2013;3:534-547. Published OnlineFirst March 26, 2013.Cancer Discovery
David Akhavan, Alexandra L. Pourzia, Alex A. Nourian, et al.
Resistance to EGFR Tyrosine Kinase Inhibitors in Glioblastoma
Transcription Promotes Acquired
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Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502
De-Repression of PDGFR?b
Acquired Resistance to EGFR
Tyrosine Kinase Inhibitors in
David Akhavan 1 , 2 , Alexandra L. Pourzia 3 , Alex A. Nourian 3 , Kevin J. Williams 3 , 7 ,
David Nathanson 2 , Ivan Babic 9 , Genaro R. Villa 1 , 2 , 9 , Kazuhiro Tanaka 2 ,
Ali Nael 2 , Huijun Yang 9 , Julie Dang 2 , Harry V. Vinters 3 , William H. Yong 3 ,
Mitchell Flagg 3 , Fuyuhiko Tamanoi 5 , Takashi Sasayama 12 , C. David James 8 ,
Harley I. Kornblum 4 , Tim F. Cloughesy 6 , Webster K. Cavenee 9 , 10 ,
Steven J. Bensinger 2 , 3 , 7 , and Paul S. Mischel 9 , 10 , 11
MAY 2013?CANCER DISCOVERY | 535
Acquired resistance to tyrosine kinase inhibitors (TKI) represents a major chal-
lenge for personalized cancer therapy. Multiple genetic mechanisms of acquired
TKI resistance have been identifi ed in several types of human cancer. However, the possibility that
cancer cells may also evade treatment by co-opting physiologically regulated receptors has not been
addressed. Here, we show the fi rst example of this alternate mechanism in brain tumors by show-
ing that EGF receptor (EGFR)-mutant glioblastomas (GBMs) evade EGFR TKIs by transcriptionally
de-repressing platelet-derived growth factor receptor β (PDGFRβ). Mechanistic studies show that
EGFRvIII signaling actively suppresses PDGFRb transcription in an mTORC1- and extracellular signal–
regulated kinase-dependent manner. Genetic or pharmacologic inhibition of oncogenic EGFR renders
GBMs dependent on the consequently de-repressed PDGFRβ signaling for growth and survival. Impor-
tantly, combined inhibition of EGFR and PDGFRβ signaling potently suppresses tumor growth in vivo .
These data identify a novel, nongenetic TKI resistance mechanism in brain tumors and provide compel-
ling rationale for combination therapy.
SIGNIFICANCE: These results provide the fi rst clinical and biologic evidence for receptor tyrosine
kinase (RTK) “switching” as a mechanism of resistance to EGFR inhibitors in GBM and provide a molecu-
lar explanation of how tumors can become “addicted” to a nonamplifi ed, nonmutated, physiologically
regulated RTK to evade targeted treatment. Cancer Discov; 3(5); 534–47. ©2013 AACR.
Authors’ Affi liations: 1 Medical Scientist Training Program, David Geffen
School of Medicine; Departments of 2 Molecular and Medical Pharmacol-
ogy, 3 Pathology and Laboratory Medicine, 4 Psychiatry and Biobehavioral
Sciences, and 5 Microbiology, Immunology & Molecular Genetics; 6 Neuro-
Oncology Program; 7 Institute for Molecular Medicine, University of Cali-
fornia Los Angeles, Los Angeles; 8 Brain Tumor Research Center, University
of California San Francisco, San Francisco; 9 Ludwig Institute for Cancer
Research; 10 Moores Cancer Center; 11 Department of Pathology, Univer-
sity of California San Diego, La Jolla, California; and 12 Department of
Neurosurgery, Kobe University Graduate School of Medicine, Kobe, Hyogo,
Note: Supplementary data for this article are available at Cancer Discovery
Corresponding Authors: Paul S. Mischel, Ludwig Institute for Can-
cer Research, University of California San Diego, 9500 Gilman Drive,
La Jolla, CA 92093-0753. Phone: 858-534-6080; Fax: 858-534-7750;
E-mail: email@example.com ; and Steven J. Bensinger, Institute for Molecular
Medicine Department of Pathology and Laboratory Medicine, 36-128
Center for Health Sciences, University of California Los Angeles,
Los Angeles, CA 90095. Phone: 310-825-9885; Fax: 310-267-6267;
©2013 American Association for Cancer Research.
The EGF receptor, EGFR , is commonly amplifi ed and/
or mutated in many types of solid cancer, including a
variety of epithelial cancers and glioblastoma (GBM) ( 1–3 ).
Despite compelling evidence for EGFR addiction in experi-
mental models, the clinical benefi t of most EGFR tyrosine
kinase inhibitors (TKI) has been quite limited. Multiple
genetic resistance mechanisms enable cancer cells to main-
tain signal fl ux to critical downstream effector pathways,
and thus evade EGFR-targeted therapy, including: (i)
acquisition and/or selection for secondary EGFR muta-
tions conferring EGFR TKI resistance ( 4 ); (ii) additional
activating mutations in downstream effectors, such as
PTEN ( 2 ), PIK3CA ( 5 ), or KRAS ( 6 ); and (iii) co-occurrence
of other amplifi ed or mutated receptor tyrosine kinases
(RTK), including C-MET and platelet-derived growth factor
receptor α (PDGFRα; refs. 7, 8 ). In addition to these genetic
resistance-promoting mutations, it is suspected that EGFR-
dependent cancers may escape targeted therapy by devel-
oping dependence on other nonamplifi ed, nonmutated
RTKs ( 9 ). However, the mechanisms by which cancers,
including GBM, evade EGFR TKI treatment by coopt-
ing other physiologically regulated receptors has not been
Herein, we integrate studies in cell lines, patient-derived
tumor cultures, xenotransplants, and tumor tissue from
patients with GBM in a phase II clinical trial to provide the fi rst
demonstration of EGFR TKI resistance mediated by transcrip-
tional de-repression of PDGFRb . We show that the persist-
ently active EGFR mutation, EGFRvIII, suppresses PDGFRβ
expression via mTORC1- and extracellular signal–regulated
kinase (ERK)-dependent mechanisms. We show in cells, mice,
and patients that EGFR TKI treatment de-represses PDGFRβ,
rendering GBMs dependent on PDGFRβ signaling for growth.
