Cell Cycle and Vascular Targets for Radiotherapy

The cell cycle checkpoints ensure that cells replicate their
genomes with high fidelity. In response to DNA-damaging
agents such as ionizing radiation or cytotoxic chemotherapy,
cell cycle checkpoints delay progression through cell cycle to
allow repair before completion of mitosis. Critical parts of the
cell cycle machinery are the evolutionarily conserved cyclindependent
kinases (Cdks), which regulate the transition from
one phase of the cell cycle to the next. The Cdks are activated
by cyclins and inhibited by naturally occurring Cdk inhibitors
(CKI). Cell cycle arrest after DNA damage is critical for
maintenance of genomic integrity, and loss of normal cell cycle
checkpoint signaling is common in cancers and is considered a
pathologic hallmark of neoplastic transformation. 1
The ability to manipulate cell cycle signaling has important
clinical implications in lung cancer. For example, modulation
of the checkpoints before the completion of DNA repair
could enhance cellular sensitivity to DNA damage agents such
as radiotherapy, leading to cell death.
This chapter focuses on the cell cycle dysregulation present
in lung cancer, the cell cycle effects of radiotherapy, and
on the role a cell cycle modulator may play in combination
therapy. The knowledge of these concepts might lead to more
efficient use of current anticancer therapies and to the development
of novel agents.
Cell Cycle Signaling Response to Ionizing
Radiation Cell proliferation results of the repeated progression
through a cycle made of four phases and regulated
by a defined set of protein complexes. 2,3 Molecular mediators
of the cell cycle include cyclins that promote cell cycle transition;
Cdks that regulate the cell cycle by forming complexes
with their cyclin catalytic partners; and endogenous CKIs
that inhibit cyclin–Cdk complexes (Fig. 14.1). The cellular
response to DNA damage is the activation of cell cycle checkpoints
that serve as natural surveillance mechanism for DNA
integrity (Fig. 14.2). Irradiation induces both single- and
double-stranded DNA breaks (DNA DSBs), the latter being
generally considered the lethal event. 4–7 Two major systems
contribute to the repair of DNA DSBs induced by radiation.
Homologous recombination, during the late S and G2 stages
of the cell cycle, is critical in cell signaling and is regulated
by the cell cycle, whereas nonhomologous end-joining is more
important during G1 and early S phases and is the predominant
mechanism of DNA DSBs repair.
Central to the signal transduction pathways are two phosphatidylinositol
3-kinase–like kinases (PIKKs), ataxia telangiectasia
mutated (ATM), and ATM and Rad3-related (ATR),
which transmit the damage response signal through phosphorylation.
8 Radiation-induced DNA damage activates ATM,
serving as proximal damage sensor, by autophosphorylation at
Ser 1981 resulting in ATM dimmer dissociation. 9–12 Evidence
suggests an activating role for the Mre11-Rad50-NBS1 (MRN)
complex that binds independently to DNA DSBs and facilitate
the signaling of ATM. 13,14 Cells with defective or lacking ATM
or mutated ATM gene are extremely sensitive to irradiation,
suggesting that ATM is a cornerstone in the DNA damage response.
15–17 ATM and ATR respond to single-stranded regions

of DNA by replicative stress. 18 Once activated, ATM can potentially
activate signaling receptors and stimulate cell cycle checkpoints,
p53 activity, and DNA repair complex function. 19–21
Cell cycle checkpoints monitor the structural integrity
of chromosomes before progression through crucial cell cycle
stages. 22 Checkpoints occur at entry into S phase (the G1/S
checkpoint), entry into mitosis (the G2/M checkpoint), as
well as during replication (intra-S checkpoints). 23,24 After
irradiation, the damage response signaling pathways facilitate
communication between damage recognition proteins and the
checkpoint machinery to effect arrest of cell cycle progression
(G1 or G2 arrest) and increase the opportunity for repair before
undertaking important events such as replication or mitosis. 25
In most solid tumor cells, G 1 arrest is dependent on the
activation of the tumor suppressor gene p53 and the downstream
CKI p21, which also plays a role in cellular senescence,
apoptosis, and DNA repair. 26,27 It is not clear whether p53
gene and G 1 arrest have an influencing role in radioresistance
in solid tumor cells. 28 In contrast, G 2 arrest, which can be
influenced by p53, may have a radioprotective function by
providing cells time to repair DNA damage, and is controlled
by the nuclear activities of the cyclin B1-CDC2 complex. 29,30
Two ATM-dependent G1/S checkpoints can be individualized.
