Therapeutic interactions of autophagy with radiation and temozolomide in glioblastoma: Evidence and issues to resolve

Abstract

Glioblastoma is a unique model of non-metastasising disease that kills the vast majority of patients through local growth, despite surgery and local irradiation. Glioblastoma cells are resistant to apoptotic stimuli, and their death occurs through autophagy. This review aims to critically present our knowledge regarding the autophagic response of glioblastoma cells to radiation and temozolomide (TMZ) and to delineate eventual research directions to follow, in the quest of improving the curability of this incurable, as yet, disease. Radiation and TMZ interfere with the autophagic machinery, but whether cell response is driven to autophagy flux acceleration or blockage is disputable and may depend on both cell individuality and radiotherapy fractionation or TMZ schedules. Potent agents that block autophagy at an early phase of initiation or at a late phase of autolysosomal fusion are available aside to agents that induce functional autophagy, or even demethylating agents that may unblock the function of autophagy-initiating genes in a subset of tumours. All these create a maze, which if properly investigated can open new insights for the application of novel radio- and chemosensitising policies, exploiting the autophagic pathways that glioblastomas use to escape death.British Journal of Cancer advance online publication, 18 February 2016; doi:10.1038/bjc.2016.19 www.bjcancer.com.

Full text

Therapeutic interactions of autophagy with
radiation and temozolomide in glioblastoma:
evidence and issues to resolve
Michael I Koukourakis*
,1
, Achilleas G Mitrakas
1
and Alexandra Giatromanolaki
2
1
Department of Radiotherapy/Oncology, Democritus University of Thrace, PO Box 12, Alexandroupolis 68100, Greece and
2
Department of Pathology, Democritus University of Thrace, Alexandroupolis 68100, Greece
Glioblastoma is a unique model of non-metastasising disease that kills the vast majority of patients through local growth, despite
surgery and local irradiation. Glioblastoma cells are resistant to apoptotic stimuli, and their death occurs through autophagy.
This review aims to critically present our knowledge regarding the autophagic response of glioblastoma cells to radiation and
temozolomide (TMZ) and to delineate eventual research directions to follow, in the quest of improving the curability of this
incurable, as yet, disease. Radiation and TMZ interfere with the autophagic machinery, but whether cell response is driven to
autophagy flux acceleration or blockage is disputable and may depend on both cell individuality and radiotherapy fractionation or
TMZ schedules. Potent agents that block autophagy at an early phase of initiation or at a late phase of autolysosomal fusion are
available aside to agents that induce functional autophagy, or even demethylating agents that may unblock the function of
autophagy-initiating genes in a subset of tumours. All these create a maze, which if properly investigated can open new insights
for the application of novel radio- and chemosensitising policies, exploiting the autophagic pathways that glioblastomas use to
escape death.
Glioblastoma is the most common primary brain tumour in adults,
and certainly one of the most lethal human tumours (Hayat et al,
2007) Its incidence is about 3 new cases per 100 000 population per
year, the median age of diagnosis is 64 years and most of the
patients have passed away 2 years following diagnosis (Ohgaki
et al, 2004). Unlike low-grade gliomas, complete surgical removal
of glioblastoma is impossible because of the infiltrative nature of
the disease and the functional vulnerability of the brain. Post-
operative radiotherapy is the standard adjuvant therapy offered
worldwide. Following surgery alone, the median survival is 6
months and increases to 12 months with the addition of
radiotherapy (Salazar et al, 1979). Because of the extent and the
location of the disease adjacent to critical brain areas or because of
the poor performance status of patients at diagnosis, surgery is
often omitted and radiation is offered as the main treatment
option.
Attempts to improve the efficacy of radiotherapy with the
addition of chemotherapy, mainly nitrosoureas (BCNU, CCNU),
go back to the early 1970s. In 1983, Eyre et al in a randomised trial
failed to show a benefit from the addition of CCNU and
procarbazine to radiotherapy. In the same year, an RTOG study
provided evidence that radiochemotherapy with BCNU or methyl-
CCNU or DTIC was superior to radiotherapy alone (Chang et al,
1983). The benefit, however, provided by nitrosoureas was limited
and questionable as stressed in subsequent randomised trials
(Hatlevoll et al, 1985; Deutsch et al, 1989; Shapiro et al, 1989;
Curran et al, 1992). The only therapeutic innovation that had an
impact in the management of glioblastomas appeared in 2005,
when a randomized study showed that temozolomide improved
the median survival of patients undergoing radiotherapy by 2.6
months (Stupp et al, 2005). In a more recent analysis of the
EORTC-NCIC trial on 573 patients, the 2-year survival was 27.2%
in the temozolomide group vs 10.9% in the radiotherapy-alone
group (Stupp, 2009). Nowadays, postoperative radiotherapy with
daily administration of TMZ is the gold standard for patients with
glioblastomas, although the efficacy of this therapy remains
disappointing and attempts to improve the median survival using
the combination of TMZ with other drugs, such as motexafin
*Correspondence: Professor MI Koukourakis; E-mail: targ@her.forthnet.gr
Received 21 September 2015; revised 21 December 2015; accepted 31 December 2015
&2016 Cancer Research UK. All rights reserved 0007 – 0920/16
MINIREVIEW
Keywords: glioblastoma; autophagy; radiotherapy; temozolomide
British Journal of Cancer (2016), 1–12 | doi: 10.1038/bjc.2016.19
www.bjcancer.com | DOI:10.1038/bjc.2016.19 1
Advance Online Publication: 18 February 2016

gadolinium (Brachman et al, 2015), has failed, with the 5-year
survival remaining o10%.