We further show that combined abrogation of EGFRvIII and
PDGFRβ potently prevents GBM growth in vivo . These results
identify a novel physiologic EGFR TKI resistance mechanism
in GBM and suggest a clinically actionable approach to sup-
EGFR Inhibition Promotes PDGFRb
Upregulation in Glioma
To better understand how malignant glioma acquires resist-
ance to EGFR inhibitors in vivo , U87 glioma cells expressing
the EGFRvIII gain-of-function mutation (designated U87-
EGFRvIII herein) were placed in the fl anks of severe combined
536 | CANCER DISCOVERY?MAY 2013 www.aacrjournals.org
Akhavan et al.
immunodefi cient (SCID) mice. EGFRvIII, the most common
EGFR mutation in GBM, arises from an in-frame genomic
deletion of exons 2 to 7, resulting in a persistently active,
highly oncogenic protein ( 10 ). Tumor-bearing mice were
gavaged with the EGFR inhibitor erlotinib (150 mg/kg) or
vehicle control, and tumor growth was assessed over 18 days
( Fig. 1A ). As expected, erlotinib treatment slowed U87-
EGFRvIII tumor growth relative to control; however, tumors
retained a substantial growth rate despite continued erlotinib
treatment ( Fig. 1B ). Immunoblots of tumor lysates confi rmed
Figure 1. A reciprocal relationship between EGFR and PDGFRβ in glioma. A, experimental design of a mouse model of EGFR inhibitor resistance. U87-
EGFRvIII cells were subcutaneously implanted in the mouse fl ank on day 0. Mice were treated with erlotinib (150 mg/kg) on day 2 and as indicated there-
after. B, tumor growth curve of U87-EGFRvIII xenografts in mice treated with erlotinib (150 mg/kg as indicated in A) or vehicle. C, immunoblot of indicated
tumor lysates determining total and phospho-PDGFRβ or EGFR from U87, U87-EGFRvIII ± eroltinib treatment and U87-EGFRvIII kinase dead xenografts
harvested on day 21. PI3K-p85 is used as a loading control in this and subsequent immunoblots. D, RTK array of 42 RTKs conducted on U87-EGFRvIII
xenograft lysates on day 21 from mice treated with erlotinib or vehicle as described in A. E, immunohistochemistry (IHC) of PDGFRβ and phospho-EGFR in
vehicle- and erlotinib-treated U87-EGFRvIII orthotopic xenografts. F, immunoblot of PDGFRα, PDGFRβ, and phospho-EGFR (pEGFR) from patient-derived
GBM neurospheres expressing EGFRvIII or wild-type EGFR as indicated. Whole-cell lysates were collected after 24 hours of erlotinib or vehicle treatment.
G, immunoblot of tumor lysates from EGFRvIII expressing GBM39 xenografts following oral gavage with vehicle or erlotinib for 10 days.
Tumor volume (mm3)
Day 10 Day 13 Day 17
150 mg/kg, 18 d
2018 1614 12 1086420
Tumor 1Tumor 2
Erlotinib (5 μmol/L)
Erlotinib (5 μmol/L)
MAY 2013?CANCER DISCOVERY | 537
An RTK Switch in Malignant Glioma
that erlotinib treatment of mice signifi cantly reduced EGFR
signaling in xenografts to a level comparable with that of
U87 tumor cells expressing kinase-dead EGFR-VIII ( Fig. 1C ).
Thus , U87-EGFRvIII–expressing tumors maintain growth
despite a signifi cant reduction in EGFR activity when treated
with erlotinib, phenocopying the results observed in human
trials ( 2 ).
We considered the possibility that erlotinib-treated U87-
EGFRvIII tumors maintain growth by acquiring neo-RTK
activity. To directly address this, we conducted a phospho-
RTK array on control and erlotinib-treated tumor lysates. As
expected, control U87-EGFRvIII tumors expressed signifi cant
levels of phospho-EGFR that were reduced in erlotinib-treated
mice ( Fig. 1D ). Erlotinib-treated U87-EGFRvIII tumors also
had considerable phospho-PDGFRβ activity ( Fig. 1D ). Height-
ened activation of the RTK AXL was also noted, albeit to a
lesser extent than PDGFRβ. Immunoblots of tumor lysates
confi rmed upregulation and activation of PDGFRβ in response
to pharmacologic or genetic inhibition of EGFRvIII ( Fig. 1C ).
Parental U87 tumors expressed signifi cant PDGFRβ ( Fig. 1C ),
which was suppressed by EGFRvIII. Furthermore, erlotinib
treatment markedly upregulated PDGFRβ expression in ortho-
topic U87-EGFRvIII GBM xenografts ( Fig. 1E ), suggesting that
EGFRvIII signaling actively represses PDGFRβ.
To determine if the reciprocal relationship could be extended
into other patient-derived glioma models endogenously
expressing EGFRvIII, or high levels of wild-type EGFR, we
examined phospho-EGFR and PDGFRβ expression in a panel
of low passage patient-derived GBM neurospheres ( 11 ). Uni-
formly, erlotinib treatment resulted in upregulation PDGFRβ
expression in EGFRvIII-expressing GBM neurospheres, as
well as GBM neurospheres expressing high levels of EGFR
( Fig. 1F ). In addition, GBM39 cells that developed erlotinib
resistance in long-term culture maintained suppression of
EGFR phosphorylation and concomitant PDGFRβ upregu-
lation (Supplementary Fig. S1A). Of note and in contrast
to PDGFRβ, erlotinib treatment had no effect on PDGFRα
expression. To assess whether erlotinib treatment similarly
elevated PDGFRβ levels in GBMs endogenously expressing
EGFRvIII in vivo , we examined PDGFRβ expression up to
10 days of treatment with erlotinib at 150 mg/kg. Consist-
ent with our proposed model, erlotinib treatment resulted in
elevated phosphorylated and total levels of PDGFRβ ( Fig. 1G
and Supplementary Fig. S1B). Single-nucleotide polymorphism
array analysis showed that PDGFRb was not amplifi ed in these
tumor cells, either at baseline or after erlotinib treatment (data
not shown). Taken together, these data indicate that EGFRvIII/
EGFR signaling negatively regulates PDGFRβ expression in
glioma models, and that inhibition of EGFRvIII/EGFR signal-
ing results in upregulation of PDGFRβ.