First, ATM activation in G1 leads to CHK2 phosphorylation
and in turn, to phosphorylation of the phosphatase CDC25A.
This increases the proteolytic degradation of CDC25A and
prevents activating dephosphorylation of CDK2 and initiation
of the G1/S checkpoint. 31,32 Second, ATM phosphorylates
the tumor suppressor p53, either directly or indirectly through
CHK2, stabilizing the protein and prolonging its half-life,
which function as a transcription factor for the CKI p21. p53
serine 15/20 phosphorylation disrupts the normal binding of
the oncoprotein MDM2 to p53, thereby inhibiting its degradation
process and prolonging its half-life. This pathway has a
role in the maintenance of the G1/S checkpoint. 33
Two distinct G2/M checkpoints can also be identified,
determined by the kind of DNA damage and by the time sequence.
34 The first checkpoint, ATM dependent, occurs rapidly
after radiation-induced damage and is controlled by CHK1-
mediated signaling that leads to inhibition of cyclin B1/CDC2
activity. It represents the failure of cells that had been in G 2
at the time of irradiation to progress into mitosis. This G2/M
checkpoint, which is dose independent, may be the transducer
element linking low-dose hyperradiosensitivity to the subsequent
process of resistance. 35 In contrast, the dose-dependent G2/M
accumulation begins to be measurable several hours after irradiation.
This mechanism is ATM independent, and represents the
accumulation of cells that were in earlier phases of the cell cycle
at the time of exposure. 34 G2/M accumulation after exposure to
radiation is not affected by the early G2/M checkpoint and is
enhanced in cells lacking the radiation-induced S-phase checkpoint,
such as those lacking NBS1 or BRCA1 function. 34 It is
initiated by the phosphorylation of checkpoint kinases, CHK1
and CHK2, and CDC25A/C, which inactivate the enzymes,
blocking activation of CDK1, and causing a G2 arrest. p53
was shown to suppress promoters of cdc2 and cyclin B, which
leads to G2/M arrest, and to have a regulation role in the sustain
of the G2/M arrest. 36 DNA damage by radiation blocks pRb
phosphorylation through p21 to maintain the pRb-regulated cell
cycle arrest to complete DNA damage repair. The further identification
of cell cycle signaling elements in response to ionizing
radiation will have important implication for the development of
anticancer targeted strategies.
Cell Cycle Alterations in Lung Cancer Molecular
analysis demonstrates that alterations in components of the
cell cycle machinery and checkpoint signaling pathways occur
in most lung tumors (Table 14.1). Alterations in the cell cycle
machinery that occur most frequently include overexpression
of cyclins, cdks, and Cdc25 phosphatases, loss or mutation of
the RB tumor suppressor, and loss of cdk expression. The most
frequently altered cell cycle checkpoint signaling molecule is
the p53 tumor suppressor.
Cyclins D1 Following a mitogenic signal during the G1 phase,
cyclin D1 assembles with cdk4 and a CDK-interacting protein
(CIP) (p21)/kinase-interacting protein (KIP) (p27) protein. This
complex enters the nucleus and phosphorylates the retinoblastoma
tumor suppressor protein Rb1, 37 promoting the release of
E2F transcription factor from the Rb1/E2F complex 38 and thus
the progression from G1 to S phase. During G1/S transition,
glycogen synthase kinase (GSK)-3 enters the nucleus and phosphorylates
cyclin D1, allowing its nuclear export by Crm1 39,40
and degradation by the 26S-proteasome. 40,41 Under certain conditions,
the cyclin D1/cdk4 complex can also act as a mediator of
programmed cell death. 42,43 Cyclin D1 can also directly interact
with several nuclear receptors and transcription factors, 44–48 by
repressing or inducing them. This transcriptional function links
cyclin D1 not only to cell cycle and apoptosis but also to migration,
invasion, differentiation, inflammation, and angiogenesis.
Cyclin D1 has been identified as a proto-oncogene, PRAD1, and
was found to be overexpressed relative to normal tissue in many
human cancers. 3,49,50 Overexpression of cyclin D1 is associated
with dysregulation of cell cycle and is reported in 25% to 60%
of non–small cell lung cancer (NSCLC), 51,52 varying from 30%
to 35% in squamous cell carcinoma (SCC) and 36% to 56% in
adenocarcinoma (ADC) in the largest series. 53–55 Cyclin D1 overexpression
is less common in neuroendocrine tumors (NE) and,
among these, more frequent in atypical carcinoids (20%) and
large cell neuroendocrine cancers (LCNE) (9.5%). Cyclin D1 dysregulation
is frequently an early event in tumorigenesis, elevated
levels being already found in bronchial 56,57 and alveolar hyperplasia.