In the era of targeted therapies, there is an increasing optimism
that the identification of key molecular pathways may lead to
major improvements in the management of this highly lethal
disease. Glioblastomas, being highly angiogenic tumours, were
thought to be susceptible to antiangiogenic therapy. Indeed, in
2009, bevacizumab (BVZ), an anti-VEGF humanised monoclonal
antibody, received accelerated approval by the FDA for the
treatment of recurrent glioblastoma following a randomised phase
II study (Kreisl et al, 2009). In the most recent randomised RTOG
0825 trial, however, the addition of BVZ (10 mg kg
1
every 2
weeks) to standard chemoradiation improved the progression-free
interval but had no effect on overall survival (Gilbert et al, 2013).
Recent studies on newly diagnosed glioblastoma patients showed
that the same results and sub-population of patients with better
response to BVZ treatment could not be predicted by the
molecular data (Poulsen et al, 2014). The benefit from BVZ
therapy seems to be confined to oedema reduction and a cut down
on the usage of corticosteroids (Thomas and Omuro, 2014).
Epidermal growth factor receptor-mediated oncogenic signal-
ling is active in most human carcinomas and has become a target
for therapeutic interventions. Indeed, anti-EGFR1 moAb and
pharmacological tyrosine kinase inhibitors have shown important
clinical activities in the colon, lung and head and neck cancer.
About half of glioblastomas bear EGFR gene amplification and
receptor overexpression (Hatanpaa et al, 2010). Mutation of EGFR
with constitutive activation of the pathway is also quite common.
Although the efficacy of such agents as single therapy is limited
(van den Bent et al, 2009), randomised trials in combination with
radiotherapy are missing.
The unique nature of the glioblastoma cells to be resistant to
apoptotic stimuli, their death occurring rather through autophagy
(Yao et al, 2003; Kanzawa et al, 2004), suggests that autophagy-
interfering agents in combination with radiation and temozolo-
mide may represent a new strategy to test. This hypothesis
becomes much more relevant under the new light shed by studies,
suggesting that autophagy is the main pathway exploited by cancer
stem cells to survive and accelerate their renewal (Jiang et al, 2007;
Gong et al, 2013; Berardi et al, 2015). Glioma stem cells is
considered the most radioresistant cell population and autophagy
targeting may assist to eradicate the disease (Shen et al, 2015;
Yu et al, 2015). This review aims to critically present our
knowledge regarding the autophagic response of glioblastoma cells
to radiation and TMZ and to delineate eventual research directions
to follow in the quest of improving the curability of this incurable,
as yet, disease.
AUTOPHAGY AND AUTOPHAGIC DEATH
Macroautophagy (herein stated as autophagy for simplicity) is a
major cell pathway dedicated to a continuous renovation of cell
constituents through the degradation and recycling of long-lived or
damaged proteins and even of entire organelles, such as ribosomes,
mitochondria, endoplasmic reticulum and Golgi apparatus
(Klionsky and Emr, 2000; Feng et al, 2014). During autophagy
double-membrane vesicles are formed, which sequester portions of
the cytoplasm and fuse with endosomal and lysosomal vesicles
(Yorimitsu and Klionsky, 2005). Beclin 1 and ULK (UNC-51-like
kinases) proteins are important autophagosome initiation proteins,
whereas LC3A, LC3B and LC3C are key structural components of
the autophagosomes, which are extensively used for the experi-
mental study of autophagy (Kabeya et al, 2000; Wong et al, 2013),
whereas the difference in expression levels and of subcellular
distribution and kinetics of these proteins may be an indication of
different roles during autophagy (Giatromanolaki et al, 2014;
Koukourakis et al, 2015).
Autophagy-related protein 8 (Atg8/LC3), an ubiquitin-like
protein, has different homologues encoded by different chromo-
somes in animals, including MAP1LC3A, B and C, as well as the
GABARAP proteins, which are the most important constituents of
the autophagosomal membranes (Shpilka et al, 2011). Most of
them are produced as a pre-LC3 25–30 kDa protein that are
cleaved by the autophagophagin (Atg4) to the soluble form of LC3,
the 18 kDa LC3-I protein. Following an ubiquitination-like
reaction, the exposed C terminus conjugates to the head group
amine of phosphatidylethanolamine (PE) through an amide bond,
becoming the so-called membrane-bound 16 kDa LC3-II form that
forms both the outer and inner autophagosome membranes. The
p62/sequestrosome protein is the main carrier of the waste material
to the LC3-II membranes that engulf the complex within a mature
autophagosome (Katsuragi et al, 2015). This is followed by fusion
with lysosomes and digestion of the autophagolysosomal content,
resulting in the disintegration of intracellular waste and at the same
time providing energy to the cell. During this process, LC3-II
protein can be delipidated by Atg4 proteases and liberated in the
cytoplasm. The different forms of LC3 proteins and the kinetics of
the p62 protein can be detected by western blot or confocal
microscopy methods, providing important tools to study the
autolysosomal flux in normal cells and cancer cells under various
stresses (Klionsky et al, 2012).
