A JOURNAL OF NEUROLOGY
Antagonism of the mammalian target of
rapamycin selectively mediates metabolic effects
of epidermal growth factor receptor inhibition
and protects human malignant glioma cells from
hypoxia-induced cell death
Michael W. Ronellenfitsch,1,2Daniel P. Brucker,2Michael C. Burger,2Stefan Wolking,1
Felix Tritschler,1Johannes Rieger,2Wolfgang Wick,3Michael Weller1,4and
Joachim P. Steinbach2
1 Laboratory for Molecular Neurooncology, Department of General Neurology, University of Tu ¨bingen, Tu ¨bingen, Germany
2 Dr. Senckenberg Institute of Neurooncology, Goethe-University Hospital, Frankfurt am Main, Germany
3 German Cancer Research Centre, Heidelberg, Germany
4 Department of Neurology, University Hospital Zu ¨rich, Zu ¨rich, Switzerland
Correspondence to: J. P. Steinbach,
Dr. Senckenberg Institute of Neurooncology,
Centre of Neurology and Neurosurgery,
60528 Frankfurt am Main,
Although inhibition of the epidermal growth factor receptor is a plausible therapy for malignant gliomas that, in vitro, enhances
apoptosis, the results of clinical trials have been disappointing. The mammalian target of rapamycin (mTOR) is a serine/
threonine kinase that integrates starvation signals and generates adaptive responses that aim at the maintenance of energy
homeostasis. Antagonism of mTOR has been suggested as a strategy to augment the efficacy of epidermal growth factor
receptor inhibition by interfering with deregulated signalling cascades downstream of Akt. Here we compared effects of antag-
onism of mTOR utilizing rapamycin or a small hairpin RNA-mediated gene silencing to those of epidermal growth factor
receptor inhibition or combined inhibition of epidermal growth factor receptor and mTOR in human malignant glioma cells.
In contrast to epidermal growth factor receptor inhibition, mTOR antagonism neither induced cell death nor enhanced apoptosis
induced by CD95 ligand or chemotherapeutic drugs. However, mTOR inhibition mimicked the hypoxia-protective effects of
epidermal growth factor receptor inhibition by maintaining adenosine triphosphate levels. These in vitro experiments thus
challenge the current view of mTOR as a downstream target of Akt that mediates antiapoptotic stimuli. Under the conditions
of the tumour microenvironment, metabolic effects of inhibition of epidermal growth factor receptor, Akt and mTOR may
adversely affect outcome by protecting the hypoxic tumour cell fraction.
Keywords: mTOR; EGFR; glioma; hypoxia; metabolism
doi:10.1093/brain/awp093Brain 2009: 132; 1509–1522 |
Received October 29, 2008. Revised March 9, 2009. Accepted March 10, 2009. Advance Access publication May 4, 2009
? The Author (2009). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
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Abbreviations: 4E-BP1=eukaryotic translation initiation factor 4E binding protein 1; CD95L=CD95 ligand; EGFR=epidermal
growth factor receptor; eIF4E=eukaryotic translation initiation factor 4E; HIF-1 a=hypoxia inducible factor-1 a; LDH=lactate
dehydrogenase; mTOR=mammalian target of rapamycin; p42/44 MAPK=p42/44 mitogen-activated protein kinase;
PI3K=phosphatidyl-inositole-3-phosphate kinase; RPS6=ribosomal protein S6; S6K1=ribosomal protein S6 kinase 1;
shRNA=small hairpin RNA
Patients with glioblastoma face a grim prognosis with a median
survival of 51 year in unselected cohorts (Ohgaki et al., 2004).
Novel therapeutic approaches are, therefore, urgently needed.
Targeting signalling downstream of activated growth factor
receptors is currently considered one of the most promising
approaches. Specifically, the epidermal growth factor receptor
(EGFR) is amplified or activated in the majority of primary
glioblastomas and can be regarded as one of the most plausible
targets for molecular therapy (Kleihues et al., 2000). EGFR signal-
ling sustains many key features of the neoplastic phenotype, e.g. it
enhances cell autonomous growth, invasion and angiogenesis and
confers protection from growth inhibitory and proapoptotic stimuli
(Mendelsohn, 2002; Steinbach and Weller, 2002).
Clinical trials with small molecule kinase inhibitors of the EGFR
in malignant glioma patients, however, have produced disappoint-
ing results (Van Den Bent et al., 2009). Insufficient suppression of
the intracellular signalling cascade downstream of EGFR has been
suggested as an important cause for the failure of EGFR inhibitors.