58,59 Cyclin D1 level has also been linked to heavy smoking
and said to constitute a marker of tobacco exposure. 52,60,61
Cdk Inhibitors
p21. p21 was the first CKI to be discovered. It is a member
of the KIP family of proteins that nonspecifically inhibit
cyclin–cdk complexes in the nucleus. 62 It can inhibit the cyclin
D/cdk4 and cyclin E/cdk2 complexes early in G1, and
it can also inhibit the cyclin A/cdk2 complex later, prior to
the S-phase/G2-phase transition. 63 p21 transcript is directly
activated by p53 and seems to be a fundamental partner for
p53. It contributes to p53-induced apoptosis 64 and blocks
the G2/M transition through cdc2 inhibition, mediating
the pro-apoptotic and cell cycle–arresting effects induced by
p53 in response to genotoxic stimuli. 65 Recent studies have
shown potential new roles for p21 and p27, as inhibitors of
apoptosis and so, as potential oncogenes. These roles have been
related to their relocalization to the cytoplasm, 66–70 whereas
the cell cycle inhibitory activity is dependent on the nuclear
localization. Although p21 is infrequently expressed in normal
lung, 71 it is overexpressed in 35% to 51% of NSCLC cases,
without significant differences between ADC and SCC. 71–75
p21 expression has been correlated with better differentiation
in NSCLC. 71,73,74 In two studies adequately controlled
for disease stage, p21 expression was associated with improved
survival, 72,75 whereas another study reached slightly different
conclusions. 76 In an analysis combining p21 and TGF- , an
improved survival was observed in early stage NSCLC when
p21 and TGF- were in concordance, presenting both high
or both low expression. 76 Absence of p21 expression has been
correlated to a worse prognosis when associated with p53 positivity
75 and absence of p16 expression. 77
p27. p27, another member of the KIP family, blocks the
activity of cyclin D1-CDK4/6 and cyclin E-CDK2 and is an
important negative regulator of the G1 to S transition. 66,78
In the normal resting cell, concentration of p27 is high and
declines in response to proliferative stimuli. p27 overexpression
leads to cell cycle arrest, whereas its loss of expression may
result in tumor development and/or progression. As described
for p21, some studies have suggested p27 to be an oncogene
by inhibiting apoptosis, and that this role is related to its cytoplasmic
relocalization. 66,79 In vivo studies have shown that
p27 expression decreases progressively during lung carcinogenesis.
80 Increased p27 proteolytic activity has been associated to
low levels of p27. 77,81 Low p27 expression has been associated
with higher stages of lung cancer, 82 as well as poor survival
in NSCLC, either by itself 77,81–83 and/or when associated to
other abnormalities like p53 mutations and/or Rb loss, 84 Ras
mutations, 85 high cyclin E levels, 79 high proliferative rates 84,85
and aneuploidy. 84 A favorable response to chemotherapeutic
agents, including drugs targeting EGFR and HER-2, has been
correlated to increased p27 expression. 86,87
p16. p16 is a member of the INK4 family of proteins
that inhibits cdk4 and cdk6 activation through a competitive
mechanism for cyclin D binding site and specifically during
the G1 phase. 88 Loss of p16 has similar effects on G1 progression
than overexpression of cyclin D and loss of Rb. The
discovery of p16 overexpression in Rb / cells, which alters
the relationships between cyclin D, cdk4, and cdk6, suggests
the existence of a feedback mechanism between p16 and Rb. 89
Decreased p16 is one of the most frequent alterations in lung
cancer. Interestingly, loss of p16 is usually found in NSCLC,
whereas loss of Rb is found in SCLC. These changes in p16 and
Rb seem to be mutually exclusive. 90–93 The strong inverse correlation
is more evident in SCC and SCLC than in ADC. 94,95
p14. p14 is an alternate transcription product from the
INK4/ARF locus shared with p16. 96–98 By its ability to antagonize
the MDM2-mediated ubiquitination and degradation
of p53, 99–102 it induces apoptosis 103,104 and growth inhibition.