An RTK Switch to PDGFR?b Occurs
in Lapatinib-Treated Patients
Intratumoral heterogeneity of RTK expression is a common
feature of malignant gliomas, but it remains unclear if this
heterogeneity refl ects co-amplifi cation of RTKs within a given
tumor cell or differences in RTK expression among tumor
cells. To distinguish between these possibilities, we examined
glioma tissue microarrays (TMA) for EGFR and PDGFRβ
expression. Similar to our model system studies, we observed
a strong inverse correlation between EGFR (total and phos-
phorylated tyrosine 1086) and PDGFRβ expression in patient
glioma tissues ( Fig. 2A ; P = 0.02). To determine if RTK expres-
sion was fi xed within a given tumor, we used patient tissues
from a cohort of patients enrolled in a biopsy-treat-biopsy
study, in which patients underwent 7 to 10 days oral treat-
ment with another EGFR TKI, lapatinib, as part of a phase II
clinical trial ( 12 ). Post-lapatinib biopsy samples were divided
into EGFR-on and EGFR-off groups following immunoblot
analysis and show striking inverse correlation between phos-
pho-EGFR status and PDGFRβ protein expression ( Fig. 2B ;
P = 0.04). Immunohistochemical (IHC ) analysis of one patient
was available before and after lapatinib treatment and showed
signifi cant reduction of phospho-EGFR after treatment, with
concomitant PDGFRβ expression in the tumor ( Fig. 2C ).
These clinical data support a model where highly active EGFR
signaling negatively regulates PDGFRβ expression in primary
brain tumors and indicate that pharmacologic inhibition of
EGFR signaling results in an RTK switch to PDGFRβ.
Suppression of PDGFR?b Expression Is
Dependent on the AKT/mTOR Signaling Pathway
EGFRvIII and, to a lesser extent, wild-type EGFR have been
shown to potently activate phosphoinositide 3-kinase (PI3K)
signaling in GBM, resulting in phosphorylation of AKT and its
downstream effector mTORC1 ( 12–17 ). Therefore, we set out
to determine whether EGFRvIII suppresses PDGFRβ through
AKT and mTORC1 signaling. To examine whether EGFRvIII
suppresses PDGFRβ through AKT, U87-EGFRvIII cells were
transfected with the constitutively active AKT1 E17K allele
( 18 ). Ectopic expression of AKT1 E17K fully abrogated the
upregulation of PDGFRβ in response to erlotinib, confi rming
that EGFRvIII suppresses PDGFRβ through AKT ( Fig. 3A ).
Previous work has identifi ed mTOR as a negative regulator
of PDGFRβ expression in mouse embryonic fi broblasts ( 19 ),
leading us to hypothesize that EGFRvIII signaling to AKT
suppresses PDGFRβ expression through mTORC1. To test
this, we determined PDGFRβ expression in U87-EGFRvIII
cells transiently transfected with siRNA targeting the mTORC
proteins Raptor and Rictor. Immunoblot analysis of U87-
EGFRvIII cells transiently transfected with siRNA targeting
the mTORC proteins Raptor and Rictor indicated that the
inhibition of mTORC1, and to a lesser extent mTORC2, led to
increased levels of PDGFRβ expression ( Fig. 3B ). Conversely,
transfection of a constitutively active mTOR (S2215Y) allele
( 20 ) abrogated erlotinib-dependent upregulation of PDGFRβ
( Fig. 3C ). Furthermore, genetic depletion of the mTORC1 effec-
tor p70 S6Kinase by siRNA knockdown similarly upregulated
PDGFRβ ( Fig. 3D ). Confi rming mTOR-dependent repression
of PDGFRβ, rapamycin robustly upregulated PDGFRβ protein
expression in GBM cell lines in vitro and in vivo ( Fig. 3E and
F ). These results show that EGFR signals through AKT and
mTORC1 to suppress PDGFRβ.
EGFR Signaling Represses
Transcription of the PDGFR?b Gene
Next, we sought to determine if the infl uence of mTOR
signaling on PDGFRβ expression was regulated at the tran-
scriptional level. To that end, U87-EGFRvIII cells were treated
with erlotinib or vehicle, and mRNA was collected up to
538 | CANCER DISCOVERY?MAY 2013 www.aacrjournals.org
Akhavan et al.
36 hours after treatment. Quantitative real-time PCR (qRT-
PCR ) showed that PDGFRβ mRNA was upregulated by
8 hours after the addition of erlotinib, and expression pro-
gressively increased over a 24-hour period ( Fig. 4A ; P < 0.001).
To determine if the increase in PDGFRb expression was a func-
tion of increased transcription at the PDGFRβ gene locus, we
assessed the expression levels of PDGFRβ primary transcripts.
qRT-PCR studies revealed that the expression pattern of
PDGFRβ primary transcript mirrored that of PDGFRβ mRNA
following EGFR inhibition ( Fig. 4A ; P < 0.001). Treatment
and washout studies revealed that PDGFRβ primary tran-
script was dynamically regulated by the addition or removal
of erlotinib, further suggesting that expression of PDGFRβ
is an active transcriptional process ( Fig. 4B ). Correspond-
ingly, transcriptional reporter studies using the PDGFRβ pro-
moter upstream of luciferase indicated that knockdown of
EGFR or Raptor signifi cantly increased luciferase activity
in U87-EGFRvIII cells ( Fig. 4C ; P < 0.001). Finally, chroma-
Patient surgery #1 (S1)
10 d lapatinib
Patient surgery #2 (S2)
Western blot analysis of S2 tumors
P value P = 0.0206
P value P = 0.0425
EGFR-off tumors EGFR-on tumors
Figure 2. PDGFRβ expression is suppressed in EGFR-activated GBMs. A, IHC staining for phospho-EGFR and PDGFRβ in clinical GBM tissues. The P
value indicated was calculated using Fisher’s exact test. B, representative immunoblot of PDGFRβ and EGFR in clinical GBM tumors samples treated with
lapatinib. Patients were treated with lapatinib for 10 days following initial diagnosis. Second tumor samples were obtained following recurrence. Tumor
lysates were prepared and grouped according to phospho-EGFR status (EGFR-off/on). The P value was calculated using Fisher’s exact test. C, IHC stain-
ing for PDGFRβ and phospho-EGFR in pre- and post-lapatinib–treated GBM tissue.
