Tumor Vasculature: The Achilles` Heel of Cancer ?
Given its pivotal role in growth and survival, the tumor vasculature represents an attractive target for anticancer therapy. Over the last few decades, rapid progress has been achieved in the understanding of tumor angiogenesis including signaling pathways and their regulation. This has enabled the development of numerous potentially effective vasculature-targeted anticancer drugs (VTAD), which are being tested in the clinical setting. In this review I will focus on the most promising and advanced drugs targeting the tumor vasculature, briefly summarizing their mechanism of action and the clinical results so far obtained.
Tumors require a functioning vascular network to provide tumor cells with oxygen and other nutrients and also to remove toxic waste products associated with cellular metabolism. This vasculature can be acquired, in part, by the incorporation of existing host blood vessels. Nonetheless, for continued growth and development, tumors must generate their own networks of microvessels through the process of neovascularization. In fact, it is widely accepted that no solid tumor can grow larger than a critical size of 1 mm3 without developing a blood supply network. The neovasculature that is formed in tumors differs greatly from what is found in most normal adult tissue. It is primitive in nature, morphologically and functionally abnormal and typically unable to keep pace with the rapidly growing tumor cell mass. Thus, the vascular network inevitably fails to meet the nutritional needs of the tumor cells and this failure results in nutrient deprivation, oxygen deficiency and highly acidic conditions in certain regions within the tumor. Given its pivotal role in growth and survival, the tumor vasculature represents an attractive target for anticancer therapy. Two key approaches to targeting the tumor blood vessel network have been developed (Tortora et al., 2004). The first approach aims to inhibit the tumor-initiated angiogenic process itself (antiangiogenic drugs). Strategies that have been tested include the use of agents that interfere with the delivery or export of angiogenic stimuli; antibodies that inhibit or inactivate angiogenic factors after their release; and inhibitors of receptor activity, tumor invasion, or endothelial cell proliferation. Many of these agents currently are undergoing clinical evaluation. The alternative approach involves the use of therapeutic agents to preferentially destroy the established tumor vessel network. These vasculature-disrupting drugs (VDD) differ from antiangiogenic agents not only in their mode of action but also in their therapeutic application. Whereas VDD are used in intermittent doses, antiangiogenic treatment is administered continually over months or years. At present, clinical trials involving lead VDD also are being conducted.
Tumor Vascularization and Angiogenesis
Like in healthy tissues, tumor neovascularization may include angiogenesis, vasculogenesis and intussusception. Angiogenesis represents the process of new blood vessels sprouting from existing vessels. Recently, vasculogenesis, recruitment of circulating endothelial cells differentiated from stem cells into the newly formed blood vessels, has been found to be critical for tumor neovascularization (Lyden et al., 2001). In addition, intussusception, a process of splitting large mother blood vessels into smaller daughter vessels, has been reported to participate in tumor vessel growth. Despite their ability to stimulate new blood vessel growth from the host, there are fundamental differences between tumor vessels and host tissue vessels. Morphologically, tumor vessels are irregular, heterogeneous and leaky. These features are considered as hallmarks of destruction of normal blood vessel integrity. The endothelial cells are disorganized and irregularly shaped, sometimes overlapped each other and luminal projections, which lead to abluminal sprouts. It has been reported that blood vessels in tumors consist of mosaic cell types including tumor cells (Folberg et al., 2000). Although mural cells have been found on tumor vessels, they have unusually loose associations with endothelial cells. In addition, tumor vessels contain an abnormal basement membrane including changes in matrix protein composition, assembly and structures. Unlike normal blood vessels in a healthy tissue, there is no clear distinction between arterioles and veinules among tumor vessels. As a result of this abnormal vessel architecture, blood flow in tumor vessels is chaotic. For example, a single vessel transports blood to distal tumor cells and removes it from the tumor tissue. Thus, the tumor tissue is relatively hypoxic with poorly oxygenated blood. The unusual leaky features of tumor vessels result in increased interstitial pressure that would further restrict fresh blood flow into the tumor tissue. Thus, normalization of tumor vessels and blood supply would improve drug delivery into the tumor tissue. It is also possible that normalization of leaky tumor vessels could prevent cancer metastasis by limiting the access of tumor cells into the circulation.
