vß3.
In addition, angiogenesis induced
with either basic fibroblast growth
factor or vascular endothelial cell
growth factor can be disrupted by
adenoviral delivery of a mutationally
inactive form of Ras. These findings
indicate that the Ras/MAP kinase
pathway plays a key role in
angiogenesis and suggest that
gene-delivery strategies may be an
effective means of regulating
angiogenesis in vivo.
MULTIPLE ROLES FOR THROMBIN IN ANGIOGENESIS
The coagulation
cascade is often dysregulated in
cancer patients, but its role in the
progression of cancer is not clear.
In fact, tumors are often surrounded
by fibrin, which is a ligand for
vß3.
We have shown that fibrin, which is
generated from fibrinogen by the
coagulation protease thrombin,
promotes the survival of endothelial
cells in vitro in a 3-dimensional
extracellular matrix and that this
survival depends on the expression of
vß3.
The fact that survival of endothelial
cells is critical for angiogenesis in
vivo prompted us to examine whether
fibrin at tumor sites provides a
survival signal for invading
angiogenic endothelial cells.
We found that
thrombin, which promotes formation of
fibrin, also directly potentiates
angiogenesis when added to the
surface of chick chorioallantoic
membranes. This effect can be
duplicated with a peptide agonist of
the G protein--coupled thrombin
receptor. Together, these findings
suggest that thrombin has at least 2
functions in tumor-induced
angiogenesis: to generate a
compatible extracellular matrix
environment for invasion by blood
vessels and to ultimately activate
endothelial cells to form new blood
vessels.
DISRUPTION OF ANGIOGENESIS BY PEX
Angiogenesis
depends on both cell adhesion and
proteolytic mechanisms. In fact,
matrix metalloproteinase 2 and
vß3
are functionally associated on the
surface of angiogenic blood vessels.
A fragment of the metalloproteinase
that makes up the C-terminal
hemopexin-like domain, termed PEX,
prevents binding of the enzyme to
vß3
and blocks cell-surface
collagenolytic activity. PEX blocks
activity of matrix metalloproteinase
2 on chick chorioallantoic membranes
and disrupts angiogenesis and tumor
growth. A naturally occurring form of
PEX can be detected in vivo in
conjunction with expression of
vß3
in tumors and during developmental
retinal neovascularization. Levels of
PEX in these vascularized tissues
suggest that it interacts with
endothelial cell
vß3
and acts as a natural inhibitor of
the activity of the
metalloproteinase, thereby regulating
the invasive behavior of new blood
vessels.
ROLE OF ANGIOGENESIS IN ARTHRITIS
Rheumatoid
arthritis is an inflammatory disease
associated with neovascularization.
We showed that
vß3
on synovial endothelial cells is an
effector of angiogenesis in a rabbit
model of rheumatoid arthritis and
that a cyclic peptide antagonist of
this integrin blocks synovial
angiogenesis. This blocking resulted
in reduced synovial cellular
infiltration, decreased pannus
formation, and prevention of
cartilage erosion. These results
substantiate neovascularization as a
central factor in arthritic disease
and implicate
vß3
as a therapeutic target in rheumatoid
arthritis.
REGULATION OF CELL MIGRATION
The interaction
of cells with extracellular matrix
proteins or cytokines and growth
factors promotes signaling events
that regulate cell migration. We
showed that ligation of integrin or
cytokine receptors activates 2
distinct signaling pathways necessary
for cell movement. One pathway
involves activation of Ras/MAP kinase.
Activated MAP kinase influenced the
motility machinery of cells by
directly phosphorylating myosin
light-chain kinase, a situation that
led to increased phosphorylation of
myosin light chains. The second
pathway involves the molecular
coupling of the adaptor proteins c-crk
and CAS (crk-associated substrate
protein). Expression of c-crk and CAS
in cells promoted a crk-CAS
association and activation of the
small GTPase Rac. This pathway
promotes migration by inducing actin
organization and membrane ruffles at
the leading edge of migratory cells.
Moreover, activation of these
signaling pathways is associated with
induction of tumor cell invasion and
pulmonary metastasis in vivo.
PUBLICATIONS
Boudreau, N.,
Andrews, C., Srebrow, A., Ravanpay,
A., Cheresh, D.A. Regulation of the
angiogenic phenotype by Hox D3. J.
Cell Biol. 139:257, 1997.
Brooks, P.C.,
Montgomery, A.M.P., Cheresh, D.A. Use
of the 10-day-old chick embryo model
for studying angiogenesis. Methods
Mol. Biol., in press.
Brooks, P.C.,
Silletti, S., von Schalscha, T.L.,
Friedlander, M., Cheresh, D.A.
Disruption of angiogenesis by PEX, a
non-catalytic metalloproteinase
fragment with integrin binding
activity. Cell 92:391, 1998.
Cheresh, D.A.
Death to a blood vessel, death to a
tumor. Nature Med. 4:408, 1998.
Eliceiri, B.P.,
Strömblad, S., Klemke, R., Cheresh,
D.A. Integrin requirement for
sustained MAP kinase activity during
angiogenesis. J. Cell. Biol.
140:1255, 1998.
Felding-Habermann, B., Silletti, S.,
Mei, F., Siu, C., Yip, P., Brooks,
P.C., Cheresh, D.A., Ginsberg, M.H.,
Montgomery, A.M.P. A single
immunoglobulin-like domain of the
human neural cell adhesion molecule
L1 supports adhesion by multiple
vascular and platelet integrins. J.
Cell Biol. 139:1567, 1997.
Klemke, R.L.,
Leng, J., Molander, R., Brooks, P.C.,
Vuori, K., Cheresh, D.A. CAS/crk
coupling serves as a "molecular
switch" for induction of cell
migration. J. Cell. Biol. 140:961,
1998.
Li, E., Stupack,
D., Klemke, R.C., Cheresh, D.A.,
Nemerow, G.R. Adenovirus endocytosis
and cell motility require distinct
signaling pathways mediated by
v
integrins. J. Virol. 7:2055, 1998.
Nicolaou, K.C.,
Trujillo, J.I., Jandeleit, B.,
Chibale, K., Rosenfeld, M.,
Diefenbach, B., Cheresh, D.A.,
Goodman, S.L. Design, synthesis, and
biological evaluation of nonpeptide
integrin antagonists. J. Bioorg. Med.
Chem., in press.
Radar, C.,
Cheresh, D.A., Barbas, C.F. A phage
display approach for rapid antibody
humanization: Designed combinatorial
V gene libraries. Proc. Natl. Acad.
Sci., U.S.A. 95:8910, 1998.
Sipkins, D.A.,
Cheresh, D.A., Kazemi, M.R.,
Bednarski, M.D., Li, K.C.P. Detection
of tumor angiogenesis in vivo by
vß3-targeted
magnetic resonance imaging. Nature
Med. 4:623, 1998.
van der Zee, R.,
Murohara, T., Passeri, J., Kearney,
M., Cheresh, D.A., Isner, J.M.
Reduced intimal thickening following
vß3
blockage is associated with smooth
muscle cell apoptosis. Cell Adhes.
Commun., in press.
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