VASCULAR TARGETS FOR RADIOTHERAPY

One of the most important promoters of angiogenesis is the vascular
endothelial growth factor (VEGF), which binds to specific
receptors including VEGF-receptor 2 to stimulate blood vessel
formation, growth, and permeability. Clinical correlative studies
have established that tissue VEGF expression is correlated with
a poorer prognosis in NSCLC. 231–235 VEGF, fibroblast growth
factor (FGF), and platelet-derived growth factor (PDGF) receptors
are tyrosine kinase receptors that are expressed on the surface
of endothelial, stromal, and tumor cells, and play important roles
during tumor angiogenesis. Studies of these factors and their receptors
have led to the development of antiangiogenic agents that
target the function of VEGF, FGF, and PDGF.
Radiotherapy promotes expression of angiogenic activators,
VEGF, FGF, and PDGF. 236–238 Radiation-induced VEGF
activation subsequently attenuates vasculature damage and
lead to a reduced tumor cytotoxicity. 239 Serving as a paracrine
proliferative stimulus, VEGF also instigates the growth of previously
dormant microtumors located out-of-field with respect
to the radiation treatment field. 240 In addition, radiation triggers
phenotypic changes favorable for tumor angiogenesis. 241
Recently, the combination of radiotherapy with antiangiogenic
agents has been found to ameliorate the problem of vascular radioresistance.
Antiangiogenic agents act on the endothelial cells
by suppressing the radiation-induced release of proangiogenic
factors. Just as with radiation alone, antiangiogenic agents used
as a monotherapy results in objective response rates of 10% or
less 242 with a gradual loss of activity and efficacy. 243–245 This
is a result of the protean nature of tumor cells in being able to
activate secondary angiogenic pathways when the primary angiogenic
pathway is inhibited. 246 When antiangiogenic agents
and radiation are combined, however, their antitumor effects
become additive or synergistic under the principles of nonoverlapping
toxicities and spatial cooperation. 247 This phenomenon
has been shown in most preclinical studies, suggesting that the
combination of antiangiogenic agents with radiation treatment
has the potential to enhance tumor cytotoxicity and prevent
the development of distant metastases.
Radiotherapy also activates initiator of prosurvival signaling
pathways, such as phospholipases, lipid kinases, and phosphatases,
thereby increasing viability of vascular endothelial
cells. Therefore, this section will focus on potential targets for
radiation therapy, including VEGF inhibitors, as well as the
eicosanoid, sphingolipid, and ceramide pathways. Only representative
antiangiogenic agents are discussed (Table 14.3 provide
a more comprehensive list).
VEGF Inhibitors VEGF is a ligand with a central role
in controlling tumor blood vessel development and survival.
248–250 Numerous evidence indicate that targeting VEGF
and its signaling pathway, when combined with radiation,
could significantly enhance tumor toxicity in preclinical models.
238,251–255 Interestingly, VEGF inhibition does not increase
the hypoxic cell fraction in tumors. 251,252 These results
are encouraging because hypoxia is associated with enhanced
radioresistance and malignant progression in tumors of the
uterine cervix, head and neck, and sarcomas. 256 Furthermore,
emerging concepts, such as the tumor vascular normalization,
suggest that the combination of antiangiogenic agents and
radiotherapy could engender a transient normalization of the
abnormal tumor vasculature and a time period of increased
oxygenation. 257 Anti-VEGFR-2 antibody can create such a
time dependent enhancement of radiation-induced tumor
regression. 258 Alternatively, Lee et al. 251 attributed this transient
oxygenation to the capacity of radiation therapy to ablate
mainly immature tumor vessels and oxygen-consuming cells.
Nevertheless, the two therapeutic modalities combined are superior
than either alone.
VEGF inhibitors include VEGF receptor TKI VEGF trep
and anti-VEGF antibody. When combined with radiation,
these inhibitors have produced promising antitumor efficacy
in preclinical studies. Three of these agents, bevacizumab,
sorafenib, sunitinib, are currently in clinical trials. Many inhibitors
of VEGFR TKI have proved to be potent radiosensitizers
in mouse models of lung cancer. One such example is
AZD2171, an orally available pan-VEGFR TKI with activity
against PDGF receptor and c-Kit. 259 AZD2171 has been shown
to induce a synergistic tumor growth delay when given on days
4 to 6 prior to fractionated RT. 258,260 Alternatively, the administration
of ZD6474 (Zactima), an EGFR and VEGFR TKI,
on xenograft models of lung cancer showed greater antitumor
effects when given 30 minutes after radiotherapy (36 1 days,
p 0.001 vs. radiation alone or the concurrent schedule). 261
The results regarding sequence optimization vary across studies,
and might be dependent on many factors including the drug’s
intrinsic properties, total radiation dose, dose per fraction, and
overall treatment time. Conversely, a recent report showed that
the sequencing of ZD6474/radiotherapy had little impact on
treatment outcomes in a human colorectal carcinoma model,
although this combined strategy had a clear therapeutic advantage.