Signaling Pathways in Cancer Angiogenesis
A finely tuned equilibrium exists between physiological anti-angiogenic
and pro-angiogenic factors (Carmeliet, 2003). Angiogenesis is mostly mediated
by vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)
under hypoxic conditions due to tumor progression. Low pH, hypoglycemia and
inflammation induced by tumor proliferation also promote angiogenesis. The angiogenic
tumor phenotype is characterized by high microvessel density, elevated VEGF
levels and is correlated to poor prognosis. During angiogenesis, endothelial
cells are stimulated by various growth factors by binding to membrane receptors
such as tyrosine kinase receptors (TKR). TKR that are directly involved in angiogenesis
include receptors for VEGF, FGF, PDGF, Ang-1, angiopoietin-2 (Ang-2), hepatocyte
growth factor (HGF), Eph and receptors belonging to the epithelial growth factor
family. VEGF is the most potent inducer of angiogenesis and there are four VEGF
receptors (VEGFR). Like the other TKR involved in angiogenesis, these receptors
are expressed by endothelial cells and not by the tumor cells secreting the
growth factors. TKR are not only activated by their own ligands but also by
hormones, neurotransmitters and lymphokines.
VEGF and VEGF Receptors
Angiogenesis is a fundamental developmental and adult physiological process,
requiring the coordinated action of a variety of growth factors and cell-adhesion
molecules in endothelial and mural cells. So far, VEGF-A and its receptors are
the best-characterized signaling pathway in developmental angiogenesis (Ferrara
et al., 2003). Loss of a single VEGF-A allele results in embryonic lethality.
This pathway also has an essential role in reproductive and bone angiogenesis.
Much research has also established the role of VEGF-A in tumor angiogenesis.
VEGF-A binds to two receptor tyrosine kinases (RTK), VEGFR-1 (Flt-1) and VEGFR-2
(KDR, Flk-1). Of the two, it is now generally agreed that VEGFR-2 is the major
mediator of the mitogenic, angiogenic and permeability-enhancing effects of
VEGF-A. The significance of VEGFR-1 in the regulation of angiogenesis is more
complex. Under some circumstances, VEGFR-1 may function as a decoy
receptor that sequesters VEGF and prevents its interaction with VEGFR-2. However,
there is growing evidence that VEGFR-1 has significant roles in haematopoiesis
and in the recruitment of monocytes and other bone-marrow-derived cells that
may home in on the tumor vasculature and promote angiogenesis. In addition,
VEGFR-1 is involved in the induction of matrix metalloproteinases (MMP) and
in the paracrine release of growth factors from endothelial cells. Thus the
VEGFR-1-selective ligands VEGF-B and placental-like growth factor (PlGF) may
also have a role in these processes. Furthermore, in some cases VEGFR-1 is expressed
by tumor cells and may mediate a chemotactic signal, thus potentially extending
the role of this receptor in cancer growth. VEGF-A gene expression is upregulated
by hypoxia: in particular, he transcription factor hypoxia inducible factor
(HIF), which operates in concert with the product of the von Hippel-Lindau (VHL)
tumor suppressor gene (under normo-oxic conditions, the VHL protein targets
HIF for ubiquitination and degradation), has a major role iuch regulation. Iitu
hybridizatiotudies demonstrate that VEGF-A messenger RNA is expressed in many
human tumors. Renal cell carcinomas have a particularly high level of VEGF-A
expression, consistent with the notion that inactivating VHL mutations occur
in about 50% of such tumors, thus providing a further explanation for the responsiveness
of this tumor type to a VEGF-A blockade. However, VEGF-A upregulation in tumors
is not only linked to hypoxia or VHL mutations. Indeed, a very broad and diverse
spectrum of oncogenes is associated with VEGF-A upregulation, including mutant
Ras, ErbB-2/Her2, activated EGFR and Bcr-Abl. In 1993, investigators reported
that a murine anti-human VEGF-A monoclonal antibody inhibited the growth of
several tumor cell lines in nude mice, whereas the antibody had no effect on
tumor-cell proliferation in vitro (Kim et al., 1993). Subsequent
studies have shown that many additional tumor cell lines, regardless of the
tumors origin, are inhibited in vivo by the same anti-VEGF monoclonal
antibody or other strategies, as described in the second part of this essay.