99 p14 is decreased in 50% of NSCLC, 97,101 where it has
an inverse relationship with MDM-2 expression. 101 p14 can
be lost secondary to losses at the p16 locus, and also because of
the gene promoter methylation. 103,105
p53 TUMOR SUPPRESSOR. p53, mapped on chromosomic
region 17p13, is a tumor suppressor gene that, after sublethal
DNA damage, mediates cell cycle arrest in the G1 phase. 106,107
In this pathway, p53 interacts with several genes. The first one
is mdm2: Levels of p53 are kept low by its association with the
mdm2 oncogene product, which binds p53 and shuttles it out
of the nucleus for proteolytic degradation, establishing an autoregulated
feedback circuit. The second gene target is gadd45,
that belongs to a gene family implicated in cellular growth
arrest. 108,109 The third target is the gene coding for p21, which
develops inhibitory action on multiple cyclin–cdk complexes
and also complexes with PCNA, a protein playing a key role
in DNA reparation processes. 110 The fourth target is the gene
coding for Bax, which promotes apoptotic mechanisms and
forms a heterodimer with Bcl-2 gene. 111 As a key regulator of
cell growth and cell death, p53 is activated by several kinases
that regulate the DNA damage checkpoint following many
environmental stimuli, including DNA-damaging agents such
as ionizing radiation, and is crucial in preventing the propagation
of mutations in normal cells. 67 Activated p53 induces cell
cycle arrest (p21 Cip1/Waf1 ) to allow cells to repair the damage
or apoptosis if the damage is too severe and/or irreparable. 112
During carcinogenesis, p53 is frequently inactivated by multiple
mechanisms. The most common mechanism is mutation
at the p53 gene, which occurs in more than 50% of all human
cancers. In response to DNA damage, some p53 mutants show
less capacity to bind and initiate transcription from their target
genes, such as p21, Mdm2, Bax, and cyclin G, and so, some of
the p53-mediated effects are blunted, 113 resulting in insensitivity
to growth-inhibitory signals as well as evasion of apoptosis.
Mouse models have confirmed cooperation between mutated
p53 and mutative active K- ras in NSCLC development 114 and
between p53 and Rb in SCLC development. 115
p53 mutation is one of the most common genetic alterations
in lung cancer, with about 70% of SCLC and 50% of
NSCLC presenting mutations in one allele of p53, often accompanied
with loss of the wild-type allele. 116 In NSCLC,
p53 mutations are more frequently found in SCC than
ADC. 117 The p53 mutational spectra of lung cancer are different
from those of other cancers with an excess of G:C to
T:A observed, 118 these ones especially linked to exposure to
tobacco carcinogens such as benzopyrene. 119 Several studies
investigated the association of p53 abnormalities with prognosis
in lung cancer, with discordant results. Some of them
showed an association of p53 overexpression (mutants form
have a longer half-life and lead to the detection of high levels
of protein by IHC) with shorter survival, 120–122 others failed
to find such an association. 123,124 Concurrent p53 and p16
abnormalities have been associated to a worse prognosis. 125,126
Many reports have linked the presence of p53 mutations to resistance
to DNA-damaging chemotherapeutics agents, 127 and
this is not surprising as we know that most chemotherapeutics
agents act by stimulating apoptosis.
Cell Cycle as Target for Combined Treatment with
Radiotherapy Cellular radiosensitivity varies along the
different phases of cell cycle. The majority of cells surviving
after a first dose of radiation were most likely in a less sensitive
(G1 phase) or in a resistant phase of the cell cycle (late S phase).
Those cells will then progress into a more sensitive cell cycle phase,
that is at or close to mitosis, which represents a more ideal time for
delivering radiation therapy. Several cell cycle regulatory proteins
are potential molecular targets for cancer therapy, and agents can
be combined to radiation to enhance its effects on lung cancer.