Under energy-demanding stress conditions, such as starvation
or hypoxia, autophagy is accelerated (Koritzinsky and Wouters,
2013; Bernard et al, 2015). On the one hand, excessive
intensification of this very process may lead to intolerable
cytoplasmic digestion, leading to cell exhaustion and death. On
the other hand, accumulation of autophagosomes under stress,
which block their fusion with lysosomes (e.g., hydroxychloroquine
or 3-methyadenine), may also lead to autophagic death (Kimura
et al, 2013; Saetre et al, 2015). Autophagy is called type II
programmed cell death pathway, which is a distinct pathway
compared with the apoptotic type I death pathway triggered by
DNA or membrane damage followed by release of caspases from
the mitochondria. Autophagy is, therefore, a balance that weighs
between renovation, energy acquisition, survival and cell death,
according to the external or internal stimuli received.
Cancer cells have an increased autophagic activity as shown in a
series of clinicopathological studies, where LC3 and autophagy-
initiating proteins are overexpressed, a feature linked with
aggressive clinical behaviour (Sivridis et al, 2010; Giatromanolaki
et al, 2014; Koukourakis et al, 2015). In a recent study, we noted
that overexpression of LC3A in the form of stone-like cytoplasmic
structures was linked to poor postradiotherapy prognosis in a
series of patients with glioblastoma (Mehta et al, 2015). Since
several decades we know that an inert apoptotic machinery is a
very common phenomenon in cancer cells, allowing them to
escape death after exposure to cytotoxic chemotherapy and
radiotherapy (Hickman, 1996). Evasion from apoptosis is a well-
established phenomenon in gliomas and glioblastomas (Krakstad
and Chekenya, 2010). Therefore, autophagic death is an alternative
pathway to pursue in an attempt to eradicate glioma cells exposed
to cytotoxic stress (Zois and Koukourakis, 2009).
RADIOTHERAPY AND AUTOPHAGY IN GLIOMAS/
GLIOBLASTOMAS
Several experimental studies have confirmed that glioma and
glioblastoma cells exhibit important resistance to apoptosis after
exposure to ionising radiation. Expression status of apoptosis-
related genes such as PTEN may be involved in the phenomenon
(Lee et al, 2011), and interference with various signaling pathways
BRITISH JOURNAL OF CANCER Autophagy, radiation and temozolomide
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such as STAT3 and EGFR may restore the ability for apoptotic
death (Gao et al, 2010).
The study by Yao et al (2003) is probably the first to show that
irradiation of human glioblastoma cells results in enhanced
autophagy, whereas no apoptosis was evident in radiosensitive or
radioresistant cell lines. Ito et al (2005) subsequently confirmed
that ionising radiation induces cell cycle arrest and autophagic
death, but not apoptosis, in glioma cell lines. Moreover, Jo et al
(2014) suggested that autophagy is a pathway to cell death after
glioma irradiation. In 2005, a study from the MD Anderson Cancer
Center showed that DNA-protein kinase (DNA-PK)- (enzyme
involved in the repair of DNA double-strand breaks) deficient
glioma cells suffered massive autophagic death even after low doses
of radiation (Daido et al, 2005). Intact DNA-PK pathway
prevented autophagic death, but cells still exhibited a low apoptotic
tendency. Of interest, induction of autophagy using siRNA against
DNA-PK sensitised glioblastoma cell lines (Zhuang et al, 2011a, b).
An additional study using particle radiation, which mainly kills
through DNA double-strand damage, supports the suggestion that
DNA repair disabling is a potent stimulus not only for apoptotic
death but also for autophagic death in gliomas (Jinno-Oue et al,
2010).
The Akt/m-TOR is recognised as a major pathway regulating
autophagy. Inhibitors of Akt/m-TOR activity, such as rapamycin
analogues, intensify the autophagic process (Martelli et al, 2011).
This pathway is often upregulated in tumours, including glioma
(Akhavan et al, 2010). Akt inhibitors induce autophagic death, but
not apoptotic death, in both radioresistant and radiosensitive U87
glioma cell lines and enhance sensitivity to radiation (Fujiwara
et al, 2007). In the same cell line, Benzina et al (2008) found that
high LET radiation kills cells through autophagy. Mehta et al
(2015) further confirmed that Akt inhibition radiosensitises
primary human glioblastoma stem-like cells. Moreover, silencing
of EGFR, an activator of PI3K/Akt/mTOR pathway, with
simultaneous induction of autophagy led to better response to IR
and suppressed migration in the T98G cell line (Palumbo et al,
2014). These results are further supported by Gini et al (2013), who
showed that the growth of EGFRvIII-activated glioblastoma was
blocked after treatment with CC214-1 and CC214-2, which are
inhibitors of mTORC1 and mTORC2. The study by Chiu et al
(2009) suggested that arsenic trioxide also enhances the activity of
radiation in glioma cell lines by augmenting the autophagic cell
death, which is also supported by Carmignani et al (2014), who
showed that glioblastoma stem cells differentiated into non-
tumourigenic cells as a result of autophagy induction, after
inhibition of PI3K/Akt and stimulation of mitogen-activated
protein kinase pathway using arsenic trioxide and metformin,
respectively. Rapamycin has also been shown to induce differ-
entiation of glioma-initiating cells and increase their radio-
sensitivity by activating autophagy (Zhuang et al, 2011a, b).
Furthermore, research related to NF-kB, a factor that suppresses
autophagy in response to TNFa (Djavaheri-Mergny et al, 2007),
showed that treatment of glioma cells with pitavastatin (inhibitor
of NF-kB) resulted in autophagic death but not apoptotic death,
and empowered the activity of radiation (Tsuboi et al, 2009).