Phosphorylation of Akt, in particular, represents a negative
predictive marker for the response of malignant glioma patients
to EGFR inhibitors (Haas-Kogan et al., 2005). The serine/threonine
kinase mammalian target of rapamycin (mTOR) is a key mediator
of phosphatidyl-inositole-3-phosphate kinase (PI3K) and Akt sig-
nalling. It integrates growth-inhibitory signals such as deprivation
of glucose and amino acids, ATP depletion, hypoxia and lack of
growth factors (Wullschleger et al., 2006) in order to generate
adaptive cellular responses primarily by altering the translation of
specific proteins. Deregulated mTOR signalling sustains prolifer-
ation of malignant cells by antagonizing these physiological
starvation signals (Peng et al., 2002). It therefore appears plausible
that the commonly observed activation of mTOR signalling in
glioblastoma (Choe et al., 2003) contributes to the typical pattern
of glioblastoma pathology with necrotic cores and aggressive
growth at the tumour margins. The translational effects of
mTOR signalling are mediated by phosphorylation of its two
direct target molecules, eukaryotic translation initiation factor 4E
binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1
(S6K1), at specific sites. Phosphorylation of 4E-BP1 occurs
stepwise and relieves its repression of eukaryotic initiation factor
4E (eIF4E) (Hara et al., 1997), resulting in activation of translation
initiation. In this process, phosphorylation of the residues Thr37/
46 is the initial step in eukaryotic translation initiation factor 4E
binding protein 1phosphorylation. It is also a prerequisite for phos-
phorylation of residues Thr70 and Ser65, whose phosphorylation
alone is not sufficient to block binding to eukaryotic initiation
factor 4E (Gingras et al., 2001). The different phosphorylation
states of 4E binding protein 1 are evidenced by three differently
migrating bands in immunoblot analyses: (i) a fastest migrating
hypophosphorylated a band; (ii) a b band of intermediate mobility;
and (iii) a slowest migrating hyperphosphorylated g band. Thr37/
46 phosphorylated 4E-BP1 can be detected in all three bands and
is relatively insensitive to serum deprivation and mTOR inhibition.
However, the pattern of phosphorylated 4E-BP1 shifts to the
hypophosphorylated faster migrating bands following mTOR
inhibition (Brunn et al., 1997). Phosphorylation of S6K1 results
in activation of its kinase activity and phosphorylation of ribosomal
protein S6 (RPS6), which, among other proteins, regulates trans-
lation of ribosomal proteins and elongation factors (Hay and
Sonenberg, 2004). With the availability of rapamycin and related
drugs that specifically inhibit mTOR signalling, considerable
interest has been generated to investigate antagonism of mTOR
as an anti-glioma strategy (Hidalgo and Rowinsky, 2000; Huang
and Houghton, 2001). In preclinical models, mTOR inhibition has
been reported to result in apoptotic and non-apoptotic cell death
(Rao et al., 2005; Takeuchi et al., 2005), cell cycle arrest (Tanaka
et al., 2007) and decreased angiogenesis (Del Bufalo et al., 2006).
mTOR inhibition is particularly effective against some cell lines
with mutant PTEN (Neshat et al., 2001).
Augmentation of the efficacy of EGFR inhibition by mTOR
inhibition therefore appears plausible (Doherty et al., 2006;
Reardon et al., 2006). However, we have previously identified
an undesirable consequence of interference with EGFR function
that offers an alternative explanation for the poor clinical perfor-
mance of EGFR antagonistic strategies and may also adversely
affect the efficacy of mTOR inhibition. Altered cellular metabolism
and energy expenditure, which are enhanced by EGFR signalling,
are at the core of this phenomenon which results in the protection
of malignant glioma cells from hypoxia when EGFR is inhibited
(Steinbach et al., 2004, 2005). In these experiments, EGFR
inhibition and resistance towards hypoxia were associated with
decreased phosphorylation of the mTOR target ribosomal protein
S6. Given the physiological role of mTOR in the regulation of
metabolic demands, these results suggested that EGFR inhibition
confers protection against hypoxia in a mTOR-dependent fashion.
We report theresultsof
cell-autonomous effects of mTOR antagonism on CD95 ligand-,
chemotherapy- and hypoxia-induced cell death in human malig-
nant glioma cells employing rapamycin and hairpin RNA-mediated
gene suppression of mTOR (Brummelkamp et al., 2002) and a
comparison of mTOR inhibition with EGFR inhibition or combined
inhibition of mTOR and EGFR.
Materials and Methods
Reagents and cell lines
U0126, an inhibitor of MEK 1 and 2, the PI3K inhibitor LY294002,
Akt inhibitor VIII and EGF were purchased from Calbiochem
Brain 2009: 132; 1509–1522M. W. Ronellenfitsch et al.
by guest on January 1, 2016
which will be conducted by the EORTC brain tumour group
(W. Wick, personal information).
Supplementary material is available at Brain online.
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