Hindawi Publishing Corporation
Journal of Signal Transduction
Volume 2012, Article ID 483040, 15 pages
1Department of Pharmacology, CNR Institute of Neuroscience, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy
2Neuromuscular Diseases and Neuroimmunology, Neurological Institute Foundation Carlo Besta, Via Celoria 11, 20133 Milan, Italy
Correspondence should be addressed to Carlo Sala, email@example.com and Chiara Verpelli, firstname.lastname@example.org
Received 9 January 2012; Revised 27 April 2012; Accepted 2 May 2012
Academic Editor: Laura Cerchia
Copyright © 2012 Valentina Cea et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Currently, antiangiogenic agents are routinely used for the treatment of patients with glioma. However, despite advances in
pharmacological and surgical therapy, glioma remains an incurable disease. Indeed, the formation of an abnormal tumor
vasculature and the invasion of glioma cells along neuronal tracts are proposed to comprise the major factors that are attributed
to the therapeutic resistance of these tumors. The development of curative therapeutic modalities for the treatment of glioma
requires further investigation of the molecular mechanisms regulating angiogenesis and invasion. In this review, we discuss the
molecular characteristics of angiogenesis and invasion in human malignant glioma, we present several available drugs that are
used or can potentially be utilized for the inhibition of angiogenesis in glioma, and we focus our attention on the key mediators of
the molecular mechanisms underlying the resistance of glioma to antiangiogenic therapy.
Angiogenesis and tumor cell invasion play a critical role in
glioma development and growth, even during the earliest
phases . Indeed, the formation of abnormal tumor
vasculature and glioma cell invasion along white matter
tracts are proposed to be the major causes of the therapeutic
resistance of these tumors; thus, glioma remains a fatal
disease despite advances in surgical and medical therapy.
Glioma tumors are an example of highly vascularized
tumors, which induce angiogenesis by upregulating vascular
endothelial growth factor (VEGF) and its downstream
pathways. Indeed, several molecular abnormalities have been
described in glioma that promote angiogenesis, such as
mutations and/or upregulation of PI3K/Akt and the VEGF
receptor (VEGFR) in the glioma endothelium . Interest-
ingly, each of these signaling pathways involves alterations
that can be therapeutically targeted . Evaluation of drugs
that target these pathways requires novel preclinical and
clinical experimental trial design to define the optimal
drug dose and delivery times to avoid toxicity during the
first months of treatment [4, 5]. Furthermore, whether
these agents can be used in combination with classical
response, and whether they can be potentiated by such
combinatorial treatments are important issues that remain
to be explored.
In this paper, we first discuss the molecular charac-
teristics of angiogenesis and invasion in human malignant
glioma. Secondly, we discuss the commercially available
drugs that are currently used or might be potentially utilized
for the inhibition of angiogenesis in glioma. Thirdly, we
focus our attention on the key mediators of the molecular
antiangiogenic therapy. Finally, we highlight the necessity for
further investigation of the clinical utility of antiangiogenic
therapies and the development of novel strategies for the
treatment of glioma.
Angiogenesis, the formation of new blood vessels, is a critical
step during tumorigenesis and represents a pathological
hallmark of cancer.
When a solid tumor, such as a brain tumor, grows
larger than a critical size (1-2mm in diameter), it must
recruit new blood vessels to supply the required oxygen and
nutrition levels necessary for its survival and proliferation.
This process comprises the formation of new blood vessels
2 Journal of Signal Transduction
from preexisting ones and is a crucial step in the progression
of cancer from a small and localized neoplasm to a highly
aggressive tumor. The major in vitro and in vivo models
to study tumor angiogenesis are summarized and critically
discussed in Table 1.
2.1. Mechanisms of Neoangiogenesis in Glioma. Angiogenesis
requires three distinct steps: (1) blood vessel breakdown,
(2) degradation of the vessel basement membrane and the
surrounding extracellular matrix (ECM), and (3) migration
of endothelial cells and the formation of new blood vessels.
Angiogenesis plays a crucial role in glioma development
and growth . Gliomas are highly vascularized tumors
and neovascularization in and around the tumor are well
characterized. Holash et al. reported also vascular cooption
before the real agiogenesis in an experimental model of
glioma . The level of angiogenesis is correlated with
the aggressiveness of gliomas and is often associated with
prognosis. Glioblastomas (GBM) are the most lethal cancer
and the most vascularized brain cancer, with the highest
degree of vascular proliferation and endothelial cell hyper-
plasia . Patients with high tumor microvascular densities
low microvascular densities [30, 31]. Perivascular migration,
proliferation, and angiogenesis are closely associated and
progress concurrently in gliomas.
2.2. Angiogenic Factors. Angiogenesis results from a balance
between proangiogenic factors and antiangiogenic factors.
During tumor progression, there is a shift towards angio-
genic factors that stimulate uncontrolled and disorganized
vascular growth. These molecular factors can be secreted
by cancer, endothelial, stromal, and blood cells and by the
extracellular matrix [32, 33].