tin immunoprecipitation (ChIP) experiments revealed that
rapamycin (5 nmol/L ) treatment results in recruitment of
RNA polymerase II to both the transcriptional start site and
exon 1 of PDGFRβ ( Fig. 4D ; P < 0.01). Taken together, these
studies support a model where EGFR signaling dynamically
regulates transcription of PDGFRβ in an mTOR-dependent
manner. However, we cannot rule out the possibility that
additional factors such as increased stability of the PDGFRβ
mRNA pool or heightened translation also contribute to
ERK Signaling Contributes to the
Regulation of PDGFR?b
The mitogen-activated protein kinase (MAPK) pathway
is also activated by EGFRvIII signaling ( Fig. 5A ); thus, we
investigated whether the MAPK signaling pathway also con-
tributes to the regulation of PDGFRβ expression. The MAP–
ERK kinase (MEK) inhibitor U0126 upregulated PDGFRβ
MAY 2013?CANCER DISCOVERY | 539
An RTK Switch in Malignant Glioma
Figure 3. EGFRvIII suppresses PDGFRβ through AKT and mTORC1 signaling. A, immunoblot of PDGFRβ and indicated proteins in U87-EGFRvIII cells
expressing constitutively active AKT1 (E17K) treated with erlotinib (5 μmol/L) for 24 hours. B, immunoblot of lysates from U87-EGFRvIII cells with
transient knockdown of mTOR complex proteins Raptor or Rictor and treated with erlotinib (5 μmol/L) as indicated. C, immunoblot of U87-EGFRvIII cells
expressing constitutively active (S2215Y) or wild-type mTOR and treated with erlotinib (5 μmol/L) for 24 hours as indicated. D, immunoblot of PDGFRβ
levels in response to transient knockdown of EGFRvIII, or S6 kinase 1 in U87-EGFRvIII cells. E, PDGFRβ levels in U87-EGFRvIII and U251 cells treated
with vehicle or rapamycin (5 nmol/L) for 24 hours. F, IHC of PDGFRβ in intracranial U251 GBM tumors following 3 days of rapamycin (2 mg/kg/d) or
2 mg/kg/d (3 d)
expression, although to a lesser extent than erlotinib ( Fig.
5A ), which was not abrogated by overexpression of wild-
type S6K1 or constitutively active S6K1 or S6K1 and S6K2
alleles ( Fig. 5B ). Taken together, these results show the pres-
ence of a parallel pathway by which EGFRvIII/EGFR signal-
ing regulates PDGFRβ through a MAPK ( Fig. 5C ). Other
RTKs such as MET have been shown to engage PI3K signal-
ing to confer resistance to erlotinib in GBM ( 7 ). Therefore,
we asked whether MET signaling, which can activate both
AKT/mTORC1 and MAPK pathways, could similarly pro-
mote PDGFRβ upregulation. In U87-EGFRvIII or GBM39
neurospheres, the MET inhibitor PHA-665752 (PHA, 0.05–
4 μmol/L) was not suffi cient to promote PDGFRβ upregula-
tion as erlotinib did ( Fig. 5D and E ). However, the addition of
exogenous hepatocyte growth factor (HGF) ligand promoted
AKT and ERK phosphorylation and suppressed erlotinib-
mediated upregulation of PDGFRβ in a dose-dependent
fashion ( Fig. 5F ). These results suggest that HGF-mediated
activation of MET can also repress PDGFRβ by engaging
AKT/mTOR and MAPK signaling.
PDGFR?b Is Dispensable for EGFRvIII-Driven GBM
Growth but Becomes Required for the Growth of
Next, we asked if PDGFRβ signaling infl uences prolifera-
tive capacity in EGFR-inhibited glioma. To that end, U87-
EGFRvIII cells were cultured with erlotinib or vehicle and
PDGF-BB (0–20 ng/mL/d) for 4 days. The addition of PDGFR
540 | CANCER DISCOVERY?MAY 2013 www.aacrjournals.org
Akhavan et al.
ligand to untreated U87-EGFRvIII cells had little effect on pro-
liferative capacity ( Fig. 6A ). As expected, treating U87-EGFRvIII
cells with erlotinib alone signifi cantly reduced both EGFR sign-
aling (Supplementary Fig. S2A) and proliferation ( Fig. 6A ). The
addition of PDGF-BB to cultures restored proliferative capacity
of erlotinib-treated cells ( Fig. 2A ) in a receptor-specifi c (Sup-
plementary Fig. S2B) and dose-dependent manner (Sup-
plementary Fig. S2C). Similarly, the addition of PDGFRβ
ligand to cultures signifi cantly restored the proliferative
capacity of U87-EGFRvIII cells transfected with siRNA-
targeting EGFRvIII ( Fig. 6B and Supplementary Fig. S2D).
Next, we asked whether PDGFRβ signaling was required for
tumor growth in vivo in GBM cells expressing EGFRvIII, and
whether abrogation of EGFRvIII rendered these tumor cells
PDGFRβ-dependent. To that end, U87-EGFRvIII and U87-
EGFRvIII-kinase dead cells were stably transduced with short
hairpin RNAs (shRNA) targeting PDGFRβ or control shRNA
and implanted in the fl anks of SCID mice. Consistent with
our in vitro studies, the silencing of PDGFRβ had little effect
on U87-EGFRvIII tumor growth ( Fig. 6C ). In contrast, silenc-
ing PDGFRβ signifi cantly attenuated the growth of tumors
expressing kinase dead-EGFRvIII ( Fig. 6D ). Immunoblots of
xenograft lysates confi rmed an inverse relationship between
PDGFRβ and EGFR activation in tumors ( Fig. 6E ).
To determine whether PDGFRβ signaling could abro-
gate the growth-inhibitory effects of erlotinib in GBM
cells endogenously expressing EGFRvIII or high levels of
wild-type EGFR, we examined the effect of PDGFRβ signal-
ing on the proliferative capacity on patient-derived GBM
neurospheres. The PDGFR kinase inhibitor AG1295 (2
μmol/L) alone had no antiproliferative effect on GBM39
(EGFRvIII positive, PTEN intact) or HK250 (high-level
wild-type EGFR, PTEN-defi cient) cells ( Fig. 6F and G ). In
contrast, in the presence of erlotinib, the addition of the
Figure 4. EGFR signaling regulates transcription of PDGFRb gene. A, time course of PDGFRb primary transcript and mRNA expression in U87-EGFRvIII
cells treated with erlotinib (5 μmol/L) or vehicle for up to 32 hours. B, determination of PDGFRb mRNA expression in response to erlotinib treatment or media
washout over 60 hours. C, luciferase assay comparing PDGFRb promoter activity in U87-EGFRvIII cells transfected with scrambled siRNA to siRNA against
Raptor or EGFRvIII. D, ChIP of RNA polymerase II at the promoter or exon 1 of PDGFRβ gene following treatment with vehicle or rapamycin (5 nmol/L) for
24 hours. **, P < 0.01; ***, P < 0.001. n.s., no statistical signifi cance.