Efforts have been made to sensitize cancer cells to the cytotoxicity
of DNA damage by anticancer agents as early as four decades ago
with compounds such as caffeine, which resulted in abrogation
of the G2 cell cycle checkpoint. Many malignant cells, including
lung cancer, have defective G1 checkpoint mechanisms and
depend far more on G2 checkpoint than normal cells. More recently,
several potent agents have been studied in preclinical and
clinical trials. Among them is flavopiridol, the first CKI to enter
clinical trials as a potential anticancer agent that exerts both cytostatic
and cytotoxic actions on cancer cells. Another well-known
drug is UCN-01, a potent prototypical chk1 inhibitor, which
function as a CKI at high concentrations. Such compounds seek
to force cells to enter mitosis without allowing adequate time for
DNA repair, increasing the likelihood that cell death will occur
by accumulation of DNA lesions. Treatment strategies consisting
in abrogation of G2 checkpoints can also be achieved with
the use of microtubule-targeting compounds, such as taxanes and
epothilones to stall cells in G2/M (by preventing mitosis, thereby
trapping cells in the phase of the cell cycle where ionizing radiation
have the greatest effects). Microtubule-stabilizing agents
will be discussed as mitotic targets in combination with ionizing
radiation. Finally, Aurora kinases, part of the spindle checkpoint,
are recent agents that can also be used in combined anticancer
therapy (Table 14.2).
Cyclin-Dependent Kinases (CDK) Modulators Cell
cycle regulatory proteins such as CDKs are potential molecular
targets for radiation therapy. 128–130 The rationale for targeting
the cell cycle and the CDKs in lung cancer therapy has been
based on the frequency of their perturbations in lung tumors
(overexpression of cyclins, and/or absent or diminished levels
of CKI). 54,55,131,132 For example, overexpression of cyclin D1
and loss of p16 gene expression has been associated with the
development of lung cancer (see previous discussion). These
defects in tumor cells lead to uncontrolled proliferation as a
result of the loss of checkpoint integrity. In addition, cell cycle
arrest by CKI has been shown to induce apoptosis. 1,133–135
FLAVOPIRIDOL. Flavopiridol is a semisynthetic flavone that
directly competes with the ATP substrate and reversibly inhibits
kinase activity of multiple classes of CDKs, including cdk1, cdk2,
cdk4, cdk6, cdk7, and cdk9 at submicromolar concentrations
(IC50 values of 100 to 400). 136 This pancyclin inhibitor causes
arrest at both G1 and G2/M phases of the cell cycle by several
mechanisms: direct inhibition of cdks 1, 2, and 4, and indirect
inhibition by downregulating cyclin H-cdk7, 137–140 as well as
tumor growth arrest in most solid tumors and xenografts. 140–144
Flavopiridol also promotes a decrease in the level of cyclin D1, 135
which is commonly overexpressed in many cancers including
lung cancer where it has been described as a poor prognosis
marker. 51–55,131,145–149 However, at considerably higher concentrations
than necessary to inhibit CDKs, flavopiridol inhibits the
activity of several other protein kinases 150,151 including signal
transducing kinases protein kinase A (PKA), PKC, and Erk-1,
the receptor tyrosine kinase EGFR, and receptor-associated protein
kinases such as c-Src. 152 In addition, although described as
cytostatic, flavopiridol has been shown to be also cytotoxic to
many lung cancer cell lines 143,144,153,154 by induction of apoptosis
except in the A549 lung carcinoma cell line. 154 Normally,
DNA-damaging agents induce p53, which in turn transcriptionally
induces p21 and Mdm-2, the later binds p53 and targets it
for degradation. 155 Although upregulating p53, flavopiridol was
shown to inhibit transcription of p21 and Mdm-2 and to inhibit
cell proliferation in A549 lung cancer cells. 153
Despite promising preclinical data, flavopiridol has not
demonstrated in trial significant clinical activity as a single
agent in patients with metastatic lung cancer. 129 The unexpected
and significant toxicity of this agent given as single cancer
therapy in all these clinical trials have so far discouraged its
use in monotherapy.
Another approach for the use of flavopiridol in anticancer
therapy is to take advantage of its potential to augment
cytotoxic actions of chemotherapeutic agents and radiation.
This strategy of combining flavopiridol with chemotherapy
has been investigated in several studies showing promising results.
Flavopiridol enhanced the cytotoxic effects of many chemotherapeutic
agents in vitro (12, 13, 14, 15, 16, and 17) as
well as in vivo (8, 13, and 15), in a sequence-dependent manner.
Similar results were observed in the combined approach
with ionizing radiation both in vitro, 156,157 and in vivo 130 in
various human cancer types. 156,158–160 Flavopiridol sensitized
human cancers to radiation in a dose-dependent manner, by
cell cycle redistribution, by inhibiting cellular repair from radiation
damage, and possibly by effects on angiogenic factors.