Proangiogenic factors involve a variety of angiogenic
factors including vascular endothelial growth factor (VEGF),
acidic fibroblast growth factor, basic fibroblast growth
factor, placental growth factor, angiopoietin-2, and inter-
leukins, whereas antiangiogenic factors include angiostatin,
endostatin, thrombospondin 1, and endothelial monocyte-
activating polypeptide 2 [33, 34]. In addition, enzymes
including serine proteinases and metalloproteinases degrade
the extracellular matrix, which plays an important role in
both the induction and the suppression of angiogenesis
. For example, Raithatha et al.  reported that
MMP-9 might regulate angiogenic remodeling. Autocrine
or paracrine factors of the glioma microenvironment and
PDGF also contribute to angiogenesis in gliomas .
The endothelial cells that are stimulated by angiogenic
factors then migrate and proliferate, resulting in neovascular
formation . This paracrine loop constitutes an extended
foothold that allows tumor cells to migrate.
2.3. The VEGF/VEGFR Pathway. Accumulating evidence
indicates that the VEGF and VEGFR signaling pathways play
a major role in tumor angiogenesis in malignant glioma,
similar to most other solid tumors.
VEGF-A is upregulated in glioblastoma and is produced
by multiple cell types, including the tumor, stromal, and
inflammatory cells . The expression levels of VEGF are
regulated by the following mechanisms: (1) low oxygen
concentrations in growing gliomas induces upregulation of
HIF that increases VEGF mRNA levels; (2) EGFR signaling
stimulates VEGF gene expression via an HIF-independent
mechanism; (3) FoxM1B transcription factor is upregulated
in glioblastoma multiforme and stimulates VEGF expression
independently of HIF ; (4) upregulation of the HuR
protein that suppresses the posttranscriptional degradation
of VEGF-A mRNA under hypoxia leads to a further increase
in VEGF levels ; (5) brain-derived neurotrophin factor
can enhance the expression of VEGF, increasing the levels
of hypoxia-inducible factor-1 expression ; (6) integrin-
linked kinase 1(ILK1) is an important regulator of tumor
angiogenesis because it increases VEGF expression by stimu-
lating HIF-1α via AKT phosphorylation on Ser473.
The two tyrosine kinase receptors, VEGFR-1 and
VEGFR-2, are both highly expressed in gliomas . They
are activated by VEGF-A but are differentially linked to
angiogenesis and glioma growth in vivo. VEGFR-2 is mainly
expressed in vascular endothelial cells, where it directly
transduces most of the mitotic signals that result in angio-
genesis. In contrast, VEGFR-1 is expressed not only in
vascular endothelial cells but also in monocyte/macrophage
lineage cells. These macrophages can act as proangiogenic
and protumorigenic cells, similar to the tumor-associated
macrophages [44, 45].
ways including activation of Ras/Raf/mitogen-activated pro-
tein kinase [46, 47] and phospholipase C-γ/protein kinase C
, which regulate endothelial cell proliferation and migra-
complexes and perturbing the adherens junctions between
endothelial cells [49, 50]. Another important signaling
cascade activated not only by VEGFR but also by other
proangiogenic stimuli, including platelet-derived growth
factor, neurotrophins, insulin-like growth factor, epidermal
growth factor, and integrins, is the phosphatidylinositol-3
kinase/AKT pathway [51, 52], that is, fundamentally altered
during brain tumor angiogenesis .
Notably, brain tumor blood vessels are tortuous, dis-
organized and highly permeable, resulting in irregular and
inefficient blood flow [54–57] and vasogenic brain edema.
This irregularity and inefficiency are strongly associated with
the action of VEGF.
One of the insidious features of gliomas is their ability
to metastasize and to establish numerous microtumors at a
distance from the primary tumor. In all types of gliomas,
the potential of single cells to invade normal brain tissue is
closely related to angiogenesis.
The formation of abnormal tumor vasculature and
glioma cell invasion along white matter tracts are believed to
be the major factors responsible for the resistance of gliomas
to treatment. Therefore, further investigation of the mecha-
nisms underlying angiogenesis and invasion in glioblastoma
is essential for the development of a curative therapy.
Journal of Signal Transduction3
Table 1: A critical summary of the major in vitro and in vivo models to study tumor angiogenesis.
There are a total of 60 cell lines representing nine distinct tumor types. However, this
model does not reflect the complexity of the real tumor environment.
are differentially expressed in cells grown as multicellular spheroids versus 2D cultures.
The capacity for spheroid outgrowth in 3D matrices is an interesting parameter to study
the migratory behavior of tumor spheroid cells; however, this parameter can only be used
for rapidly migrating cells (e.g., glioblastoma spheroids).
Endothelial cell spheroids are increasingly used for evaluating the pro- and anti-
angiogenic potential of drugs.
Cospheroids of HUVEC and human fibroblasts are used for angiogenesis studies.
Tumoral spheroids cocultured with endothelial cells potentiate tumor angiogenesis by
upregulating proangiogenic factors that are absent in multicellular tumor spheroids alone
or in monolayers.