Platelet-derived Growth Factor (PDGF) and Angiopoietins
Other signaling molecules that have an established role in the development
and differentiation of the vessel wall such as PDGF-B/PDGFR-31 and the angiopoietins
(Ang), the ligands of the Tie2 receptor, may also be therapeutic targets (Yancopoulos
et al., 2000). PDGF-B is required for recruitment of pericytes and maturation
of the microvasculature. Inhibition of PDGFR-signalling has been reported to
result in a tumor microvascular tree that is particularly dependent on VEGF-mediated
survival signals. Withdrawal of VEGF-A leads to endothelial apoptosis and vascular
regression. In this context, newly formed vessels, whether they are tumor-associated
or not, are particularly vulnerable to VEGF-A blockade, whereas mature vessels,
covered by extracellular matrix and pericytes may be resistant to VEGF inhibitors
and other antiangiogenic agents. Furthermore, recent studies have emphasized
the significance of tumor-derived PDGF-A (and potentially PDGF-C) and PDGFR-signaling
in the recruitment of an angiogenic stroma that produces VEGF-A and other angiogenic
factors. Therefore, combining PDGF and VEGF inhibitors is an attractive anti-vascular
and anti-tumor strategy. Ang-1 is required for further remodeling and maturation
of the initially immature vasculature. Unlike mouse embryos lacking VEGF-A or
VEGFR-2, embryos lacking Ang-1 or its receptor Tie2 develop a rather normal
primary vasculature, but this vasculature fails to undergo effective remodeling.
The generally accepted view is that Ang-1 is the major agonist for Tie2, whereas
Ang-2 may act as an antagonist or a partial agonist. However, more recent evidence
indicates that, unexpectedly, Ang-2 has a positive role, at least in tumor angiogenesis.
Administration of Ang-2 inhibitors to tumor-bearing mice has been reported to
result in delayed tumor growth, accompanied by reduced endothelial cell proliferation,
consistent with an antiangiogenic mechanism. Therefore, inhibitors of Ang-2
may be candidates for clinical development.
Recently, the role of axon-guidance receptors and ligands in developmental
angiogenesis has received much attention (Klagsbrun and Eichmann, 2005). There
are four main families: the neuropilins (NRP)/semaphorins, the ephrins, Robo/Slit
and netrin/Unc5. Although the significance of these pathways in tumor angiogenesis
is far from clear, there is emerging evidence that they have a role iome cancer
models and therefore may be potential therapeutic targets. NRP1 and NRP2, previously
shown to bind the collapsin/semaphorin family and implicated in axon guidance,
are also receptors for the heparin-binding isoforms of VEGF-A and appear to
potentiate the activation of VEGFR-2 by VEGF165. Therefore, NRP may participate
in tumor angiogenesis as positive modulators of VEGF signaling in endothelial
cells. Furthermore, NRP1 and NRP2 are expressed on the cell surface of several
tumor cell lines that bind VEGF165 and display a chemotactic response to this
ligand, suggesting a pro-tumor activity of NRP, with or without the involvement
of VEGF RTK signaling. The ephrins and their tyrosine kinase Eph receptors are
a large family, initially implicated in neuronal guidance during development
and subsequently found to have activities in other cell types, including vascular
cells. The earliest evidence for a role of this family in angiogenesis was that
ephrin A1 mediates TNF-induced angiogenesis in vivo. Recent studies suggest
a role for Eph/ephrin interactions in malignant tumor progression and angiogenesis.
Soluble EphB4-expressing human melanoma A375 cells growubcutaneously in nude
mice showed reduced tumor growth compared with control tumors. Interfering with
EphA signaling has been also reported to result iome inhibition of angiogenesis
in tumor models. Slits are secreted proteins that function as chemorepellents
in axon guidance and neuronal migration through the Roundabout (Robo) receptor.
Investigators reported that Slit2 is expressed ieveral tumor cell types and
that Robo1 is localized to vascular endothelial cells. Moreover, recombinant
Slit2 protein attracted endothelial cells and promoted tube formation. Finally,
neutralization of Robo1 reduced microvessel density and growth of A375 cells
transplanted in nude mice.