In addition, studies also demonstrated the effects of flavopiridol
in enhancing apoptosis and tumor regression. 160–163 The
therapeutic ratio of radiotherapy in the in vivo tumor models
was increased by flavopiridol. 130,156 More specifically, the radiosensitizing
action of flavopiridol was recently determined in
zebrafish embryos using cyclin D1 (CCND1) downregulation
by antisense hydroxylprolyl-phosphono peptide nucleic acid
oligomers compared to control. 164 This study demonstrated
that the specific sensitizing effects of flavopiridol in response
to radiation were in part caused by the inhibition of cyclin
D1, one of its primary pharmacologic targets. 158 Another
study analysed the sequence-dependent effects of flavopiridol
when combined to radiation and showed that the maximum
radiopotentiation and apoptosis were observed when the
lung cancer cells were treated with the sequence of docetaxel,
then radiation, and finally flavopiridol both in vitro and in
vivo. Therefore, the combined, sequence-dependent strategy
radiation/flavopiridol has the potential to enhance outcome in
many types of cancer and needs to be further investigated in a
well-designed clinical trial.
Chk1 Inhibitors
UCN-01. Staurosporine is a potent nonspecific protein
and tyrosine kinase inhibitor (TKI). UCN-01 or 7-
hydroxystaurosporine, an analogue of staurosporine, was
found to be a nonspecific inhibitor of CDKs, protein kinase
C (PKC) isoenzymes, and causes cell arrest in G 1 and
G 2 phases in different cell types. 165 UCN-01 is a checkpoint
protein kinase inhibitor that promotes cellular G 1 arrest by
inhibiting the activity of CDK-1 and CDK-2, thereby downregulating
cyclins and increasing the expression of CKI, p21.
Interestingly, UCN-01 abrogates the G2 checkpoint induced
by radiation, leading irradiated cells into early mitosis and
the onset of apoptotic death. 29,166 The mechanisms by which
the cells enter mitosis include activation of cdc2 kinase, and a
direct inhibiting effect on the G 2 -associated checkpoint protein
kinase 1 (Chk1). 167,168 UCN-01 inhibits the cdc25C regulatory
pathway and interferes with DNA damage-mediated
inhibition of cdc2-cyclin B1-kinase. It targets more strongly
Chkl than Chk2 (IC50 20 nM vs. 1 pM). 29,168,169 It has
been shown that Chk1 is required for the radiation-induced
G 2 checkpoint. 170,171 Chk1 may control the G 2 checkpoint
via phosphorylation-mediated negative regulation of Cdc25A,
Cdc25C, and Cdc25B. 170,172,173 Thus, the potential of Chk1
inhibitors would most likely be as sensitizer to other anticancer
treatments such as radiotherapy because inhibiting Chk kinases
forces the cell to enter mitosis with DNA damage accumulation.
Cancer cells could likely be particularly sensitive to such
treatment, since they commonly lack normal G 1 checkpoint
control and may rely more on the S and G 2 checkpoints compared
to normal cells. 174 In vitro, UCN-01 exhibits a potent
radiosensitivity effect in mutated p53 carrying NSCLC cell
models. 175,176 Despite encouraging preclinical results, combination
studies of UCN-01 with radiotherapy have not yet
been opened, whereas UCN-01 alone has undergone phase I
and II clinical trials in several cancer types. 177–179
Microtubule-Stabilizing Agents In all eukaryotic cells,
microtubules are essential components of the cytoskeleton,
and crucial in the successful process of mitosis and cell division,
via uniquely rapid dynamics in the mitotic spindle. 180
Microtubule-stabilizing agents, which interfere with microtubule
dynamics, have been particularly effective as anticancer
therapy and are a well-known strategy to induce G2/M
arrest. 181 These agents suppress cancer cell growth by promoting
accelerated assembly of excessively stable microtubules,
resulting in G2/M cell cycle arrest and cell death. The abrogation
of the G2 cell cycle checkpoint will lead to the forced
accumulation of tumor cells in the radiosensitive G2/M phase
of the cell cycle, where irradiation could achieve a higher
rate of unrepaired chromosomal aberrations during mitosis.