Nuclear factor-kB is a transcription factor negatively regulating
apoptotic death. Early phase blockers of autophagy, such as 3-
methyladenine (3-MA), counteracted the cytotoxic effect of
radiation and of the combination with pitavastatin.
A variety of researches focused on glioma radiosensitisation via
autophagy suppression. Yuan et al (2015) exhibited that suppres-
sion of ATG5 using siRNA or suppression of autophagy using 3-
methyladenine increased the radiosensitisation effect of gliomas
after STAT3 inhibition. Moreover, according to Ye et al (2013), the
resistant clones of glioma stem cells bear high expression levels of
early growth response 1 that induce autophagy. Additionally,
mitochondrial isoenzyme of NADP
þ
-dependent isocitrate
dehydrogenase siRNA-transfected A172 glioma cells were sensi-
tised after inhibition of autophagy (Kim et al, 2013).
There is, therefore, important evidence that in glioma cells
autophagic death is the most common pathway exploited by
radiation compared with apoptosis. Although reactive autophagy
may occur in the context of a cell survival response to radiation,
which may represent a radioresistance pathway, augmenting the
autophagic response through combination with Akt/mTOR
inhibitors would shift the balance to death. DNA repair enzyme
inhibitors seem also to facilitate radiation-induced autophagic
death, and at the same time restoring to a certain extent the
apoptotic pathway. The fact that early phase autophagy inhibitors
also sensitise glioma cells to radiation (Lamonaco et al, 2009) is not
contradictory to the whole concept, as abrogating low-level
autophagy that keeps the balance to the survival side is expected
to increase vulnerability to radiation. Late phase autophagy
inhibitors that block autophagolysosomal formation are also
expected to be radiosensitisers as they abrogate cytoprotective
autophagy and load the cell with waste material, resulting in
autophagy-mediated death.
TEMOZOLOMIDE AND AUTOPHAGY IN GLIOMAS/
GLIOBLASTOMAS
One of the first studies regarding the role of autophagy in
malignant gliomas showed that TMZ at clinically relevant
concentration of 100 mM induces autophagy rather than apoptosis
in malignant glioma cells, as shown by the accumulation of the
LC3 protein on autophagosomal membranes. Lee et al (2015)
suggest that TMZ in combination with chloroquine could inhibit
the growth and apoptosis of glioblastomas, whereas autophagy
suppression leads to the abolishment of the combination effect of
TMZ and chloroquine (Lee et al, 2015). Moreover, blockage of
autophagy at an early step by 3-MA prevented the accumulation of
autophagosomes and suppressed the cytotoxic effect of TMZ.
Zanotto-Filho et al (2015), however, suggested that autophagy has
a protective role in gliomas as TMZ/curcumin treatment in
combination with 3-MA leads to a reduction of cell viability.
Nevertheless, bafilomycin that blocks autophagy at a late step, by
preventing the fusion of mature autophagosomes with lysosomes,
sensitised glioma cells to TMZ by inducing apoptotic rather than
autophagic death.
These studies suggest that autophagy and drug interaction is a
very complex process. Blockage of autophagosome formation
abrogates the autophagic death induced by TMZ. On the contrary,
non-functional accumulation of autophagosomes (induced by
TMZ and blocked by late phase autophagy drugs) releases an
apparent pre-existing obstacle in the apoptotic pathway, driving
cells to apoptotic death. Lefranc and Kiss (2006) suggested that as
glioblastoma cells are resistant to apoptosis, blocking targets that
keep autophagy suppressed, such as mTOR, may enhance the
activity of TMZ-induced autophagic death (Zhivotovsky et al,
1999).
An important link between TMZ-induced autophagic death and
cell metabolism has been subsequently brought forward by
Katayama et al (2007). In experiments in multiple glioma cell
lines, TMZ consistently induced autophagy in parallel with an
increase of ATP production. This ATP surge could not be blocked
by glucose starvation but was blocked by agents blocking early
steps of autophagy (methyladenine and beclin 1 siRNA). As this
inhibition resulted in micronucleation, it was suggested that
autophagy-induced ATP surge counteracts the TMZ-induced
autophagic death. Indeed, administration of pyruvate abrogated
the activity of early phase autophagy inhibitors and enhanced
glioma cell survival. Small molecules, such as dasatinib, which
antagonise the ATP binding pocket of abl, kit or EGFR proteins,
Autophagy, radiation and temozolomide BRITISH JOURNAL OF CANCER
www.bjcancer.com | DOI:10.1038/bjc.2016.19 3

induce autophagic cell death in glioblastoma cells and have
synergistic effect with TMZ (Milano et al, 2009). Moreover, the
association between metabolism and mitochondria with the
efficacy of TMZ is supported by a several recent studies. You
et al (2013) showed that the expression of ATAD3A, a
mitochondrial protein ATPase (ATAD3A), is a prognostic factor
for glioblastomas and a predictor of TMZ or radiation resistance
(You et al, 2013). Another study that supports the hypothesis of
association between the function of mitochondria and TMZ
showed that metformin, which inhibits mitochondrial electron
transport chain complex I, enhances the cell cytotoxicity of TMZ
(Sesen et al, 2015).