Endogenous Negative Regulators of Angiogenesis
Angiogenesis is a tightly regulated process and seems to be under the control
of both positive and negative regulatory factors. Although several potential
negative regulators of angiogenesis have been identified, relatively little
is known about their role in the physiological regulation of angiogenesis (Sund
et al., 2005). Thrombospondin, a large multifunctional glycoproteiecreted
by most epithelial cells in the extracellular matrix, inhibits angiogenesis
associated with tumor growth and metastasis. Several fragments of larger proteins
have been described as endogenous inhibitors of angiogenesis including endostatin,
tumstatin and vasostatin. The most recently described endogenous inhibitor of
angiogenesis is vasohibin, which seems to be derived from the endothelium and
to operate as a feedback regulator. The precise mechanism of action of these
proteins remains to be more clearly defined, although several hypotheses have
been proposed, including that they bind to specific integrins in the case of
endostatin and tumstatin.
Bone-marrow-derived Cells and Angiogenesis
An intensively debated issue in the oncology field is the role of bone-marrow-derived
endothelial progenitor cells (EPC) to angiogenesis (Peters et al., 2005).
However, there is little doubt that bone-marrow-derived cells participate in
angiogenic processes, at least as a source of angiogenic factors. After their
isolation from human peripheral blood on the basis of cell-surface expression
of CD34 and other endothelial markers, EPC were reported to differentiate in
vitro into endothelial cells and appeared to be incorporated at sites of
active angiogenesis in various animal models of ischaemia. These findings suggested
that incorporation into the lumen of bone-marrow-derived endothelial precursor
cells contributes to the growing vessels, complementing resident endothelial
cells iprouting new vessels. Also, ischaemia and various cytokines, including
VEGF and granulocyte-macrophage colony-stimulating factor (GM-CSF), were reported
to mobilize EPC into sites of neovascularization. However, the precise contribution
of these cells in various pathophysiological circumstances was not clearly defined.
Subsequent studies have suggested that the contribution of such cells to angiogenesis
is dependent on the experimental system employed. In the angiogenic-defective,
tumor-resistant Id-mutant mice, EPC accounted for a large proportion of endothelial
cells associated with xenografted tumors. Investigators proposed that mobilization
of EPC from bone marrow requires angiogenic-factor-mediated activation of MMP-9,
which leads to the release of the soluble KIT ligand. This ligand would in turn
promote proliferation and motility of EPC within the bone-marrow microenvironment,
thus creating permissive conditions for their mobilization into the peripheral
circulation. However, ipontaneous tumors occurring in Id -deficient mice in
the tumor-prone PTEN +/- genetic background, the contribution of EPC was less
significant. Furthermore, other investigators suggested that the percentage
of EPC that are truly incorporated into a growing vessel wall is very low and
that the majority of bone-marrow-derived cells homing in on the tumor vasculature
are adherent perivascular mononuclear cells, which may contain angiogenic factors.
Vasculature Targeted Anticancer Drugs
Large numbers of vasculature targeted anticancer drugs (VTAD) with various mechanisms of action are currently under clinical development and in this review we will focus on the most promising and advanced drugs targeting the tumor vasculature briefly summarizing their mechanism of action and the clinical results so far obtained.
Inhibition of the VEGF Pathway
Tyrosine Kinase Inhibitors
This compound has a wide spectrum of activity and inhibits the tyrosine
kinase activity of PDGFR, FGFR1 and VEGFR2. It permits the regression of human
tumor xenografts in mice with complete histological response following the rapid
apoptosis of tumor microvessels. The maximum tolerated dose of SU6668 given
orally, thrice daily under fed conditions, is 100 mg m-2. Because
of the low plasma levels reached at this dose level, the efficacy of SU6668
as a single agent is not to be expected (Kuenen et al., 2005).
SU011248 inhibits VEGFR1, PDGFR and c-Kit, a receptor of the stem cell growth
factor (SCF) implicated in malignant blood diseases. At higher concentrations,
it inhibits another angiogenesis TKR, FGFR1. SU011248 was synergistic with radiotherapy
in mouse models attaining tumor responses and sustained tumor control. In a
phase II study, 63 patients with renal cancer after failure of a cytokine received
SU011248 that resulted in 21 (33%) partial responses and 23 (37%) stabilizations
(Mancuso and Sternberg 2005); moreover, the 1-year survival was 65%. Interestingly,
SU011248 has also achieved clinical activity in gastrointestinal stromal tumors
(GIST). GIST are sensitive to STI-571 a specific c-Kit tyrosine-kinase inhibitor.