Additionally, antimicrotubule agents have the capacity to induce
apoptosis, mediated through inactivation of bcl-2 protooncogene,
even in p53-mutated and radioresistant cells. 182–184
It has been increasingly recognized that the combined use of
microtubule-stabilizing agents such as taxanes in combination
with radiotherapy is a clinically interesting strategy to improve
anticancer treatment. 185
TAXANES. Taxanes, such as paclitaxel and docetaxel, have
already been proved to have a potent anticancer activity against
many cancer types. 186 In addition, they are able to block cells
at the radiosensitive G2/M phase of the cell cycle, suggesting
the rationale for the use of taxanes to enhance radiotherapy. 185
Several studies showed the effectiveness of taxanes in enhancing
tumor radiosensitivity in vitro and increasing significantly
tumor growth delay in vivo. 187,188 Paclitaxel mechanism of
action, however, is not limited to G2/M accumulation and
results from direct cytotoxic effect in addition to the cell cycle
effects as well. Furthermore, taxanes showed significant clinical
efficacy for several tumor types, including non–small cell
lung, breast, and ovarian cancers. The specific molecular mechanisms
of taxanes antitumor activity in lung cancer have been
extensively reviewed. 189 The use of taxanes in cancer treatment,
however, is often limited by resulting toxicities, such as neutropenia
(which can be very severe), and peripheral neuropathy, 190,191
and by multidrug resistance (MDR). 192 This has lead to the
development of novel microtubule-stabilizing agents, such as
epothilones, with improved overall safety and broader antitumor
activity, particularly in MDR. 193,194
EPOTHILONES. Epothilones are natural products fungicidal
macrolides, originally isolated from the myxobacterium
Sorangium cellulosum . They were found to exert strong microtubule-
stabilizing effects similar to that of paclitaxel, even
though structurally unrelated to taxanes. 193,194 Epothilone B
(Patupilone, EP0906) and epothilone A (demethylated epothilone
B) are both natural products, whereas semisynthetic
epothilones were developed including ixabepilone (BMS-
247550, the lactam analog of epothilone B) and epothilone
D (KOS-862). Epothilone and its analogs can sensitize both
paclitaxel-sensitive as well as paclitaxel-resistant cells to ionizing
radiation at low concentration both in vitro and in vivo. 52,60–63
BMS-247550 has been characterized for its radiosensitizing potential
against human paclitaxel-sensitive lung carcinoma cells
showing additive apoptosis and enhanced tumor growth delay
when used in combination with ionizing radiation. 79 Similarly,
epothilone B sensitized P-glycoprotein–overexpressing p53-
mutated colon ADC to radiation in vitro and in vivo. 80 Kim
et al.195 reported the radiosensitizing effects of BMS-247550
in human lung carcinoma model. The study also demonstrated
that the enhancement was correlated with the percentage of
G2/M arrest induced by epothilone and with the induction of
apoptosis. These preclinical results support the concept of combining
epothilones with radiation to enhance lung cancer response
to therapy. To date, only a phase I study of epothilone B
administered concurrently with radiotherapy is ongoing for
cancer patients presenting advanced solid tumors or recurrent
disease for which there is no standard therapy or tumors have
failed standard therapy. Objectives of the trial are to evaluate
the safety and toxicity profile of epothilone B, and tumor response,
when combined to radiation therapy.
Aurora Kinase Inhibitors Aurora kinases represent a novel
family of serine/threonine kinases crucial for cell cycle control. In
mammals, there are three types of Aurora kinases: Aurora A, B,
and C. 196 Thus far, very limited data are reported on Aurora C,
which expression is limited to the testes. 197 Despite the fact that
they share 70% homology in their catalytic domain, Aurora
A and Aurora B have different subcellular localization and functions
during mitosis. 198 Aurora A is localized at the centrosome
during duplication and to the spindle poles in mitosis. Aurora A
kinase activity is regulated by TPX2, a protein required for spindle
assembly. Silencing of Aurora A leads to abnormal spindle
morphology in human cells. 199 Aurora A reaches the maximum
activity at the G 2-M transition. 200 At the G 2 -M transition, centrosome
separation requires functional Aurora A. 199 Aurora A
contributes also to transition from G 2 to M phase. Suppression
of Aurora A by RNA interference results in G 2 -M arrest of HeLa
cells and promotes apoptosis. 201 It was shown that ectopic expression
of Aurora A results in a bypass of the G 2 -M checkpoint
induced by DNA damage. 202 Inhibitors of Aurora kinases were
developed to take advantage of these targets to modulate the
abnormal cell cycle regulation for cancer therapy and tested in
cultured cells and xenograft models. 203,204
We and others reported on the effects of combining Aurora
kinase inhibition with ionizing radiation. 205–207 ZM447439 is
one of such Aurora A and B inhibitors, 208 but is known to produce
cellular phenotypes consistent with inhibition of Aurora B
rather than A, including the Aurora B–specific inhibition of
histone H3 phosphorylation on serine 10. 208,209 ZM447439
caused p53-dependent multinucleation, growth inhibition in
a colony-forming assay with a marked toxicity toward proliferating
cells, and increased the amount of apoptotic cells.