Another advantage is the possibility to use tumor spheroids from biopsies. This is useful
initiating cells and tumor progenitors stem cells in tumor spheroids.
The CAM tumor model could allow for a prescreening of drugs and subsequently reduce
the number of animals used for in vivo experiments. This model is much faster than
tissue that strongly resembled clinical specimens of human tumors. The CAM model
to human disease that is impossible to achieve with other nonanimal models; it combines
the advantages of an in vivo environment with the simplicity of an in vitro experiment.
The duration of the follow-up period is limited due to the hatching of the chick 21 days
Tumors were produced by Wechsler in Koestner’slaboratory by the i.v. administration
of a single dose of ethyl-nitrosourea (50mg/kg b.w.) to a pregnant CD Fischer rat on
the 20th day of gestation. The isolated clones retain individual characteristics, including
the differentiation status, despite repeated propagations in vitro, elevated mitotic index
and an increased nuclear-cytoplasmic ratio consistent with glioma cells in culture. When
injected, these tumors have been refractory to chemotherapy and radiotherapy and
adaptive to immunotherapy and exhibit an infiltrative pattern of growth within the brain.
These characteristics closely resemble those of human glioblastoma.
Tumors obtained from the direct implantation of the human cell lines or patient tumor
biopsies are models that allow the monitoring of tumor growth. However, growth can be
too slow; in xenografted models, the microenvironment and host immune responses are
altered, and this may influence the tumor response.
This model mimics the morphology, growth characteristics of clinical disease and
metastatic processes more efficiently. There are several studies that report differences in
the therapeutic responses between subcutaneous and orthotopic models.
The traditional orthotopic models for brain tumors did not aggressively invade healthy
all of the human GBM features.
Spontaneous canine glioblastoma approximates the human disease characteristics.
However, it is not trivial to study a large number of spontaneous canine glioblastomas.
The orthotopic xenograft implant of the two GBM cell lines, J3T-1 and J3T-2, into
phenotypes:angiogenesis-dependent andangiogenesis-independent invasionobservedin
Human tumor cell lines
membrane tumor assay
RG2 and F98 rat cell
implanted human tumor
J3T-1 and J3T-2
orthotopic mice and rat
The HGA murine models with Pten, Rb1, or Tp53 deletion are relevant to human disease,
reflecting a spectrum of tumor histology and molecular features. Thus, molecular and
other complex processes including specific contributions of the tissue microenvironment,
4Journal of Signal Transduction
Table 1: Continued.
tumors of the VM
The inbred VM mouse strain is unique in exhibiting a relatively high incidence (1.5%) of spontaneous
brain tumors. The VM-M3 brain tumor arose spontaneously in the forebrain of a VM mouse and
expresses properties of microglia/macrophages similar to that seen in several types of invasive cancers
of neural origin. Similar to high-grade human gliomas, the VM-M3 tumor cells, highly invasive, can be
grown in the syngeneic VM mice with reproducible growth rates and have genetic similarities to human
GBM. In addition, the tumor cells are labeled with the firefly luciferase gene allowing for noninvasive
detection and quantitation of tumor growth.
GBM is the most common primary brain tumor in dogs and brachycephalic breeds such as Boston
provide a valuable large animal model for the investigation of novel delivery and therapeutic strategies for
intracranial tumors. The presence of pseudopalisading necrosis and endothelial proliferation that closely
resemble those found in human GBMs suggests the presence of a hypoxic environment in canine GBM.
The large size of the canine brain compared to the rodent brain would be more useful for preclinical
assessment of doses, comprising more relevant volumes needed to implement novel therapies. However,
spontaneous GBM in dogs is not a tumor model that is as easily accessible as the rodent GBMs. These
models have variable penetrance, resulting in lack of synchrony in tumor development. The variability in
time to progression represents a limitation in its use for drug testing.
This tumor is produced by the administration of a single i.v. dose of ethylnitrosourea to a pregnant
rat. It has been classified as an anaplastic and undifferentiated glioma. It is refractory to chemotherapy
and radiotherapy. This model is effective for the evaluation of survival rate. However there are serious
limitations in directly applying data from rat tumor models to any clinical treatment for human brain
F98 rat glioma
Tumors require nutrients and oxygen in order to grow,
and new blood vessels provide these requirements. GBM
cells are characterized by their invasive abilities and striking
angiogenic potential. The blood vessels formed by tumor
cells are structurally and functionally abnormal: the blood
vessels are leaky and dilated, the endothelial cells exhibit
aberrant morphology, the pericytes are loosely attached or
absent and the basement membrane is incomplete .
These abnormalities lead to an abnormal tumor microen-
vironment that is characterized by interstitial hypertension,
hypoxia and acidosis. The abnormal vasculature represents a
barrier to the delivery and efficacy of anticancer therapeutic
agents. These observations suggest that if the structure and
function of tumor vessels could be “corrected,” then the
tumor microenvironment might be normalized, ultimately
improving the efficacy of cancer treatments.