After failure of STI-571, 98 patients (92 evaluated) received SU011248. Of those
patients, 7 (8%) partial responses (PR) and 53 (58%) SD were observed. Efficacy
of SU011248 is higher in patients without mutation in c-Kit or PDGFR-A, or a
mutation in the exons 9, 13 and 14 of c-Kit or PDGFR-A. The efficacy of SU011248
is limited to patients carrying mutations in the exons 11 and 17.
PTK-ZK inhibits the tyrosine kinase activity of VEGFR1 and VEGFR2 and is
given orally. Preclinical studies demonstrated activity through the inhibition
of tumor vasculature, alone or in combination with chemotherapy or radiotherapy.
Pre-irradiated vasculature may be more sensitive to PTK-ZK compared to vasculature
not irradiated. Phase I studies have shown good PTK-ZK tolerance and efficacy
in various tumors. The most commonly reported adverse events were nausea (59%),
fatigue (41%), vomiting (35%), dizziness (29%) and headache (24%). The first
study in 20 patients with relapsing glioblastoma yielded four stabilizations
and one partial response in the 15 tested patients (Zakarija and Soff, 2005).
Magnetic resonance imaging depicted a decrease in vascular permeability of tumors
after intake of the drug that was correlated with clinical response. The second
study enrolled 35 patients with advanced colorectal cancer and patients were
treated every 14 days with oxaliplatin, 5-FU and folinic acid in combination
with continuously escalated doses of PTK-ZK (500-2,000 mg day). No increased
oxaliplatin/5-FU toxicity was seen and PTK-ZK was tolerated to doses of 1,500
mg day. In 28 patients, one complete response (4%), 14 partial (50%), nine SD
(32%) and four progressive disease (14%) were obtained. For all patients, the
median overall survival was 16.6 months (Arora and Scholar, 2005). Similar results
were obtained when PTK-ZK was combined with irinotecan-5-FU-folinic. In the
third study, 45 patients (37 evaluated) demonstrated the efficacy of PTK-ZK
in metastatic renal cancer. Two patients experienced dose-limiting toxicity
(grade 3 headache and grade 3 hypertension). Seven patients (19%) achieved a
measurable response (1 partial and 6 minor) with a median time to progression
of 5.5 months, 17 (46%) achieved SD and 5 (14%) experienced disease progression.
Rapid disease progression (within 3 months) occurred in only 28% of the patients
treated with at least 1,000 mg/day compared with an expected rate of 49.7% based
on cytokine therapy in a similar patient population. One-year overall survival
was 63.7% (95% CI = 41.9, 85.5%).
Bevacizumab is a humanized antibody designed to target VEGF and not its
receptor like the other agents discussed in this review. This antibody has shown
tumor growth inhibition in preclinical and early clinical studies with good
tolerance. Phase I studies did highlight potential risk of hypertension or thromboembolism
however its clinical efficacy has since been demonstrated. A phase III trial
investigated bevacizumab (5 mg kg-1 every two weeks) versus placebo
in combination with irinotecan, 5-FU and folinic acid (FA) as first-line therapy
for metastatic colorectal cancer in 925 randomized patients. Adding bevacizumab
to chemotherapy resulted in increased mediaurvival (20.3 months vs 15.6, p =
0.00003), progression-free survival (10.6 months vs 6.24, p<0.00001), a higher
response rate (45% vs 35%, p = 0.0029) and a longer duration of response (10.4
months vs 7.1, p = 0.0014) as compared with chemotherapy plus placebo. Adding
bevacizumab did not modify the toxicity of chemotherapy but only increased grade
3 hypertension (10.9 vs 2.3%), which was easily managed with oral medications
(Hurwitz et al., 2004). The inhibition of angiogenesis was demonstrated
with a single infusion of bevacizumab that decreased tumor perfusion, vascular
volume, microvascular density, interstitial fluid pressure and the number of
viable, circulating mature and progenitor endothelial cells in patients with
rectal carcinoma. A randomized, double blind, phase II trial was conducted comparing
a placebo (40 patients) with bevacizumab at doses of 3 mg kg-1 (37
patients) and 10 mg kg-1 (39 patients), given every two weeks in
116 patients with metastatic renal cancer. There was a significant prolongation
of the time-to-progression (TTP) in the high-dose antibody group as compared
with the placebo group (hazard ratio, 2.55; p<0.001). The probability of
having progression-free tumors for patients given the high-dose antibody, the
low-dose antibody and the placebo was 64, 39 and 20%, respectively, at four
months and 30, 14 and 5%, respectively, at eight months. There were no significant
differences in overall survival between the groups but primary endpoints were
TTP and the response rate (Yang et al., 2003). Another phase II trial
investigated the adjunction of bevacizumab to gemcitabine as first-line therapy
in 21 patients with metastatic pancreatic cancer (Kindler et al., 2005).