262 Further studies examining the sequencing of therapies
are needed to ascertain the most favorable schedule of VEGF
antagonist with radiation therapy.
Other VEGFR TKIs investigated to date with radiation in
preclinical models include SU6668 and SU11248. With SU6668,
a synergistic tumor growth delay was observed with concurrent
radiation therapy, and it was found to inhibit Akt phosphorylation
and activation in mouse models with Lewis lung carcinoma
or glioblastoma multiforme (GL261). 263 SU11248 (sunitinib)
is a low nM-selective inhibitor of multiple angiogenic RTKs including
VEGFR1, VEGFR2, VEGFR3, c-KIT, PDGFR- , and
PDGFR- . 264 When given as a maintenance dose after concurrent
radiation therapy, sunitinib effectively prevented tumor regrowth
and significantly prolonged local tumor control. 265
PTK787/ZK222584 (valatinib) is a small molecule-
VEGFR TKI shown to enhance tumor hypoxia in a range that is
associated with enhanced radioresistance when given in monotherapy
but was reverted by concurrent ionizing radiation in
vivo. 266 The bimodality treatment resulted in a supra-additive
growth delay of tumor allografts and was associated with the
highest apoptotic rate and the lowest tumor cell proliferation
index. This study suggests that the risk of treatment-induced
hypoxia by antiangiogenic agents exists but is minimized by
concurrent radiotherapy, thus providing a mechanistic basis
for the combination of antiangiogenic agents with radiation
for cancer therapy. A study by Zips et al. 267 used different
combination schedules of a PTK787/ZK222584 with irradiation
of human SCCs in nude mice. Short-term neoadjuvant
and simultaneous administration showed no effect on tumor
growth delay, whereas long-term inhibition of angiogenesis
after radiotherapy significantly reduced the growth rate of local
recurrences but did not improve local tumor control. These
results suggest that recurrences after irradiation depend on
VEGF-driven angiogenesis, whereas surviving tumor cells retain
their clonogenic potential during adjuvant treatment with
PTK787/ZK222584. In addition, irradiated vessels appear to
be more sensitive to VEGF inhibition, which is supported by
the observation that in vitro–irradiated endothelial cells show
an increased VEGFR2 expression. 268 In this setting, however,
the radioprotective function of VEGF will not be counteracted
during radiation therapy.
The study performed by Kozin et al. 252 on human xenograft
tumors treated with the anti-VEGFR-2 antibody DC101
and irradiation showed promising results. In the 54A and U87
tumor models, the combined treatment resulted in a statistically
significant decrease of the dose necessary for local tumor
control. Specifically, TCD 50 (radiation dose yielding 50%
tumor cure) values were decreased by 41% in 54A carcinoma
and by 24% in U87 tumors. Finally, AEE788, a dual TKI of
both epidermal growth factor receptor (EGFR) and VEGFR,
was shown to improve tumor control when combined with
radiation in prostate cancer cells, especially highly EGFRexpressing
tumors. 269 These studies indicate the potential
of anti-VEGF strategies to improve the outcome of curative
radiotherapy.
Eicosanoid and Lysophospholipid Signaling Pathways
The phospholipase A2 (PLA 2 ) superfamilly produces
arachidonic acid, precursor of the eicosanoid metabolites and
lysophosphocholine (LPC). PLA 2 is activated in irradiated endothelial
cells. 270
Eicosanoid Pathways PLA 2 catalyzes the hydrolysis
of the sn-2 position of glycerophospholipids-releasing arachidonic
acid, which in turn is metabolized to prostaglandins by
the cyclooxygenase (COX) pathway. COX is the rate-limiting
enzyme in the conversion of arachidonic acid to prostaglandins.
Two isoforms of COX were described, COX-1 and
COX-2. Whereas COX-1 is constituvely expressed in most tissues,
COX-2 is induced in pathological states such as inflammatory
processes and cancer. 271 Overexpression of COX-2 is
frequently present in lung cancer and may play a significant
role in carcinogenesis. 272,273 Upregulation of COX-2 and its
major metabolite, prostaglandin E2 (PGE2), has been implicated
in angiogenesis, tumor growth, invasion, metastasis,
apoptosis resistance, and suppression of antitumor immunity.