It seems, therefore, that although exacerbation of autophagy is a
death pathway exploited by TMZ, the energy released by this very
process counteracts TMZ efficacy. Overactivity of the autophagic
pathway in the context of a cell-cytoprotective mechanism against
TMZ can be at the same time the cause of temozolomide-induced
cell death. Abrogation of the end point of such an autophagic-
protective mechanism, which is energy production, inevitably shifts
the balance to increased autophagic death. In pathological studies,
the majority of glioblastoma cases examined had an upregulated
autophagic pathway at diagnosis (Aoki et al, 2008; Pirtoli et al,
2009), with overexpression of LC3, beclin 1 and ULK proteins
(Giatromanolaki et al, 2014), presumably as an indispensable
pathway for energy acquisition under the stressful metabolic
demands of an accelerated growth. Any stressor, like TMZ, that
further accelerates autophagy may result in autophagic death by
disrupting a delicate balance between energy replenishment and
suicide autophagy. The synergism found between TMZ-induced
autophagy and autophagy-mediated killing effect of oncolytic
adenoviruses (Yokoyama et al, 2008; Ulasov et al, 2009) may also
be explained under this point of view.
An interesting observation from the clinicopathological studies
is that a glioblastoma group with low levels of LC3 and beclin 1 has
a poorer prognosis and may be more resistant to TMZ, although
this hypothesis demands further confirmation in larger studies.
A study by Fu et al (2009) provides important insights into
this phenomenon. Following separation of CD133-positive
and -negative glioblastoma cells from a freshly resected tumour,
positive cells (representing glioblastoma stem cells) had lower
LC3-II and beclin 1 levels compared with negative ones and were
more resistant to TMZ (Fu et al, 2009). Both cells, however, were
resistant to caspase-3 induction following incubation with TMZ.
An important hypothesis that emerges from these studies is that,
although autophagic death is a main pathway of glioma cell death
after exposure to TMZ, glioma stem cells may have an autophagic
machinery refractory to the activity of the drug and, therefore,
more easily survive under exposure to TMZ, leading subsequently
to tumour repopulation and growth.
The above data contrast the study by Lomonaco et al (2009)
where CD133-positive cells seem to upregulate autophagy more
potently than negative cells after exposure to radiation and to the
finding that autophagy inhibition sensitises the stem cell sub-
population to radiation. Similar results have been reported by
Winardi et al (2014), who supports that high level of CD133
population and high rate of autophagy lead to a poor prognosis in
astrocytomas (Winardi et al, 2014). Although further studies are
demanded to elucidate this discrepancy that may be cancer cell line-
dependent, differences in the mechanism of autophagy induction
between radiation and TMZ can also underlie. In addition, it is
stressed that for currently applied experimental techniques, it is
difficult to distinguish between autophagosomal or autolysosomal
non-functional accumulation vs intensified autophagy. Moreover, as
LC3A and LC3B are distinct proteins with different response at
various stimuli, studying the autophagic response with nonspecific
LC3 subtype markers may provide confusing results and mask
specific response patterns that may be important to TMZ activity.
In any case, restoration or protection of the autophagic flux may
be important in sensitising cells to TMZ. Methylation of the
promoter of Beclin 1 gene has been reported to be a common event
in breast cancer (Li et al, 2010). Whether hypomethylating
agents can be of value to restore autophagic responsiveness in
glioma stem cells and sensitivity to TMZ is a sound hypothesis.
5-Aza-20-deoxycytidine, a potent demethylating agent, is an
autophagy promoter (Chen et al, 2011). Although it is unknown
whether demethylating agents can restore autophagy in glioblas-
tomas, we know that they restore the expression of genes involved
in apoptosis (Eramo et al, 2005) like caspase-8, which is
hypermethylated in stem cell-like glioma cells (Capper et al,
2009). However, differentiation-inducing agent inhibitors of
histone deacetylase, such as valproic acid (Park et al, 2011), are
potent inducers of autophagy in glioma cells, enhancing auto-
phagic cell death but not apoptosis (Fu et al, 2010). Whether such
agents can target TMZ-resistant glioma stem cells is another
hypothesis to test. Carbamazepine is also another drug that
increases the autophagic flux and protects normal tissues against
radiation (Kim et al, 2011), presumably due to restoration of the
radiation-induced autophagy blockage (Zois et al, 2011).
RESTORING APOPTOSIS
Another important approach aside to autophagy death pathway
manipulation is to interfere with the molecular pathways aiming to
overcome the relative resistance of glioma cells to apoptotic
death. Both radiotherapy and TMZ are DNA-damaging agents.
Radiotherapy induces both DNA single- and double-strand breaks,
the kinetics and fidelity of repair of which define cell survival and
clonogenic ability (Mirzayans et al, 2013; Morgan and Lawrence,
2015). Whether apoptosis, mitotic catastrophe or senescence
follows defective DNA repair is an outcome defined, at least
partially, by the oncogene and apoptosis-related gene activity in
cancer cells (Vakifahmetoglu et al, 2008; Mirzayans et al, 2013).
Gene therapy or small molecules restoring p53 function increase
the radiation-induced apoptosis in cancer cells, as well as in
glioblastomas (Villalonga-Planells et al, 2011; Pflaum et al, 2014).
Temozolomide is also a DNA-damaging agent. Following its
conversion to 5-aminoimidazole-4-carboxamide, it delivers methyl
groups to DNA, resulting in the formation of O
6
-methylguanine,
which mispairs erroneously with thymine (instead of cytosine)
during DNA replication, resulting in DNA double-strand break
formation (Fukushima et al, 2009). The death pathway that follows
depends on the gene expression profile of the cancer cell, the p53,
PUMA and bcl-2 family of protein expression status being decisive
for the activation of the apoptosis pathway under exposure to
temozlomide, even of glioma stem cells (Gratas et al, 2014; Miao
et al, 2015).