Among evaluated patients, 6 PR (38%) and 7 SD (44%) were observed. Mediaurvival
was not reached and the estimated 1-year survival rate was 54% and TTP 5.5 months.
Pretreatment VEGF levels ranged from 0 to 586 pg mL-1 and were not
correlated with response, TTP, or survival.
IMC-1C11, a chimeric IgG1 antibody against VEGFR2 and blocks ligand-receptor
binding and inhibits phosphorylation. A phase I study in 14 patients with metastatic
colorectal cancer resulted in prolonged stabilization but presence of anti-chimeric
antibodies in 7 patients (2 had neutralizing antibodies) was detected (Posey
et al., 2003). Antibodies against VEGFR such as IMC-1C11 may have theoretical
advantages compared to antibodies against soluble ligands that are frequently
over produced leading to treatment resistance.
Other Mechanisms to Inhibit the VEGF Pathway
Using VEGFR-specific CD8+ cytotoxic lymphocytes as opposed to antibodies
is an original approach. A retroviral vector allows the transfection of VEGFR
sequences in CD8+ lymphocytes, which then become endowed with cytotoxic activity
against VEGFR-expressing cells. These lymphocytes are able to inhibit tumor
growth in mice in vivo. This inhibition is more powerful when administered
in combination with the anti-angiogenic agent TNP-470 (Niederman et al.,
Ribozymes are catalytic RNA molecules capable of cleaving mRNA at specific
sequences. Anti-FLT-1 ribozyme is a nuclease-resistant synthetic molecule targeting
VEGFR1 mRNA. In a phase I/II study enrolling 28 patients with refractory solid
tumors, the ribozyme was subcutaneously administered daily. Tolerance was good
apart from some reactions at the inoculatioite (Weng et al., 2005). SeventeeD
lasting 1-6 months and 2 minor responses were observed. Phase II studies testing
ribozyme alone or in combination with chemotherapy are awaited.
Aplidine is a cyclodepsipeptide that seems to decrease VEGFR1 expression
and to induce cell cycle arrest in the G1 phase. It has demonstrated promising
activity in vitro against several human tumors. Aplidine is currently
under clinical development in phase I studies and tumor responses have been
reported particularly in leukemia patients (Jimeno et al., 2004).
Neutralizing antisense oligonucleotides for VEGF mRNA have been developed
and exhibit an anti-angiogenic potential (Kamiyama et al., 2002). A neutralizing
antisense oligonucleotide for angiopoietin-1 was tested in vitro with HeLa tumor
cell lines and showed a marked decrease in tumor growth.
VEGF-trap is a fusion protein consisting of human VEGFR1 (flt-1) segments
and VEGFR2 (KDR) extracellular domains fused to the Fc portion of human IgG1.
It acts by binding to and inactivating circulating VEGF that found in tissues.
VEGF-trap has great affinity for VEGF similar to that of the high-affinity receptor
VEGFR1 (Km = 1-5 pM). This results in approximately 100-fold tighter binding
than that achieved with anti-VEGF monoclonal antibodies, while retaining pharmacokinetic
properties similar monoclonal antibodies. This compound could potentially achieve
greater efficacy at lower doses compared to antibodies. Furthermore, in contrast
to humanized monoclonal antibodies, it contains only human amino acid sequences.
A dose-escalation phase I trial tested a single subcutaneous dose of VEGF-trap
in patients with relapsed or refractory tumors, followed 4 weeks later by 6
weekly injections. Fourteen patients were treated at four dose levels (25, 50,
100 and 200 μg kg-1). The VEGF-trap/VEGF complex in plasma had
an apparent elimination half-life of approximately 17 days. No grade 3 or 4
toxicities were observed and no anti-VEGF-trap antibodies were detected. Grade
1 and 2 toxicities included reversible proteinuria, fatigue and constipation.