As a key mediator of angiogenesis, VEGF and its
to conventional therapies. Targeting the cells that support
tumor growth, rather than the actual tumor cells, represents
a relatively new approach to cancer therapy. This approach is
callystableand thereforeless likely to develop mutations that
will allow them to develop drug resistance in a rapid manner.
A significant challenge for antiangiogenic therapy is
to design combination protocols that can counteract the
diverse angiogenic stimuli produced by the tumor and its
VEGF signaling inhibitors have been shown to signif-
icantly suppress or delay tumor growth in several animal
models  and in clinical trials. The humanized mono-
clonal anti-VEGF antibody bevacizumab is the first VEGF-
targeting drug approved for use in patients with metastatic
colorectal cancer , metastatic breast cancer, lung cancer,
renal cell carcinoma, and glioblastoma multiforme .
VEGF expression is regulated by intrinsic and extrinsic
factors. Hypoxia and hypoglycemia are major stimulators
of VEGF expression . Factors that can potentiate VEGF
production and stimulate angiogenesis include tumornecro-
sis factor and transforming growth factor.
Several approaches have been used to eliminate the
hypoxic cells within tumors .
3.1. Antiangiogenic Strategies. Angiogenesis inhibitors have
been divided into two classes: direct and indirect .
Direct angiogenesis inhibitors, such as endostatin, target
the microvascular ECs, preventing their response to various
proangiogenic stimuli and thereby enhancing the effects of
In contrast, indirect angiogenesis inhibitors interfere
with the proangiogenic communication between the tumor
cells and the endothelial cell compartments. Antiangiogenic
therapies act predominantly by blocking the binding of
VEGF to its receptor and comprise neutralizing antibodies
against the ligand or the receptor, soluble receptors, or
activity of the VEGF receptors.
Due to the potential of tumor “escape” when specific,
delivered individually, appropriate combination protocols
employing these agents are required for maximal benefit
. Abdollahi and coworkers show that the treatment of
tumor xenografts with a combination of endostatin and
Journal of Signal Transduction5
with VEGF blockers results in an enhanced therapeutic
effect, which may be attributed to the endostatin-mediated
downregulation of many regulators of proangiogenic
pathways and suppression of alternative angiogenic mecha-
nisms that might be upregulated by VEGF blockade .
Here, we focus on several molecules that interfere with
the VEGF/VEGFR signaling pathway, which have been
evaluated in clinical trials for solid tumors. In Table 2, we
summarize the available treatments and the relative clinical
phases and results.
3.2. Indirect Antiangiogenic Drugs. As mentioned previ-
ously, Bevacizumab (Avastin) is a humanized neutralizing
monoclonal antibody that blocks the binding of human
VEGF to its receptors. A significant tumor response was
observed in response to Bevacizumab treatment: the 6-
month progression-free survival was 32% in GBM patients
VEGF therapy, resulting in rapid tumor progression without
improvement in overall survival [85, 86].
In recent study demonstrated that anti-VEGF therapies
can significantly reduce the vascular supply, as demonstrated
by a decrease in intratumoral blood flow and a strong
reduction of large- and medium-size blood vessels, however
these events were also shown to be accompanied by a
strong increase in infiltrating tumor cells in adjacent brain
parenchyma . Finally, a preclinical study  and a clin-
ical trial  suggest that high doses of bevacizumab could
directly enhance the invasiveness of human glioblastoma cell
lines and that dosages lower than those currently used might
improve patient outcome.
In the endothelial cells of normal animals, VEGF-A
treatment results in the upregulation of both integrins α1β1
and α2β1. The functional blocking of these integrins impairs
angiogenesis in vitro and reduces VEGF-A-induced angio-
genesis and tumor growth in vivo [90, 91]. αvβ3 integrins
are highly expressed by proliferating and activated vascular
endothelial cells. Therefore, they are a major contributor to
the formation of vasculature by supporting the migration
and survival of endothelial cells . The blockade of αvβ3
integrins inhibits tumor angiogenesis as well as blood vessel
formation in in vivo models [93, 94]. Consequently, αvβ3
might represent a potential target in antiangiogenic therapy.
Antagonizing integrins has generally included the targeting
of the receptor binding sites or other nearby sites, although
new alternative approaches target downstream signaling
Cilengitide is a cyclic RGD-peptide inhibitor of αvβ3 and
αvβ5 integrins. Blocking αvβ3 integrin inhibits blood vessel
formation in vivo . In a phase II trial, cilengitide was
associated with a median survival of 10 months in recurrent
glioma patients . Cilengitide is currently in clinical phase
III studies for the treatment of glioblastomas and is in phase
II studies for the treatment of several other tumor types,
including breast cancer, squamous cell cancer, nonsmall cell
lung cancer, and melanoma [97, 98].
Other drugs targeting integrins include the following
It blocks integrin binding to vitronectin and fibrinogen, pre-
venting cell adhesion, migration, proliferation, and integrin-
mediated cell signaling .