PDGFRB primary transcript
60 h 48 3624120
P < 0.001
P < 0.001
MAY 2013?CANCER DISCOVERY | 541
An RTK Switch in Malignant Glioma
Figure 5. ERK signaling contributes to the regulation of PDGFRβ. A, immunoblot of PDGFRβ and indicated proteins from whole-cell lysates of U87-
EGFRvIII cells treated with MEK inhibitor U0126 (5 μmol/L), erlotinib (5 μmol/L), or vehicle for 24 hours. B, determination of PDGFRβ protein levels
from U87-EGFRvIII cells transfected with empty vector (pcDNA), wild-type S6K1, or constitutively active S6K1 (T412D) and S6K2 (T401D). In addition,
cells were treated with MEK inhibitor U0126 (10 μmol/L) or vehicle for 24 hours as indicated. C, a schematic of the signals downstream of EGFRvIII
regulating PDGFRβ protein expression. D and E, PDGFRβ protein levels from U87-EGFRvIII cells (D) or patient-derived neurosphere GBM39 (E) treated
with erlotinib (5 μmol/L) or MET inhibitor PHA at the indicated dose. F, immunoblot of PDGFRβ and indicated proteins from U87-EGFRvIII cells treated
with erlotinib and MET ligand HGF (50–100 ng/mL) for 24 hours as indicated.
HGF (50 ng/mL):
U0126 (10 μmol/L):
PHA 0.1 μmol/L
PHA 0.4 μmol/L
PHA 4 μmol/L
PHA 0.05 μmol/L
PHA 0.1 μmol/L
Erlotinib + PHA
542 | CANCER DISCOVERY?MAY 2013 www.aacrjournals.org
Akhavan et al.
Tumor volume (mm3)
Cell number × 1,000
si-control + PDGF
si-EGFRvIII + PDGF
DMSO + PDGF
Erlotinib + PDGF
AG1295 + erlotinib
AG1295 + erlotinib
Tumor volume (mm3)
Day 20Day 24
Day 20 Day 24
Figure 6. PDGFRβ is dispensable for EGFRvIII-driven GBM growth but is required for the optimal growth of EGFR-inhibited tumors. A, proliferation of
U87-EGFRvIII cells over 4 days treated with erlotinib (5 μmol/L) or PDGF-BB ligand (20 ng/mL) alone or in combination as indicated. Erlotinib was added
on day 0 of culture, and PDGF-BB was added daily at 20 ng/mL thereafter. B, growth of U87-EGFRvIII cells transiently transfected with control scrambled
siRNA or siEGFRvIII and treated with PDGF-BB as described in A. C and D, growth curve of xenografts subcutaneously implanted with U87-EGFRvIII,
U87-EGFRvIII/shPDGFRβ, U87-EGFRvIII kinase dead, or U87-EGFRvIII kinase dead/shPDGFRβ cells as indicated. E, immunoblot of phospho-PDGFRβ and
EGFR from lysates harvested on day 24 from tumors as described in C and D. F and G, proliferation of EGFRvIII-expressing patient-derived neurospheres
GBM39 and HK-250 treated with erlotinib (5 μm) and PDGFRβ inhibitor AG1295 (3 μmol/L) alone or in combination as indicated. **, P < 0.01; ***, P < 0.001.
n.s., no statistical signifi cance.
PDGFR kinase inhibitor AG1295 signifi cantly suppressed
tumor cell proliferation ( Fig. 6F and G ; P < 0.01). Of note,
and in contrast to our studies on U87-EGFRvIII–engi-
neered cells, the patient-derived neurosphere cultures did
not require the addition of exogenous PDGFR ligand,
consistent with the role of autocrine and paracrine PDGF
signaling in GBM ( 21, 22 ). Taken together, these data sug-
gest a physiologic RTK switch to the PDGFRβ to maintain
the growth of EGFRvIII/EGFR–activated GBMs in response
to EGFR TKIs ( Fig. 7 ).
Acquired drug resistance presents a signifi cant challenge for
personalized cancer therapy. In principle, upfront sequencing
may guide successful combination TKI therapy by defi ning
MAY 2013?CANCER DISCOVERY | 543
An RTK Switch in Malignant Glioma
Rapamycin or ERK inhibitor
Figure 7. Model of proposed RTK-switch. Under conditions of heightened growth receptor signaling (e.g., EGFRvIII mutation), PDGFRβ expression is
repressed by downstream ERK and mTOR activity. Inhibition of these growth pathways, as with EGFR or mTOR inhibitors, results in the transcription of
the PDGFR b gene and the upregulation of PDGFβ receptor.
both the druggable kinase mutations and the potential “seeds”
of resistance—second site mutations, downstream effector
mutations, and coamplifi cation of multiple RTKs. However,
nongenetic adaptive resistance mechanisms complicate this
paradigm. Identifying the ways that cancer cells “rewire” their
circuitry through pathway cross-talk and release of inhibitory
feedback loops to evade treatment may be critical for develop-
ing more successful combination approaches. By integrating
studies of cells, mice, and tumor tissue from patients treated
with EGFR inhibitors in a clinical trial, we provide the fi rst
experimental evidence that EGFR TKI resistance can be medi-
ated by transcriptional de-repression of another, physiologi-
cally regulated RTK: PDGFRβ.
TKI-mediated release of inhibitory feedback loops is emerg-
ing as a frequent, nongenetic mechanism of targeted cancer
drug resistance. In colorectal cancer cells bearing the BRAF
V600E mutation, resistance to the BRAF inhibitor PLX-4032
(vemurafenib) is mediated by reactivation of EGFR signaling
through the MAPK pathway ( 23, 24 ), although upregulation
of EGFR itself does not seem to be involved. In breast cancer
cells, AKT and mTOR inhibition reactivates PI3K signaling
through release of an inhibitory feedback loop in a process that
seems to involve multiple RTKs ( 25, 26 ). Most recently, tran-
scriptional upregulation of the RTK AXL has been shown to
promote erlotinib resistance in non–small cell lung cancer
( 9 ), although the mechanism underlying AXL upregulation
is not known. It is interesting to note that we too observed
AXL upregulation in response to erlotinib treatment ( Fig. 1 ),
suggesting that it may also play a role in mediating erlo-
tinib resistance in GBM. However, we focused on PDGFRβ
because of the dominance of its signal across all in vitro and
in vivo models and in all patient samples we studied.
In contrast to the recognized importance of PDGFRα
alterations ( 7 , 27–29 ), the role of PDGFRβ in malignant
gliomas has not been clearly defi ned. PDGFRβ amplifi cations
and/or mutations are exceedingly rare events in GBM ( 27 ).