Disease stabilization was obtained in patients with renal or colon cancer (Bergsland,
Other Strategies to Target Tumor Vessels
The production of VEGF and other angiogenic factors calls into service a
cascade of intermediate signals, transcription and regulation factors and among
the prostaglandins produced by cyclooxygenases (COX), the COX-2 isoform. COX-2
induction has been reported in numerous solid tumors (colon, prostate, lung,
breast, pancreas, skin, head and neck) but not in corresponding normal tissues.
COX-2 was detected in tumor cells, tumor vascularization and pre-existent adjacent
capillaries. Tumors transplanted into COX-2 deficient mice showed that COX-2
is not only crucial for the production of VEGF, but that endogenous COX-2, produced
by vascular endothelium or by fibroblasts, contributed markedly to the growth
of transplanted tumor cells. The detection of COX-2 in neovasculature was described
as a characteristic of epithelioma. In colon cancer, the number of cells expressing
COX-2 was found to correlate with tumor stage, size and mediaurvival. Pre-clinical
studies have demonstrated that anti-tumor activity of selective COX-2 inhibitors
have a good safety profile. The anti-tumor properties of COX-2 inhibitors may
be attributed to the inhibition of angiogenesis mediated by PGE and VEGF, however
COX-2 may also play a role in the migration and survival of endothelial cells,
vessel permeability and the suppression of immune responses. Collectively, these
events contribute to tumor growth and may eventually be potent therapeutic targets
as assessed by several phase II studies in different tumor types. A recent trial
tested the benefit of adding celecoxib (400 mg bid) to a chemotherapy cocktail
containing irinotecan and capecitabine in the treatment of metastatic and unresectable
colorectal cancer. Twenty-three patients were included (17 evaluated) and 9
PR (53%), 10 SD (56%) and 3 progressive disease (17%) were observed and celecoxib
appeared to decrease the toxicity of capecitabine (Fayette et al., 2005).
A further study evaluated the combination of celecoxib (400 mg p.o. bid continuously)
and paclitaxel (80 mg m-2 i.v. weekly for six weeks followed by a
two-week rest) in 27 patients (16 evaluated, mostly with adenocarcinomas) as
second line therapy in non-small cell lung cancer (NSCLC) after failure of a
first line with platinum compounds (Altorki et al., 2003). Four PR (25%),
6 SD (37%) and 6 progressive disease (37%) were observed with acceptable toxicity.
Another study evaluated the benefit afforded with celecoxib (400 mg ·
2/d) combined with two preoperative cycles of paclitaxel and carboplatin in
29 patients (stages IB to IIIA). All patients completed preoperative chemotherapy
and 26 completed preoperative celecoxib. The overall clinical response rate
was 65% (48% PR, 17% complete responses). Twenty-eight patients were explored
and underwent complete resection of their tumors. No complete pathological responses
were observed but seven patients (24%) had minimal residual microscopic disease
(Fayette et al., 2005). Interestingly, it has recently beeuggested that
high COX-2 gene expression in completely resected NSCLC is correlated with poor
prognosis (RR = 3.848; 95% CI, 1.500-9.874) and limited benefit with UFT-based
adjuvant therapy. Indeed, in the setting of UFT postoperative adjuvant chemotherapy,
patients with low COX-2 gene expression have a higher 5-year survival as compared
to those with high COX-2 gene expression (93% vs 67%, p = 0.045) (Fayette et
al., 2005). A pilot study in 12 patients demonstrated the potential efficacy
of celecoxib (200 mg • 2/day) in a biochemical relapse of prostate cancer
(indicated by increased prostate-specific antigen (PSA) levels) after radiotherapy
or radical prostatectomy. PSA levels were significantly modified in 8 patients
after 3 months of treatment (declined in 5 and stabilized in 3). Of the remaining
4 patients, 3 had a marked decrease in their PSA doubling time. The short-term
responses at 3 months also continued at 6 and 12 months (Pruthi et al.,
Vitaxin is a humanized monoclonal antibody against the αVβ3 integrin
and is currently being tested in phase I trials where it has yielded PR or SD
iarcoma. EMD 121974, an αVβ3 and αVβ5 integrin antagonist,
produced responses in melanoma and brain tumors in preclinical studies. A phase
I trial resulted in good tolerance (Eskens et al., 2003). Another humanized
IgG1 (Medi-522) targeting αVβ5 integrins was tested in a phase I study
which accrued 19 patients (13 evaluated). Tolerance was acceptable and 7 patients
achieved prolonged SD with 2 lasting more than 9 months (McNeel et al.,
Endogenous Angiogenesis Inhibitors
Endostatin and angiostatin are peptides derived from natural proteins that
have been shown to exhibit antiangiogenic properties. These agents have a short
half-life and repeated, or even, continuous injections are required. Results
are modest but prolonged SD was observed, particularly in patients with advanced
neuroendocrine tumors who were enrolled in a phase II study. All the 41 patients
(25 carcinoid and 16 pancreatic tumors) were initially treated with 60 mg m-2
day-1 of rhEndostatin, self-administered by subcutaneous injection
in divided doses, at 12 h intervals. Minimal toxicity was observed during this
treatment. Doses were escalated if the therapeutic effect was insufficient but
not for progressive disease. Twenty-three (62%) SD and 12 (32%) progressive
disease were observed. The median TTP was 39 weeks and mediaurvival was not
reached (Eder et al., 2002). Thrombospondin (TSP-1) is another natural
angiogenesis inhibitor and ABT-510 is a substituted nanopeptide that mimics
the anti-angiogenic activity of the endogenous protein. It was tested in a phase
IB study that enrolled 36 patients (34 evaluated) with various tumors. Patients
received daily subcutaneous escalating doses of ABT-510 with acceptable tolerance.