Volociximab is a chimeric human-mouse monoclonal
antibody that binds to α5β1 integrins. It induces cell death
and prevents capillary tube formation in vitro. In vivo,
volociximab exhibits antitumor and antiangiogenic effects
Increased matrix metalloproteinase (MMP) levels are
associated with glioma invasion and angiogenesis. Marimas-
tat reduces MMP levels in patients with gliomas . Phase
II clinical trials evaluating the administration of marimastat
results (the progression-free survival after six months was
39%), although further investigation is needed for the
associated therapy-induced joint pain .
Sorafenib (Nexavar) is a multi-kinase inhibitor of
VEGFR2-3, PDGFR, Raf kinase, and c-Kit. It is currently
approved for the treatment of advanced HCC and renal cell
carcinoma. Phase II trials evaluating the efficacy of sorafenib
in patients with malignant glioma are currently ongoing
. Hypertension is a specific side effect of sorafenib
and of most antiangiogenic agents due to the decreased
production of nitric oxide and prostacyclins in vascular
endothelial cells .
Cediranib (Recentin) is a potent inhibitor of both
VEGFR-1 and VEGFR-2. It also exhibits activity against c-
correlation was found between cediranib dose- and time-
dependent treatment and soluble VEGFR-2 .
Sunitinib (Sutent) is a multi-kinase inhibitor of VEGFR
1-3, RET and PDGFR, approved for treatment of RCC,
imatinib-resistant gastrointestinal stromal tumors (GIST)
and pancreatic neuroendocrine tumors (pNET) [105–107].
A recent preclinical study  shows that after starting
sunitinib treatment, there is a period when tumor oxy-
genation is higher in treated compared to untreated mice.
The improved oxygenation suggests that the residual blood
vessels had improved function in terms of delivering oxygen
and nutrients. A synergistic delay in tumor growth was
observed when radiation was applied during the enhanced
tumor oxygenation after 4 days of sunitinib administration.
Imatinib is a kinase inhibitor of PDGFR, c-kit, and bcr-
abl. Administration of imatinib at low concentrations can
act as a cytostatic agent, whereas at high concentrations, it
predominantly behaves as a cytotoxic agent . Imatinib
monotherapy has failed due to the limited penetration of
the drug across the BBB, and for that reason, the inhibition
of PDGFR alone is insufficient to prevent the growth of
malignant gliomas .
Antiangiogenic therapies are integrated into the treat-
ment strategies for many different tumor types. However,
not all patients respond to therapy; only a few benefit with
progression-free survival. In most tumors, antiangiogenic
treatment is combined with chemotherapy. Furthermore,
a major problem of this therapy is the development of
resistance. Extensive evidence indicates that antiangiogenic
therapy might actually enhance tumor progression by
6 Journal of Signal Transduction
Table 2: Summary of the available treatments and the relative clinical phase and results.
DrugTarget Clinical phaseResults
Significant and clinical improvement in response
rate, median time to tumor progression, and clini-
In May 2009, the FDA approved Avastin as a single
agent for the treatment of recurrent GBM based
on the demonstration of objective response rates
in two single-arm trials: AVF3708g and NCI 06-C-
Phase II trial in conjunction with chemotherapy
and radiation: EMD 121974 in 2010 phase II trial
in recurrent glioblastomas. The efficacy of the
cilengitide alone is modest, but it is adequately
addition of cilengitide to standard chemoradio-
therapy demonstrated promising activity in GBM
Well tolerated with no evidence of immunogenic-
ity . Does not improve the effect of dacar-
bazine in a phase II trial of metastatic melanoma
Despite insufficient clinical activity in the refrac-
tory patient population to continue the study,
weekly volociximab was well tolerated. A bet-
ter understanding of the mechanism of action
of volociximab will inform future development
Treatment with marimastat in SCLC and GBM
patients does not improve survival [73, 74].
Interfere with the proangiogenic
action of growth factors
Phase III 2005
Bevacizumab (Avastin)Monoclonal antibody anti-VEGF Approved in 2004
Cilengitide Selective inhibitor of αv integrins
Orphan drug by
agency in 2008
antibody direct against the
human αvβ3 integrin
Phase II/phase I
Chimeric monoclonal antibody
that binds to and inhibits αvβ1
Small molecular inhibitor of
several tyrosine protein kinases
(VEGFR and PDGFR) and Raf
Approved in 2007 for
liver and kidney
Phase I and II trials for brain tumors. Sorafenib
can be safely administered [75, 76].
CediranibPotent inhibitor of VEGFRPhase I, Phase II
Modest single-agent activity [77, 78]. Cediranib
monotherapy yielded encouraging responses in
recurrent glioblastoma in a phase II study .
Multi-target receptor tyrosine
Approved for renal
cell carcinoma and
Approved in 2011 for
ten different cancer
Single-agent sunitinib exhibited insufficient activ-
ity in patients with recurrent glioblastoma in a
phase II study .
Specific inhibitor of receptor
In brain tumors, it did not show clinically mean-
ingful antitumor activity in phase II and phase III
promoting an invasive phenotype that allows for tumor cells
to escape angiogenic inhibition.