In mouse genetic models, PDGF-B ligand overexpression
can promote gliomagenesis by enhancing cellular prolifera-
tion ( 30–34 ). Recently, PDGFRβ has been shown to promote
glioma stem cell self-renewal, suggesting a more defi nitive
role in tumorigenesis and/or maintenance ( 35 ). In addition,
a PDGF signaling class of GBMs, characterized by PDGFRβ
phosphorylation and a lack of EGFR signaling, among other
features, has been identifi ed ( 36 ). Yet the contribution of
PDGFRβ signaling to drug resistance remains incompletely
Here, we provide the fi rst demonstration that mTORC1
inhibition mediates EGFR TKI resistance in GBM through
transcriptional regulation of PDGFRb , a mechanism which
could also be active in other cancer types. PDGFRβ has
been shown to mediate vemurafenib resistance through tran-
scriptional upregulation in melanoma ( 37 ). However, the
mechanism underlying this event is not known. In mouse
embryonic fi broblasts, PDGFRβ was shown to be a target of
mTOR-dependent negative transcriptional downregulation
( 19 ). However, its role in mediating EGFR and/or mTOR TKI
resistance has not previously been recognized. In addition,
we identify a parallel pathway ( 38 ) by which ERK signaling
also suppresses PDGFRβ. Our data defi nitively show that
EGFR inhibitors de-repress PDGFRβ transcription, provid-
ing a potent mechanism underlying RTK switching. These
fi ndings have broad implications for understanding acquired
resistance to EGFR TKIs, and potentially mTOR inhibitors
as well, across multiple cancer types. Future studies will be
necessary to address this possibility more fully. In addition,
future studies will be needed to identify the transcriptional
machinery linking mTOR/S6K with PDGFRβ.
EGFR amplifi cation and mutation presents perhaps the
most compelling druggable target in GBM. Genetic and/
or functional PTEN loss ( 2 , 39 ), cooccurrence of c-MET
and PDGFRα gene amplifi cation ( 7 , 28 , 29 ), and pharma-
cokinetic considerations ( 40 ) all contribute to EGFR TKI
resistance, indicating a broad repertoire of resistance mecha-
nisms that can be cotargeted. However, the role of nongenetic
544 | CANCER DISCOVERY?MAY 2013 www.aacrjournals.org
Akhavan et al.
“rewiring” in mediating drug resistance remains to be
defi ned. Here , we have identifi ed a transcriptional repressive
mechanism by which EGFRvIII regulates PDGFRβ, shown
that EGFR-inhibited GBMs become PDGFRβ-dependent
for survival through mTOR-dependent transcriptional de-
repression, and showed that abrogation of EGFRvIII and
PDGFRβ stop tumor growth, providing a strong rationale for
combination therapy. These results provide the fi rst clinical
and biologic evidence for the concept of RTK “switching”
as an EGFR TKI resistance mechanism in GBM, and pro-
vide a molecular explanation for how tumors can become
“addicted” to a nonamplifi ed, nonmutated, physiologically
regulated RTK to evade targeted treatment.
Cell Lines and Media
U87 and isogenic U87-EGFRvIII, U87-EGFRvIII kinase dead,
U87-EGFRvIII/shPDGFRB, and U87-EGFRvIII kinase dead shPDGFRβ
cell lines were cultured in Dulbecco’s modifi ed Eagle’s medium
(DMEM; Cellgro) supplemented with 10% FBS (Omega Scientifi c)
and penicillin streptomycin-glutamine (PSQ; Invitrogen) in a humid-
ifi ed atmosphere of 5% CO 2 at 37°C. U87-EGFRvIII kinase dead cells
were a gift from W.K. Cavenee (Ludwig Institute for Cancer Research,
UCSD). U87-EGFRvIII-shPDGFRβ cells were generated by plasmid-
mediated transfection of shPDGFRβ into U87-EGFRvIII cells fol-
lowed by selection for stable clones. Neurosphere cell lines (GBM6,
GBM12, GBM39, HK296, HK242, and HK250) were cultured in
DMEM/F12 (Cellgro) supplemented with EGF, fi broblast growth
factor, heparin (Sigma), GlutaMax, and PSQ (Invitrogen). Long-term
erlotinib-resistant GBM39 neurospheres were cultured in the pres-
ence of erlotinib for 30 days until cells were resistant.
U87 cells were obtained from American Type Culture Collec-
tion and engineered to express EGFRvIII in the Mischel laboratory.
GBM6, GBM12, and GBM39 were obtained from coauthor Dr. C.D.
James at UCSF and authenticated by DNA fi ngerprinting. HK296,
HK242, and HK250 were obtained by Dr. H.I. Kornblum (UCLA) and
authenticated by immunoblot studies.
Isogenic human malignant glioma cells were implanted into
immunodefi cient SCID/Beige mice for subcutaneous xenograft stud-
ies as follows. SCID/Beige mice were bred and kept under defi ned-
fl ora pathogen-free conditions at the Association for Assessment of
Laboratory Animal Care–approved Animal Facility of the Division of
Experimental Radiation Oncology, UCLA. For subcutaneous implan-
tation of U87 tumor cells (or indicated isogenic U87-EGFRvIII,
U87-EGFRvIII kinase dead, U87-EGFRvIII/shPDGFRB, and U87-
EGFRvIII kinase dead shPDGFRB) or GBM39 xenografts, single-cell
suspensions were injected subcutaneously at 600,000 cells/150 μL in
a solution of Dulbecco’s PBS (dPBS) and Matrigel (BD Biosciences).
Tumor growth was monitored with calipers by measuring the per-
pendicular diameter of each subcutaneous tumor. For intracranial
xenograft studies, U251 cells were injected intracranially in rats as
described previously ( 41 ). Rapamycin was administered for 3 days at
2 mg/kg/d. All experiments were carried out in accordance with the
Animal Research Committee of UCLA.
Cell Proliferation Assays
Absolute viable cell counts were determined by Trypan blue exclu-
sion and counted on a hemocytometer. Relative cell proliferation
was determined using the Cell Proliferation Assay Kit (Chemicon),
as per manufacturer’s specifications. Briefly, cells were incubated
1.5 hours (5% CO 2 , 37°C) after the addition of tetrazolium salt WST-1
tetrazolium, monosodium salt], and the absorbance was then
measured in a microplate reader (Bio-Rad) at 450 nm, with a
background reading at 650 nm subtracted. For assays using erlo-
tinib or AG1295, small molecules or dimethyl sulfoxide (DMSO)
vehicle were added at the indicated doses on day 0 of assays. In
assays with PDGF-BB ligand, cultures were stimulated daily with
PDGF-BB ligand at 20 ng/mL for the indicated days. Neurospheres
were plated in laminin-coated 96-well dishes in neurobasal media
supplemented with 5% charcoal-stripped FBS (Omega Scientific)
and treated with indicated drug as above. For transient siRNA
knockdown of EGFRvIII, cells were incubated overnight with tran-
sient siRNA of EGFRvIII (Ambion) or scrambled siRNA (Ambion)
at 10 nmol/L with RNAiMax Lipofectamine reagent (Invitrogen)
and Opti-MEM (Invitrogen). Cells were then plated in 12-well
plates and were stimulated with PDGF-BB ligand or vehicle in
medium containing 2% charcoal-stripped FBS (Omega Scientific)
and counted as above.