Gene therapy or novel therapeutic agents that restore the
apoptotic machinery in gliomas may prove to be of importance in
reactivating apoptotic death induced by both ionising radiation
and TMZ. Inhibitors of the PDGFR seem to initiate the apoptotic
pathway in apoptosis-resistant glioblastoma cells (Ziegler et al,
2008). Anti-EGFR therapy has been reported to sensitise CD133-
positive glioma cells to radiation, presumably by restoring their
apoptotic ability (Diaz Miqueli et al, 2009). Specific inhibitors of
the checkpoint kinases Chk1 and Chk2 reverse the radioresistane
of these cells (Bao et al, 2006). Drugs targeting the mitochondrial
pore, such as lonidamine, have been shown to induce apoptotic
death in TMZ-resistant glioma cells (Lena et al, 2009). Chang et al
(2009) found that Sirtuin 1 gene is exclusively expressed in CD133-
positive radioresistant stem cells and that silencing this gene
improves curability in experimental models. Agonist antibodies
of the TRAIL death receptor 5 that induce apoptosis are
BRITISH JOURNAL OF CANCER Autophagy, radiation and temozolomide
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also shown to improve the efficacy of radiotherapy and TMZ
(Fiveash et al, 2008). Gene therapy approaches, such as introduc-
tion of the caspase-8 gene or IL-24 gene, may be also useful in
restoring apoptosis of glioma cells (Tsurushima et al, 2008; Yacoub
et al, 2008).
WHICH AUTOPHAGIC DEATH?
The above data suggest that glioma and glioblastoma cells exhibit
an autophagic response after exposure to ionising radiation and
TMZ. This assumption is based on the increased accumulation of
LC3-positive autophagosomes. Such a finding, however, does not
necessarily mean an intensified ‘functional autophagy’. In fact,
disruption of the lysosomal and autophagosomal fusion may also
result in LC3-positive autophagosomal accumulation, without
intensification of the production of new autophagosomes. This is
an important point we need to clarify regarding the actual effect of
ionising radiation on the autophagic machinery. Autophagic flux
studies in cancer cells are demanded to elucidate the phenomenon,
and such studies are absent in the literature. Our studies in normal
fibroblasts and endothelial cells suggest that radiation results in an
early blockage of the autophagic flux within the first days of
irradiation (Kalamida et al, 2015). The increase of LC3-II
membrane-bound form in the soluble fraction of cells instead of
the pellet fraction and the sharp increase of p62 protein (that
normally disintegrates in the autophagolysosomal environment
once incorporated) strongly suggest that radiation induces a non-
functional accumulation of autophagosomes in cells, inducing
autophagic death in a similar way that late phase autophagy
blockers (such as chloroquine and bafilomycin) do. In fact,
restoration of autopagic flux protects normal cells from radiation
death.
Whether this phenomenon also applies in cancer cells or
whether these sustain a resistant attitude against radiation-induced
autophagy flux blockage demands thorough investigation. Pre-
liminary experiments show that the response of cancer cells seems
to be cell line- and radiation dose-specific (unpublished data).
A biphasic relation linking radiation dose levels and the autophagic
survival/death balance in cancer cells cannot be excluded. Lower
doses, especially in radioresistant cells, could trigger a functional
autophagic response with a rather cytoprotective role providing
energy to the cells. In contrast, higher doses may disrupt the
autophagic process by downregulating autophagy gene expression
and/or by blocking autophagosome/lysosomal interaction shifting
the balance to an autophagic death because of an accumulation of
waste. This, however, is nothing more than a hypothesis that
should be thoroughly examined, as different fractionation of
radiotherapy may have a different effect on autophagy and may
synergise better with different autophagy-interfering agents.
Nevertheless, in a recent study in prostate cancer we provided a
strong evidence that such a hypothesis is valid, as a radioresistant
cell line intensified its autophagic flux after low-dose radiation,
whereas this was blocked in a radiosensitive one using the same
radiation dose (Koukourakis et al, 2015). It may be, therefore, that
radioresistant tumours continue or accelerate their autophagic flux
when exposed to low dose per fraction (i.e. within the range of
standard 2 Gy per day fractionation), whereas larger fractions
(hypofractionation) may block the autophagic flux and at least
partially overcome radioresistance. Silencing of the LC3A gene
results in important radiosensitisation, suggesting that autophagy
is eventually a pathway of survival following irradiation of
radioresistant cells and tumours (Koukourakis et al, 2015).
Nowadays, it is more than clear that autophagy is overactive in a
large fraction of tumours, including gliomas (Capper et al, 2009),
and cell death features representative of an autophagic exhaustion
of the cytoplasmic material are commonly evident in the form of
stone-like cytoplasmic structures. Indeed, we identified the
formation of the so-called LC3A-positive ‘stone-like structures’ in
various cancer tissues, showing a complete elimination of the
cytoplasm of cells and its substitution by one or more dense
structures of amorphous undigested LC3-positive material (Sivridis
et al, 2010). This feature, also evident in human glioblastomas, was
inducible under acidic conditions and glucose antagonism,
showing the existence of this type of autophagic cell death in
glioblastomas (Capper et al, 2009). This autophagic death path
presumably demands functional and overactivated autophagy to
uncontrollably consume the cytoplasmic content, shifting the
balance from autophagic cytoprotection to autophagic death. Thus,
as cancer cells use functional upregulated autophagy to survive the
hypoxic and overall unfavorable tumour environment, further
exogenous induction of functional autophagy, for example,
through Akt/mTOR inhibitors, may push the balance towards
cytoplasmic exhaustion and autophagic death.