The identification of predictive biological markers of
objective response will be critical for the assessment of the
response rates correlated with overall survival and of the
development of resistance to antiangiogenic drugs. These
ment of therapeutic efficacy or in the development of
alternative antiangiogenic therapies in the event of treatment
4.MolecularMechanisms of Resistance to
VEGF is ubiquitously expressed in almost all tumors. Tumor
cells have been demonstrated to secrete VEGF, which leads to
increased angiogenesis [111, 112].
Although antiangiogenic treatment yields survival
benefits for patients with many different types of aggressive
tumors, VEGF pathway inhibitors are nonetheless failing
to produce enduring clinical responses in most patients
Journal of Signal Transduction7
[113, 114]. Here, we discuss the different ways that tumors
can circumvent antiangiogenic therapy.
4.1. Alternative Pathway Activation by Tumoral Cells. One
way that tumor cells bypass antiangiogenic therapy is via
the activation or upregulation of alternative proangiogenic
pathways. In preclinical models and in clinical trials, over-
expression of fibroblast growth factor 1 and 2, ephrin, and
angiopoietin was found in tumors that were treated with
inhibitors of VEGF signaling .
Pericytes play an important role in the pathology of
aberrant tumor vasculature. The vessels within tumors
that survive antiangiogenic therapy are tightly covered
with pericytes, which are recruited by vascular endothelial
cells to provide VEGF, the most important survival signal
for endothelial cells . Important features of hypoxic
remodeling include the loss of small vessels and extensive
proliferation of vascular mural cells (MCS) in the surviving
vasculature. PDGF-B appears to play a significant role
in promoting the integrity of vascular networks during
conditions of environmental stress. Recruited MCs are key
contributors to the maintenance of tumor neovasculature.
PDGF-B signaling via the PDGF receptor-β (PDGFR-β)
plays a critical role in MC recruitment . Similar to
VEGF, PDGF-B expression in ECs is critically regulated by
oxygen tension, and PDGF-B overexpression is associated
with abnormal proliferation of MCs . Members of the
ephrin family have been shown to play important roles in
regulating the assembly of vascular cells.
However, increased PDGF-B expression has only been
found in recurrent xenografts. PDGFR-β was also found
in the large vessels of the recurrent tumors. Prolonged
antiangiogenic therapy significantly alters the expression of
angiogenic factors implicated in vascular MC recruitment,
causing extensive morphological changes in vessels, includ-
ing significant increases in diameter and active proliferation
of vascular mural cells .
Cancer cells can also adapt to the disruption of vessels by
extravagating into normal tissues .
An alternative mechanism of escaping from VEGF block-
ade might be attributed to the local contribution of VEGF
by the host stroma, which is sufficient to maintain persistent
vessels and to sustain tumor growth. When host-derived
VEGF is blocked, tumors exhibit extensive necrosis .
Breast cancer studies have revealed that mammary stromal
fibroblasts might produce factors that influence the growth
and malignant progression of a tumor via paracrine effects
on the tumor-associated endothelium .
4.2. The Tumor Recruits Different Types of Cells. Tumor
hypoxia caused by the loss of functional vasculature after
conventional therapy (e.g., irradiation) results in the upreg-
ulation of VEGF to stimulate vascular proliferation and
is the stimulus for the influx of BMDCs (bone-marrow-
derived endothelial cells). The two principal ways in which
a tumor can expand its vasculature as it grows is either by
angiogenesis, which involves the sprouting of endothelial
cells from nearby normal vessels, or by vasculogenesis, which
occurs by the recruitment of circulating endothelial and
other cells into the tumor. Both the pharmacological or
genetic inhibition of HIF-1α attenuates BMDC recruitment
and inhibits tumor recurrence. Such BMDC accumulation
is composed largely of CD11b+ monocytes. These cells are
highly proangiogenic, suggesting that they are attractive
targets for enhancing the response of tumors to irradiation
[123, 124]. In addition, CD11b+Gr-1+ cells (also defined as
myeloid-derived suppressor cells, MDSC) have been found
to be frequently increased in tumors and to mediate their
resistance to anti-VEGF treatments by producing several
angiogenic factors including G-CSF and Bv8 .
Other cells are important for tumor growth and angio-
genesis. For instance, there is an inverse relationship between
macrophage density and vascular density . Hypoxia
upregulates the production of proangiogenic growth factors
and cytokines by tumor-associated macrophages (TAM)
. Macrophage infiltration was demonstrated to be a
prerequisite step for the angiogenic switch, which correlates
with the transition to a malignant tumor phenotype .
TAMs secrete a number of mitogenic cytokines and growth
factors, which are involved in a range of paracrine loops
that promote tumor cell proliferation and growth. A number
of studies have shown that TAM infiltration correlates with
increased cell proliferation growth of many tumors .
The indirect role of TAMs in angiogenesis is also essential
for tumor growth, as they provide oxygen and nutrients.