272–275 It has also been associated with aggressive biological
tumor behaviour, resistance to standard cancer treatment,
and adverse patient outcome in patients with resected early
stage ADC of the lung. 274,275 More precisely, COX-2 partakes
in tumor angiogenesis via various mechanisms including
the increased expression of VEGF, generation of prostaglandins
known to stimulate endothelial cell migration, and
inhibition of endothelial cell apoptosis. 276–278 Nonselective
COX inhibitors, such as indomethacin, have been shown
to enhance tumor radiation response in vitro, 279,280 and
preclinical studies provide evidence that administration of
COX-2 inhibitors with radiation increases local tumor control.
281,282 Antitumor effects may be related to the enhancement
of irradiation-induced apoptosis, 283,284 although this
hypothesis remains controversial. 281,285 Other mechanisms
include the modulation of tumor intrinsic radiosensitivity
285 and tumor angiogenesis. 281,284 It has been suggested
that COX-2 inhibition confers radiosensitivity through the
suppression of prostaglandin production. Prostaglandins
can play a cytoprotective role against irradiation, 286,287
and the removal of COX-2 derived PGE 2 has been demonstrated
to enhance the efficacy of radiotherapy. 276 However,
a recent study by Shin et al. 288 suggests that radiocytoxicity
enhancement by COX-2 inhibitors is attributed to their
alterations of cell cycle and is unrelated to PGE 2 . In this
study, the addition of PGE 2 after the administration of celecoxib,
a COX-2 inhibitor, produced no significant radiationenhancing
effects in A549 and COX-2 transfected HCT-116
cells. They hypothesized that COX-2 inhibition might alter
the G 2 -M checkpoint after noting a correlation between
COX-2 overexpression and prolonged radiation- induced
G 2 -M arrest. Most experimental data, however, do not
support this hypothesis, and additional studies are needed
to delineate the specific mechanism of radiation therapy enhancement
by COX-2 inhibitors.
Two COX-2 inhibitors, celecoxib and SC-236, were
tested in preclinical studies and showed interesting results.
Celecoxib in monotherapy strongly inhibited neovascularization
and reduced tumor growth and metastasis. 277,289 A possible
correlation between basal COX-2 expression level and
celecoxib- induced radiation sensitivity has been suggested. 283
Interestingly, celecoxib exerted an inhibitory effect on the
EGFR-mediated mechanisms of radioresistance, specifically
by preventing both basal and radiation-stimulated nuclear
transport of EGFR. 290 SC-236 also induced significant
growth delay effects when administered oraly in irradiated
sarcoma FSA rodent model. 281 The enhanced radiation response
was associated with decreased PGE 2 levels and markedly
reduced neoangiogenesis. A greater than additive prolongation
of tumor growth was achieved by a combination of
radiation and SC-236 in human glioma U251. 285 Based on
these observations several ongoing clinical trials are currently
evaluating COX-2 inhibitors as adjuvants with radiation
therapy in patients with advanced NSCLC, and preliminary
results are encouraging. Further understanding of the mechanisms
of COX-2 in interaction with radiation may facilitate
future development of targeted strategies for lung cancer
treatment.
Lysophospholipid Pathways Schematicaly, the PLA 2
superfamilly can be divided into four main types: the cytosolic
(cPLA 2 ), the secretory (sPLA 2 ), platelet-activating factor acetylhydrolases
(PAF-AHs), and the calcium-independent cytosolic
PLA2 enzymes (iPLA 2 ). 291 Activation of cPLA 2 leads to the
increased production of lysophospholipids, such as LPC. 292,293
LPC functions as a second messenger in the signal transduction
pathways that regulate vascular proliferation, 294–296 migration,
expression of adhesion molecules, 297–299 and inflammation.
292,293 LPC stimulates proliferation in endothelial cells by
transactivating VEGFR-2 and activating Akt and ERK1/2. 295
Ionizing radiation activates prosurvival pathways in the vascular
endothelium, including PI3K/Akt (PI3K/Akt) 300–302
and MAPK pathways, 301,303 thereby regulating the cellular
response and sensitivity to radiation. Recently, Yazlovitskaya
et al. 270 identified a molecular sequence involving activation of
cPLA 2 followed by the increased production of LPC, transactivation
of Flk-1, and phosphorylation of Akt and ERK1/2 in
irradiated vascular endothelial cells, constituting an immediate
radiation-triggered prosurvival signaling pathway. These data
suggest that cPLA 2 signaling mediates radiation-dependent
prosurvival response in vascular endothelial cells and participates
in endothelial radioresistance.