It can be, therefore, suggested that autophagic death is not a
single phenomenon but is rather characterised by at least two
distinct paths: (i) the excessive autophagic activity resulting in
stone-like structure formation (functional autophagic death) by
exhaustion of the lysosomal potential and; (ii) the intolerable
accumulation of waste non-functional autophagosomes (non-
functional autophagic death) by abrogation of lysosomal fusion.
Whether this latter path represents a link of autophagy with
apoptosis, necrosis or mitotic catastrophe induction or it represents
an entirely distinct death pathway is unknown.
THE PARADOXICAL SENSITISATION BY BOTH INDUCERS
AND BLOCKERS
Nevertheless, both ‘functional’ and ‘non-functional’ autophagic
death pathways triggered by radiation and TMZ can be boosted by
early or late phase autophagy blockers and by autophagy inducers.
This paradox can be explained as follows:
(1) Early phase autophagy blockers prevent the formation of
autophagosomes, blocking therefore an important source of
energy and the cytoprotective effect of a controllable functional
autophagy. This, on the one hand, leads per se to the death of a
varying fraction of tumour cells, the survival of which
depended on functional autophagy. On the other hand, by
blocking autophagosome formation tumour cells enter a state
of energy deficit and excessive loading with damaged proteins
and organelles that radiation or TMZ produces, facilitating the
apoptotic death effect of these agents. Thus, early autophagy
blockers may activate a link with apoptosis-like pathways. For
such therapeutic approaches, apoptosis restoration policies
may secure a superadditive effect.
(2) Late phase autophagy blockers, for example, those that allow
the formation of autophagosomes but block their fusion with
lysosomes, repress by one hand the acquisition of energy
through autophagy and on the other hand load the cell with
waste non-degradable autophagic vesicles. Thus, similarly to
early phase blockers, they kill a varying percentage of tumour
cells through energy deprivation and waste overloading. If a
radiotherapy schedule and/or TMZ also induces non-func-
tional autophagosome formation, the synergistic effect with the
energy austerity effect or with the autophagosome accumula-
tion is antagonistic or at least additive. However, if a
radiotherapy schedule is given in those small fractions to
induce functional autophagy, the intensified accumulation of
autophagosomes using late blockers would result in super-
additive effect. In fact, a combination of an early phase
autophagy inducer with a late phase blocker seem an appealing
therapeutic proposal.
Autophagy, radiation and temozolomide BRITISH JOURNAL OF CANCER
www.bjcancer.com | DOI:10.1038/bjc.2016.19 5

(3) Autophagy inducers could not only target tumour cells with
increased autophagy but also tumour cells with reduced
autophagy. In tumour cells with active autophagy, augmentation
of autophagy may shift the balance to functional autophagic
death through lysosomal exhaustion and cytoplasmic degrada-
tion (stone-like death). If radiation and TMZ is scheduled to
induce functional autophagy, autophagy inducers administered
concurrently with radiation will accelerate functional autophagic
death. If radiation and TMZ are scheduled to induce non-
functional autophagosome accumulation, administration of
autophagy inducers immediately before radiochemotherapy
may enhance the non-functional autophagic death.
CLINICAL EXPERIENCE
Clinical experience with autophagy-manipulating agents in the
treatment of glioblastoma is limited. Chloroquine (Kimura et al,
2013), presumably because it is already availabile in the clinical
practice as an antimalaria agent (Hall, 1976), is the only autophagy
inhibitor studied for the treatment of glioblastoma patients. In a
phase II clinical trial, the administration of hydroxychloroquine to
patients with glioblastoma undergoing radiotherapy with temozo-
lomide confirmed an increase of autophagic vacuoles and of the
LC3-II form in the peripheral blood cells, supportive of an
antiautophagic activity, but the haematological toxicity of the regimen
was unacceptable and the benefit in terms of survival was not evident
(Rosenfeld et al, 2014). A small pilot study on the combination of
chloroquine with radiation for the treatment of recurrent glioblas-
toma confirmed the feasibility of the regimen and the authors claimed
encouraging treatment outcomes (Bilger et al, 2014). The recent
observation that the acidic extracellular pH neutralises the autophagy-
inhibiting activity of chloroquine stresses the importance of the
development of potent autophagy inhibitors, the activity of which is
independent or even better take advantage of the intratumoral
hypoxic and acidic conditions (Pellegrini et al, 2014).
Mammalian target of rapamycin inhibitors have multiple
biological activities, including the induction of autophagy
(Huang and Fingar, 2014). Such agents have been introduced in
the clinical practice for the treatment of renal carcinoma (Amato,
2011), and several clinical trials have investigated the activity
mTOR inhibitors in patients with glioblastoma. Temsirolimus, for
example, showed clinical efficacy in one-third of patients treated
for recurrent glioblastoma (Galanis et al, 2005). Sarkaria et al
(2011) combined everolimus in combination with radiotherapy
and TMZ in 18 patients, showing a metabolic effect of everolimus
in most patients, as detected with fluorodeoxyglucose PET scan.