Microglia and macrophages can be recruited either
by resident brain microglia or by activated perivascular
macrophages. Microglia are recruited to the glioma, where
they can produce cytokines to benefit glioma cell pro-
liferation and migration. The cytokines produced include
MCP-1 (monocyte chemoattractant protein-1) , G-
CSF (Granulocyte colony stimulating factor) , and
several growth factors such as EGF, VEGF, HGF, and SCF
[132, 133]. HGF, and its receptor are expressed in both
and proliferation [134, 135]. Upregulation of TGFβ might
be involved in promoting tumor proliferation and invasion,
whereas TNFα is mainly produced by microglia because it
in isolated glioma cell lines .
Initially, infiltration of microglia has been proposed to
defend the brain parenchyma against tumor cells .
However, microglia can interact with the tumor environ-
ment and, when activated by the glioma, secrete factors
including MMPs that degrade the ECM. Thus, utilizing this
strategy, glioma cells can invade and expand into the brain
Some characteristics of macrophages/microglia are also
exhibited by tumor cells. The phagocytic activities observed
in human GBM are properties of malignant macrophaes/
microglia. Subpopulations of neoplastic GBM cells
exhibit the phagocytic behavior of macrophages/microglia.
Notably, GBM tumors contain cells that are positive for both
the phagocytic macrophage/microglia marker CD68 and
tumor markers such as hTERT. As microglia are the resident
macrophages of the brain, subpopulations of the malignant
GBM cells could also arise from microglia/macrophages
8Journal of Signal Transduction
Myeloid cells have been observed to fuse with tumor
cells, producing daughter cells endowed with the invasive
properties of myeloid cells and the unlimited proliferative
potential of tumor cells (reviewed in ). More recently,
Pawelek and Chakraborty. demonstrated that the fusion
between nonmetastatic cells and macrophages can result
in cells with the ability to invade and metastasize .
Macrophage/microglial antigens are expressed on neoplastic
cells within GBM . It is possible that macrophages fuse
with tumor cells during attempts to engulf the cells but that
the resulting fusion produces more aggressive and invasive
tumor cells .
VEGF blockade might be more effective if combined
with therapies that also damage endothelial cells. Because
endothelial cells proliferate at a slower rate than tumor cells,
after the administration of a low-dose cytotoxic therapy,
normal endothelial cells might be able to survive during the
recovery period .
Anti-VEGF treatment suppresses the vasculature but
not the coopted vessels . Electron microscopic anal-
ysis of capillary formation has found that the complex
vascular structures within tumors are composed essentially
of progenitor endothelial cells. Cells with ultrastructural
features of endothelial progenitors are recruited to the tumor
periphery prior to vessel formation. Endothelial progenitors
are migratory endothelial cells with characteristic ultrastruc-
tural features and the capacity to circulate, proliferate and
differentiate into mature endothelial cells .
Vascular endothelial cells might also represent a tar-
get for cytotoxic therapy, as they might be capable of
resuming growth during the recovery period after the cyto-
toxic treatment. However, Browder et al. hypothesized that
endothelial cell recovery occurring during this treatment-
free period might support the regrowth of tumor cells. This
could increase the risk of the emergence of drug-resistant
themselves can elicit antiangiogenic effects, dosing schedules
must be carefully designed to induce maximal apoptosis
of the endothelial cells. Some chemotherapeutic agents,
in particular, exhibit maximal benefit when administered
at a low-dose for long treatment periods (metronomic
therapy). The same group has conducted a clinical trial in
which children with recurrent or progressive cancers were
treated with low-dose chemotherapy in combination with
antiangiogenic therapy. Forty percent of patients exhibited
prolonged or persistent disease-free status for all of the six
months of therapy .
Recent studies suggest that tumor cells can also be
involved in tumor angiogenesis, as neoplastic lesions have
been found to contain tumor-derived endothelial cells
(TDECs). These cells originate from the tumor-initiating
cells but not from EC progenitor cells. Through the
activation of HIF-1α, hypoxia plays an important role in
endothelial differentiation. This switch is independent of
VEGF or FGF. In this model, the VEGF inhibitor treatment
elicited no effects on tumor growth .
Tumors can adapt to treatment with angiogenesis
inhibitors by activating alternative angiogenesis-promoting
mechanisms to sustain tumor growth .
In clinical trials evaluating bevacizumab, sorafenib and
sunitinib, a minority of individuals failed to show even
transitory clinical benefit . In these cases, the tumors
exhibited preexisting resistance, which was attributed to the
activation of one or more of the aforementioned evasive
resistance mechanisms, not in response to therapy but to
the selective pressure of their microenvironment. Thus, it is
important to identify markers of resistance and to identify
new approaches for targeting angiogenesis.
Relevance of ILK1 inthe Resistance to
by inserting DLR mutations into a PF4 47–70aa fragment
from Platelet Factor 4 that exhibits strong antiangiogenesis
effects and that reduces angiogenesis and tumor growth in
a dose-dependent manner in the U87-MG model .