One PR in a soft tissue sarcoma (STS) and 14 (41%) and 5 (15%) SD was observed
in 8 and 16 weeks, respectively. Prolonged SD (>24 weeks) was seen in 1 patient
with NSCLC and 1 with STS (Hoekstra et al., 2005).
Combretastatin A-4 Phosphate (CA4P)
The phosphorylated pro-drug CA4P is a synthetic analogue of combretastatin,
a tubulin-binding agent that selectively induces apoptosis in proliferating
human umbilical vein endothelial cells in vitro and produced up to a 90% reduction
in tumor blood flow in xenografted tumor models. Phase I studies showed its
potential efficacy (Bilenker et al., 2005; Stevenson et al., 2003),
but cardiotoxicity is the major adverse side effect.
Raf-1 Pathway Inhibitors
The αVβ3-integrin is preferentially expressed by the endothelium
of neovessels and is a potential target for a gene therapy. A lipidic nanoparticle
was synthesized and bound to an αVβ3-ligand. A gene could be coupled
to this particle and delivered specifically to αVβ3expressing
cells. The selected gene was a mutated form of Raf that inhibits VEGF- and FGF-induced
angiogenesis. Experiments in mice bearing melanoma or human colon tumor xenografts
demonstrated very high anti-angiogenic activity, specifically mediated by the
mutated Raf gene delivered to αVβ3-expressing endothelial cells, with
fast and dramatic tumor regressions. Nanoparticles are much less immunogenic
than viral vectors and thus allow repeated and prolonged treatment. A specific
Raf-1 inhibitor has been developed: BAY43-9006. A phase I study showed good
tolerance. A phase II study included 397 patients among which 112 with a renal
cell carcinoma. Among them, 65 patients completed the treatment, 63 were evaluated
with 25 (39%) PR and 16 (26%) SD (Clark et al., 2005).
Over the last few decades, rapid progress has been achieved in the understanding of tumor angiogenesis including signaling pathways and their regulation. This has enabled the development of numerous potentially interesting antineoplastic agents. However, further advances in dissecting the cascade of molecular events underlying cancer angiogenesis are necessary to fully exploit the therapeutic potential of VTAD. In particular, resistance to anti-angiogenic treatments has been observed to be due to mutations of the p53 protein (such is the case in 50% of human cancers) which lower tumor cell oxygen requirements and thus their dependence on neovascularization. The induction of bcl-2, a gene involved in resistance to apoptosis, has also been observed. Cross activation between the different signaling pathways must also be further elucidated. Studies in animals show that VEGFR and EGFR inhibitors can be combined to enhance their efficacy. One of the present challenges is to determine whether it is best to combine exclusively these new anti-angiogenic agents or to combine them with conventional cytotoxics so that the survival of patients can be increased significantly. This challenge implies new models that preferably considers the cytostatic rather than the cytotoxic nature of anti-angiogenic agents, the possibility of prolonged therapy with these agents and the rationale for combining them with other cytotoxic therapies. Ongoing clinical trials are applying these concepts with the prospect of using these anti-angiogenic therapies in clinical practice.
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