More recently, the authors reported a phase II study on 100
patients, which, however, did not detect any beneficial effect in
terms of survival (Ma et al, 2015). An interesting phase II study by
Hainsworth et al (2012) administered BVZ with everolimus after
radiochemotherapy for 68 glioblastoma patients (Hainsworth et al,
2012). The progression-free survival interval compared favorably
with the authors’ previous experience.
IMPORTANT ISSUES TO RESOLVE
It is evident that although we know that radiation and TMZ kill
cells through autophagy, it is unclear which of the functional or
non-functional autophagic death pathway is followed. It is very
important to investigate the role of radiotherapy fractionation on
the autophagic pathways that this activates. Several tumours, such
as gliomas, melanomas or even prostate cancer, exhibit a higher
sensitivity to large radiotherapy fractions. It may be that small
radiotherapy fractions (around 2 Gy) can simply stimulate
functional autophagy in such cells, triggering therefore a
cytoprotective pathway during the fractionated course of radio-
therapy. Larger fractions can block autophagic flux leading to non-
functional accumulation of autophagosomes, so that in such
radiation scheme self-sensitises cells to its effect.
If such a differential effect on autophagy exists, then the choice
and sequence of autophagy-interfering agent for combined
administration should be made according to the fractionation of
radiotherapy we wish to apply. The same principle can be also
applicable for TMZ as the short schedule of 200–250 mg m
2
5
days every month or the prolonged schedule of 70 mg m
2
per day
continuously used in the clinical practice may have a different
effect on the autophagic flux of gliomas and may demand different
autophagic interference to achieve radio/chemosensitisation.
It seems that the interfering autophagy may indeed help us
make a difference in the treatment of gliomas and glioblastomas. A
hypothetical scheme for intervention targeting autophagy aiming
to radiosensitisation is shown in Figure 1. Inducers of autophagy,
like mTOR inhibitors available in the clinical practice (Houghton,
2010), can lead to a functional autophagic death when combined
with standard radiotherapy and a schedule of continuous low daily
dose of TMZ. Pretreatment of tumours with autophagy inducers,
followed by hypofractionated radiotherapy and or high daily dose
schedules of TMZ, in combination with late phase autophagy
blockers, could enhance antineoplastic efficacy by leading to non-
functional autophagic death. Whether early phase autophagy
blockers could be of value in the combination of agents that restore
apoptotic death pathways with radiotherapy or TMZ is also a
hypothesis to study. Important issues remain, however, unresolved.
One key question that has to be answered is whether radiotherapy
and TMZ trigger a functional autophagic response or they just
deregulate autophagy resulting in non-degradable autophagosome
accumulation. This demands studies of monitoring the autophagic
flux and lysosomal kinetics in cell lines after escalated doses of
radiotherapy or TMZ, as dose schedule may be a principal factor
defining the type of autophagic response.
Another important issue to resolve is the characterisation of the
autophagic activity and response tendency of the tumour itself. Not
all gliomas or glioblastomas are the same as, indeed, some of them
are more radiosensitive than others. Assessment of the expression
status of autophagy-blocking pathways such as Akt/mTOR may
help to identify tumours of defective autophagy, the radio-
sensitivity of which may increase by specific Akt/mTOR inhibitors.
As beclin 1 protein is downregulated in a large percentage of
gliomas, detection of hypermethylated Beclin 1 gene or other
autophagy-initiating proteins may reveal targets for demethylating
agent administration so that subsequent interference with
autophagy inducers may become beneficial. Extensive expression
of LC3A-positive stone-like structures may characterise tumours
with functional autophagy that may benefit from concurrent
radiotherapy with either early autophagy and DNA-repair blockers
or autophagy induction therapy with mTOR inhibitors (depending
upon radiotherapy fractionation).
Assessment of the content of CD133-positive compartment can
be easily performed in immunohistochemistry of biopsies.
Pretreatment of these patients with differentiation-inducing agents
such as histone deacetylase inhibitors may prove a useful
preradiotherapy policy. Silencing of stem-cell-specific genes, such
as Sirtuin 1, or apoptosis restoration treatments, such as caspase or
IL-24 adenoviruses, may also be of value in these cases.
CONCLUSION
Malignant glioma is probably a unique model of non-metastasising
disease that kills the majority of patients through local growth.
BRITISH JOURNAL OF CANCER Autophagy, radiation and temozolomide
6 www.bjcancer.com | DOI:10.1038/bjc.2016.19

It consists, therefore, of one of the biggest challenges in radiation
therapy, especially nowadays when novel radiotherapy techniques
allow the delivery of high dose per fraction to the tumour at the
same time reducing the dose to the surrounding normal brain
tissue. Whether hypofractionation that would block the autophagy
flux, even in radioresistant cell lines, can be effectively combined
with late autophagy blockers to exploit a non-functional autopha-
gic death is a question that deserves investigation. Temozolomide
is an important drug when combined with radiotherapy, or even
for relapsed tumours after radiotherapy, and just like radiotherapy
kills glioma cells through autophagic death. The combination of
mTOR inhibitors (intensifiers or autophagy) with low daily dose of
TMZ during standard radiotherapy or after failure of radiotherapy,
aiming to trigger functional autophagic death, may also be a
promising approach. Modulation of radiation or of TMZ
autophagic response appears one of the most promising
approaches to prolong survival and to better understand the
glioblastoma therapy riddle, but important insights demanded to
create a reliable concept are still missing.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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