This inhibitor has been widely used in human glioblastoma
models, in which it significantly inhibits tumor angiogenesis
and growth. However, prolonged treatment with PF4-DLR
alone or in combination leads to the development of drug
resistance, depending on the dose and the tumors stage at
which it is administered .
Using a proteomics approach, we identified proteins that
were differentially expressed in tumors treated with PF4-
DLR at two time points: after 10 days of treatment when
the tumors are responsive to the antiangiogenic therapy,
and after 20 days of treatment when glioblastomas are still
responsive to PF4-DLR. However, if treatment is prolonged,
glioblastomas start to activate new pathways that might
induce drug resistance. The significance of Integrin-linked
kinase 1 (ILK1) expression after PF4-DLR treatment was
investigated in greater detail. Interestingly, we found that
ILK1 expression is downregulated after 10 days of treatment
and upregulated after twenty days. This result suggested
that ILK1 expression correlates with treatment response, at
least in our experimental model. ILK1 is a protein that is
involved in intracellular signal transduction of integrins and
growth factor receptors. In some tumors, increased ILK1
levels are required for cell growth/survival, cell cycle pro-
gression, invasion and migration, and tumor angiogenesis
. In glioblastoma, a link between ILK1 and tumor
cell invasion has been proposed . Over time, ILK1
increases the expression of VEGF, implying that ILK1 might
be a key molecule for a positive signaling loop that induces
that inhibiting ILK1 with small molecule inhibitors reduces
tumor hypoxia, decreases tumor vascular mass and decreases
functional vasculature in a mouse model of glioblastoma
. The inhibition of ILK1 alone was able to delay but
could not completely inhibit tumor growth. We therefore
decided to inhibit ILK1 using siRNA in addition to PF4-DLR
administration in order to investigate whether this combina-
vivo over PF4-DLR treatment alone. Interestingly, treatment
with PF4-DLR and an anti-ILK1 siRNA resulted in decreased
Journal of Signal Transduction9
tumor mass and a reduction in the number of tumor vessels.
Our findings have important therapeutic implications and
suggest that combinatorial strategies that simultaneously
inhibit different mechanisms of tumor proliferation and
We also analyzed the ILK1 expression levels in patients with
glioblastomas, astrocytomas and oligodendrogliomas and
found that high levels of ILK1 expression correlate with poor
prognosis. Our data suggest that ILK1 could represent a
novel specific pharmacological target to be inhibited alone
or in combination with antiangiogenic therapies for gliomas
The VEGF family members, angiopoietins, Notch/Delta4 or
platelet-derived growth factor are currently the major focus
of angiogenesis research. However, many other regulatory
molecules, including chemokines, critically modulate vessel
growth. The inhibition of IL-6 and VEGF results in the
inhibition of U-87-derived experimental glioma growth on
chick CAM (chorioallantoic membrane) or in xenografts in
the brains of mice .
If a tumor depends on the activity of a single kinase,
a new approach is to target the overactive kinase using
multiple drugs. However, prolonged therapy can again select
for mutations that give rise to therapeutic resistance of the
tumor [159, 160].
An alternative strategy is to target groups of different
kinases. This approach has been demonstrated to be effective
in animal models and is undergoing clinical testing. At least
21 clinical trials are currently evaluating the combination of
a tyrosine kinase inhibitor and an mTOR inhibitor in several
different types of cancer.
The specific targeting and delivery to malignant cell
populations can be achieved by targeting cell surface recep-
tors that are either uniquely expressed or overexpressed on
cancer cells. A variety of ligands have been evaluated for this
purpose, alone or affixed to nanoparticles, antibodies and
related fragments, other proteins, peptides, or aptamers. On
the basis of the discovery that most human tumors express
a high density of specific receptors, it has been possible
to develop radiolabeled peptides that localize to these
tumors and their metastases for therapeutic targeting .
For example, NP-Apt (nanoparticle-aptamer) bioconjugates
have demonstrated therapeutic efficacy both in vitro and in
vivo against cancer cells.
Modular nanotransporters (MNTS) are recombinant
multifunctional polypeptides created to exploit a cascade of
cellular processes, from the initiation of membrane receptor
cytotoxic therapeutic agents into the cells. Slastnikova et al.
nuclei of tumor cells that express a targeted receptor, thereby
improving the specificity of the therapeutic drugs .
Aptamers are oligonucleic acid or peptide molecules that
can bind to target molecules (nucleic acids, proteins, small
molecules, and even cells) expressed on both on membrane
than antibodies because are easy to synthesize and can be
conjugated to fluorescent dyes or chemically modified to
exhibit low immunogenicity. However, a major limitation
in their clinical application is their low bioavailability after
Aptamers have been used both as a tool to find new
targets in cancer therapy and as therapeutic agents against
tumor angiogenesis . In December 2004, the US
FDA approved pegaptanib (Macugen), an anti-VEGF RNA
aptamer, for the treatment of all types of neovascular age-
related macular degeneration. Pegaptanib is the first aptamer
application in humans .
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