Transient knockdown of EGFRvIII, S6K1, Raptor, and Rictor
(Ambion) were conducted as follows. siRNA was diluted to a fi nal
concentration of 10 nmol/L in Opti-MEM and 7.5 μL Lipofectamine
RNAi-max, in serum-containing, PSQ-free media overnight in a fi nal
volume of 6 mL in a 60-mm dish. Media was changed the following
morning, and cells were incubated for 24 hours before lysate collection.
For the generation of stable knockdown cell lines, cells (5 × 10 4 ) were
seeded in 12-well plates and maintained for 24 hours, after which the
medium was replaced with fresh 5% FBS medium including polybrene
(5 μg/mL; Sigma), and shRNA lentivirus was added to cells followed by
incubation for 24 hours.
Plasmids used were pcDNA control, wild-type mTOR, mTOR
S2215Y, and AKT E17K [mTOR constructs were a gift from F. Tamanoi
(UCLA), E17K was a gift from Ingo Mellinghoff (Memorial Sloan-
Kettering Cancer Center), and pcDNA control was from Addgene].
Empty vector, mTOR constructs, and AKT E17K were diluted to 2,500
ng in Opti-MEM and incubated with Lipofectamine PLUS and LTX
reagents (Invitrogen) according to the manufacturer’s instructions.
Cells were plated in 5 mL of 5% FBS, and 1 mL of Opti-MEM/plasmid/
Lipofectamine was added to each plate. Media was changed the next
day to serum-free media, and cells were incubated with erlotinib for
24 hours before lysates were collected. For S6 kinase wild-type and
constitutively active forms, plasmids were diluted to 16 μg in Opti-
MEM and incubated with Fugene6 (Roche) according to the manu-
facturer’s instructions. Cells were plated in 5 mL 10% FBS, and 1 mL
of Opti-MEM/plasmid/Fugene6 was added to each plate. The media
was changed to serum-free media the following morning, and cells
were incubated with U0126 for 24 hours. Lysates were then collected
for immunoblotting. Plasmids used were pcDNA control, EES6K1,
and T412D/T401D S6K1/2 (S6 kinase plasmids were a gift from Ivan
Gout, University College London) .
Cultured cells or snap-frozen tissue samples were lysed and
homogenized with radioimmunoprecipitation assay buffer (buffer,
Boston Bioproducts; protease and phosphatase inhibitor, Thermo-
scientifi c). Protein concentration was determined via BCA Assay
(reagents A and B, Thermoscientifi c; standards, Biorad) and sam-
ples were subjected to 4% to 12% gradient SDS–PAGE and then
transferred to a nitrocellulose membrane (Bio-Rad Laboratories).
The membrane was then probed with indicated primary antibod-
ies, followed by secondary antibodies conjugated to horseradish
MAY 2013?CANCER DISCOVERY | 545
An RTK Switch in Malignant Glioma
peroxidase. The immunoreactivity was revealed by use of an enhanced
chemiluminescence kit (Thermoscientifi c). Antibodies used in the study
include p-EGFR 1086 (Epitomics), p-EGFR 1068 , p-AKT 473 , p-AKT T308 ,
AKT, p-S6 S235/236 , p-PDGFRβ Y751 , p-ERK T202/Y204 , ERK, p-Met Y1234 ,
p-S6K1 T389 , S6K1, PathScan cocktail, p-NDRG1 T346 , β-actin, PDGFRβ,
PDGFRα (Cell Signaling), α-tubulin (Sigma); EGFR (Upstate).
Xenografts were excised from mice treated with vehicle or erlotinib as
described earlier. A portion of the tumor was fi xed in paraformaldehyde
and ethanol and sent to the Department of Pathology and Laboratory
Medicine at UCLA tissue core for slicing and staining as required.
U87-EGFRvIII tumors from vehicle- or erlotinib-treated mice were
harvested and homogenized in NP40-containing lysis buffer, then
loaded at 2,000 μg per RTK array membrane, according to the manufac-
turer’s instructions (R&D Systems). For U87-EGFRvIII cells in culture,
lysates were generated and loaded on RTK array as described earlier.
Pilot Study of Lapatinib
North American Brain Tumor Consortium trial 04-01 titled “A
biomarker and phase II study of GW 572016 (lapatinib) in recurrent
malignant glioma” enrolled consented patients from UCLA, UCSF,
Dana-Farber Cancer Center (Boston, MA), Memorial Sloan-Kettering
Cancer Center (New York, NY), University of Pittsburgh (Pittsburgh,
PA), Neuro-oncology branch of NIH (Bethesda, MD), University of
Wisconsin (Madison, WI), and Duke University (Durham, NC; ref.
12 ). Adult patients who had a Karnofsky performance score equal to
or greater than 60, who were not on enzyme-inducing antiepileptic
agents, and who had normal hematologic, metabolic, and cardiac func-
tion were eligible for this study. In addition, patients must have been
candidates for surgical re-resection at the time of enrollment. Patients
were administered 750 mg of lapatinib orally twice a day for 7 to
10 days (depending on whether treatment interval fell over a weekend)
before surgery, the time to steady state. Blood and tissue samples were
obtained at the time of resection. After recovery from surgery, patients
resumed lapatinib treatment at the neoadjuvant dose of 750 mg twice
a day until clinical or radiographic evidence for tumor progression was
found. The fi rst cohort of patients for whom tissue was available before
and after lapatinib ( n = 10) were included in this study.
TMAs were used to analyze PDGFRβ and p-EGFR Tyr 1086 IHC
staining in 140 GBM patient samples. Two GBM TMAs were con-
structed with a 0.6-mm needle to extract 252 representative tumor
tissue cores and 91 adjacent normal brain tissue cores from the
paraffi n-embedded tissue blocks of 140 patients with primary GBM
( 12 ). These cores were placed in a grid pattern into 2 recipient par-
affi n blocks, from which tissue sections were cut for IHC analysis
of p-EGFR Y1086 and PDGFRβ. EGFR and PDGFRβ staining was
scored and calculated by Fisher’s exact test.