Recent studies have established that autotaxin (ATX), also
known as phosphodiesterase-I or nucleotide pyrophosphatase/
phosphodiesterase 2, mediates the conversion of lysophosphatidylcholine
to lysophosphatidic acid (LPA) and stimulates
tumor cell motility. 304 LPA acts on specific G-protein– coupled
receptors to stimulate the proliferation, migration, and survival
of malignant cells. 305 ATX is also strongly implicated in tumor
aggressiveness, metastasis, and angiogenesis in preclinical models,
306,307 and is overexpressed in various cancers including
lung. 308,309 Therefore, the ATX-LPA pathway is an attractive
target for anticancer and antiangiogenesis therapy.
Ceramide Signaling Pathway During recent years,
evidence has been provided that sphingolipids including ceramide,
sphingosine, and sphingosine 1-phosphate (S1P) are
more than just structural components. Sphingolipids play important
roles in cell growth as well as cell survival and death
signaling, 310–312 and ceramide has been shown to function
as a lipid second messenger. 313 The best characterized membrane
signaling pathway is initiated by radiation-induced
activation of enzymatic hydrolysis of the membrane sphingomyelin
by sphingomyelinases (SMase), into the formation
of ceramide. 312,314–317 In vitro and in vivo studies showed
the crucial roles of acid sphingomyelinase enzyme activation
and a rapid ceramide generation in radiation-induced
endothelial cell death. 314 Ionizing radiation acts directly on
bovine aortic endothelial cell membrane preparations devoid
of nuclei, proving that ceramide generation after irradiation
is independent of DNA damage and cell cycle regulation induced
by DNA DSBs. 318 Importantly, only high radiation
dose (at least 15 Gy) was shown to induce ceramide production,
as opposed to low-dose irradiation that did not result
in ceramide generation. 239 It has also been shown that cells
deficient in sphingomyelinases are more resistant to radiation-
induced apoptosis. 319,320 There are several isoforms of
SMase based on required pH for their optimal activity: acid
(ASMase), neutral (NSMase), or alkaline (Smase). Ceramide
generation via activation of SMase precedes apoptosis in response
to many different stimuli in addition to radiation,
including TNF- (tumor necrosis factor ), Fas ligand, and
exposure to glucocorticoid. 321 Ceramide-mediated response
to radiation has been shown to activate various protein kinase
cascades, including the classical mitogen-activated protein
kinase (MAPK/ERK) cascade, 322,323 and the stress-activated
protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling
pathway, leading to p53-independent apoptosis. 324
This molecular cascade triggered by ceramide is mediated
by MEKK1 and involves sequential phosphorylation and
activation of SEK1/MKK4 and SAPK/JNK that lead to the
phosphorylation of c-Jun, a transcription factor in the nuclei.
324–326 Once generated, ceramide may accumulate or
be converted into ceramide 1-phosphate by ceramide kinase
phosphorylation, 312 sphingosine by ceramidases deacylation,
and subsequent sphingosine-1-phosphate (S1P) by further
phosphorylation by sphingosine kinase. 327 Interesingly, ceramide
and S1P exert opposing functions in the regulation
of cell death and survival, hence the relative balance between
ceramide/S1P determines the fate of cells in response to specific
stimuli. 328 Modulation of sphingolipid-induced apoptosis
has been proposed as a way to increase the sensitivity
of tumors to various therapeutic agents. 328,329 Sphingosine
kinase, ceramidase, and glycosylceramide synthase, among
other enzymes important to sphingolipid metabolism, are
being studied as potential new drug targets. S1P promotes
cell growth and survival, angiogenesis, vascular maturation,
and mediates cell migration. Using a monoclonal antibody
with high affinity for S1P, Visentin et al. 330 have shown that
selective absorption of S1P is sufficient to block angiogenesis
and endothelial cell migration in response to VEGF and basic
FGF and could thus represent a promising approach to cancer
therapy. Multiple investigators have demonstrated the dependence
of radiation-induced apoptosis on ceramide, 320,331–334
and that radiation sensitivity may be augmented by addition
of exogenous sphingoid bases or modulators of endogenous
ceramide production. 335–337 Preclinical studies of existing
drugs, and the development of new drugs for novel targets in
the various sphingolipid pathways, are warranted to enhance
radiation therapy for lung cancer.