Interestingly, expression of Aurora B and survivin, two of the
proteins that constitute the chromosomal passenger complex,
was increased after irradiation of mesothelioma cells, consistent
with their known activity and levels peaking in G2/M that correspond
to the radiation-induced cell cycle arrest. 197 We found
that ZM447439 sensitized mesothelioma cells to irradiation,
and that the combination of both Aurora B and survivin inhibition
resulted in a deeper radiosensitization. 207 This effect
can be explained by the small molecule, ATP-binding nature
of ZM447439, which can disrupt cell division but not the
binding to survivin and thus the localization to centromere.
Tao et al. 205 demonstrated that inhibition of Aurora A either
by PHA680632 or by small interfering RNA (siRNA) against
Aurora A enhanced cell killing after exposure to radiation in
vitro. Moreover, they also showed that PHA680632 alone were
able to induce a marked tumor growth inhibition in vivo and
that irradiation treatment combined with PHA680632 led
to an additive tumor growth inhibition compared with each
agent alone, especially in p53-deficient cells. Although it did
not act as radiosensitizer, this study showed the potential of
PHA680632 when used in combined-modality strategy and
can serve as proof of concept for Aurora A targeting with ionizing
radiation. More encouraging results were very recently
reported using VX-680 with radiation. 206 VX-680, also known
as MK-0457, targets the ATP-binding site of Aurora kinases
for reversible inhibition, and was proven to greatly inhibit
tumor growth in three xenograft models in vivo, including leukemia,
colon, and pancreatic tumors. 210 In a laryngeal SCC in
vitro model, VX-680 upregulated p53 and effectively sensitized
tumor cells to radiotherapy, whereas overexpression of Aurora
A generated radioresistance. 206 Taken together, these promising
preliminary results suggest the possibility to use Aurora
kinases as a target for radiation therapy.
mTOR Inhibitors The mammalian target of rapamycin
(mTOR), a serine/threonine kinase, is a downstream mediator in
the phosphatidylinositol 3-kinase/Akt signaling pathway, which
plays a critical role in regulating basic cellular functions including
cellular growth and proliferation. 211 The PI3K/Akt/mTOR signaling
pathway is altered in many cancer types, including lung. 212
Rapamycin (sirolimus), and its improved analogs (rapalogs: CCI-
779 - temsirolimus, RAD001 - everolimus, and AP23573 - deforolimus),
the main mTOR inhibitors used in studies have shown
G1-phase arrest 213 and significant antitumor activity in various
radiation models. mTOR inhibition combined with radiation
showed decreased tumor vascular density in murine models and
sensitized vascular endothelium. 214 Moreover, RAD001 has
been shown to increase the radiosensitivity of breast 215 and prostate
216 cancer cells, mainly via induction of autophagy cell death.
Phosphatase and tensin homolog (PTEN)-deficient prostate cancer
cells were even more sensitive to ionizing radiation as compared
to PTEN-efficient cancer cells, 216 suggesting important
clinical implications given the frequent presence of PTEN deletions
in many types of cancer. 217–219 Interestingly, mTOR inhibitors
seems to be more effective in tumor cells than in normal
cells, possibly because transformed cells have increased activation
of the Akt/mTOR pathway. 220 RAD001 has also been shown to
further enhance the radiosensitization obtained by inhibition of
apoptosis in either lung or breast cancer cells, again by increased
autophagy. 221,222 These findings suggest potential autophagy targets
to enhance efficacy of therapeutic strategies using mTOR
inhibitors. 223 Currently, mTOR inhibitors are being evaluated in
multiple clinical trials. In phase I and II trials, mTOR inhibitors
appear to be well tolerated, with most common and transient
adverse events being skin reactions, stomatitis, myelosuppression,
and metabolic abnormalities. 224–230
Interestingly, recent data suggest a certain antitumor activity,
such as tumor regressions and prolonged stable disease,
which has been reported among patients with various malignancies,
including NSCLC. Based on these encouraging data,
a phase I/II study of RAD001 and radiation therapy in patients
with brain metastasis from NSCLC is currently ongoing
at Vanderbilt University.