In vitro , U87-EGFRvIII cells were incubated in serum-free media
with and without erlotinib for 32 hours, as well as in 10% serum
with and without rapamycin for 24 hours. At each time point,
cells were lysed in Trizol for RNA extraction. RT-PCR analysis was
conducted using primers designed to amplify either the primary
transcript of PDGFRb or the mRNA transcript. Primer sequences for
PDGFRb mRNA are AGGACACGCAGGAGGTCAT (forward) and
TTCTGCCAAAGCATGATGAG (reverse). Primer sequences for
PDGFRb primary transcripts are CATCTGCAAAACCACCATTG (for-
ward) and ACTTGCCTCTGCTGAGCATC (reverse). For the washout,
U87-EGFRvIII cells were plated in 10% FBS-containing media. The
media was changed to serum-free media and erlotinib (5 μmol/L) was
added at t = 0 hours. Media was changed at 24 hours, and erlotinib
(5 μmol/L) was added again at 48 hours. At each time point, cells were
lysed in Trizol for RNA extraction as described earlier. mRNA and
primary transcripts were normalized against 36B4 .
U87-EGFRvIII cells were transfected with Switchgear genomics
PDGFRβ promoter plasmid concurrently with control cytomegalo-
virus plasmid promoter Luciferase and Renilla and Firefl y Luciferase
control. Luciferase assay was conducted using Promega Dual Luci-
ferase Reporter Assay system.
ChIP assays were conducted in U87-EGFRvIII cells with or with-
out rapamycin (5 nmol/L) for 24 hours. Cells in 2 15-cm plates were
pooled for each replicate. ChIP was conducted as previously described
( 42 ) with minor modifi cations. Briefl y, cells were cross-linked for 5
minutes in 1% formaldehyde in PBS. After sonication (15 minutes total
sonication time in 30-second pulses), soluble chromatin from each rep-
licate was split 4 ways for overnight immunoprecipitations with 2 μg
of the following antibodies: mouse immunoglobulin G (IgG; Millipore
cat #12-371) antibody against polymerase II (Millipore clone CTD4H3,
cat #05-623, positive control). Five microliter of chromatin was used as
control. DNA–protein complexes were pulled down by incubation for
2 hours with protein G-sepharose, washed, and eluted with 1% SDS
buffer. Resulting chromatin was de-cross linked with heat and protein
digested with proteinase K, along with input controls. Genomic DNA
(gDNA) was assayed by quantitative PCR (qPCR) with primers amplify-
ing PDGFRb transcriptional start site (TSS) and a fragment upstream
of the TSS. qPCR values were normalized against the input gDNA
content for each replicate. qPCR primers are available upon request.
ERK and MET Studies
For treatment with erlotinib and the MEK inhibitor U0126 or the
MET inhibitor PHA-665752, adherent cells were plated in 10% FBS.
GBM39 cells were plated on plates coated with laminin (Sigma) as
described earlier in complete neurosphere media. The following day,
media was changed to serum-free media (U0126), 2% FBS-containing
media (U87-EGFRvIII, PHA-665752), or DMEM/F12 (GBM39, PHA-
665752). Cells were then incubated with drug for 24 hours before
being lysed for immunoblot analysis.
For HGF stimulation, U87-EGFRvIII cells were plated as described
earlier. Media was changed to serum-free media, and HGF was added
at 50 or 100 ng/mL concurrently with DMSO vehicle or erlotinib
(5 μmol/L) 24 hours before collection. Cells were stimulated again
4 hours before collection.
Fisher’s exact test was used to assess correlation between EGFR
and PDGFRβ in clinical samples. All other comparisons of cell pro-
liferation, transcript level, and tumor volume were conducted using
one-way or two-way ANOVA with Tukey’s Honestly Signifi cant Dif-
ference test as required. All results are shown as mean ± SD.
Disclosure of Potential Confl icts of Interest
P.S. Mischel and T.F. Cloughesy served on an advisory board for
Celgene’s mTOR kinase inhibitor program and collaborated with
Celgene and Sanofi through research contracts on their mTOR
kinase and PI3K/mTOR kinase inhibitor clinical trials. H.I. Korn-
blum collaborated with Celgene on a research contract for the mTOR
kinase inhibitor program. No potential confl icts of interest were
disclosed by the other authors .
546 | CANCER DISCOVERY?MAY 2013 www.aacrjournals.org
Akhavan et al.
Conception and design: D. Akhavan, A.A. Nourian, D. Nathanson,
W.K. Cavenee, S.J. Bensinger, P.S. Mischel
Development of methodology: D. Akhavan, A.L. Pourzia, A.A. Nourian,
H.V. Vinters, F. Tamanoi, H.I. Kornblum, P.S. Mischel
Acquisition of data (provided animals, acquired and man-
aged patients, provided facilities, etc.): D. Akhavan, A.L. Pourzia,
K.J. Williams, D. Nathanson, G.R. Villa, A. Nael, H.V. Vinters,
W.H. Yong, M. Flagg, T. Sasayama, C.D. James, T.F. Cloughesy,
Analysis and interpretation of data (e.g., statistical analysis,
biostatistics, computational analysis): D. Akhavan, A.L. Pourzia,
A.A. Nourian, K.J. Williams, K. Tanaka, F. Tamanoi, C.D. James,
H.I. Kornblum, T.F. Cloughesy, S.J. Bensinger, P.S. Mischel
Writing, review, and/or revision of the manuscript: D. Akhavan,
A.L. Pourzia, I. Babic, G.R. Villa, H.V. Vinters, W.H. Yong, C.D. James,
H.I. Kornblum, T.F. Cloughesy, W.K. Cavenee, S.J. Bensinger,
Administrative, technical, or material support (i.e., report-
ing or organizing data, constructing databases): D. Akhavan,
A.A. Nourian, I. Babic, K. Tanaka, A. Nael, H. Yang, J. Dang, H.V. Vinters,
M. Flagg, P.S. Mischel
Study supervision: F. Tamanoi, S.J. Bensinger, P.S. Mischel
This work was supported by grants from the Concern Foun-
dation, Margaret Early Medical Research Trust, and the Sontag
Foundation (to S.J. Bensinger), the UCLA Clinical and Translational
Science Institute (NIH NCATS UL1TR000124 to S.J. Bensinger),
NIH grants NS73831 and CA119347 (to P.S. Mischel), the Ziering
Family Foundation in Memory of Sigi Ziering (to P.S. Mischel and
T.F. Cloughesy), and the Ben and Catherine Ivy Foundation (P.S.
Mischel and T.F. Cloughesy). This work was also supported by NIH
Grant P01-CA95616 to W.K. Cavenee, who is a fellow of the National
Foundation for Cancer Research; the UCLA Tumor Immunology
Training Grant (5T32CA009120-35 to K.J. Williams); NIH CA41996
(to F. Tamanoi); and by Moores Cancer Center Core grant NCI
Received October 31, 2012; revised March 4, 2013; accepted
March 8, 2013; published OnlineFirst March 26, 2013.
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