Published OnlineFirst April 22, 2014; DOI: 10.1158/1541-7786.MCR-13-0650
Molecular
Cancer
Research
Cell Death and Survival
PI3K and Bcl-2 Inhibition Primes Glioblastoma Cells to
Apoptosis through Downregulation of Mcl-1 and
Phospho-BAD
Fresia Pareja1, David Macleod1, Chang Shu1, John F. Crary1, Peter D. Canoll1, Alonzo H. Ross2, and
Markus D. Siegelin1
Abstract
Glioblastoma multiforme (GBM) is a highly malignant human brain neoplasm with limited therapeutic options.
GBMs display a deregulated apoptotic pathway with high levels of the antiapoptotic Bcl-2 family of proteins and
overt activity of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Therefore, combined interference of
the PI3K pathway and the Bcl-2 family of proteins is a reasonable therapeutic strategy. ABT-263 (Navitoclax), an
orally available small-molecule Bcl-2 inhibitor, and GDC-0941, a PI3K inhibitor, were used to treat established
glioblastoma and glioblastoma neurosphere cells, alone or in combination. Although GDC-0941 alone had a
modest effect on cell viability, treatment with ABT-263 displayed a marked reduction of cell viability and induction
of apoptotic cell death. Moreover, combinatorial therapy using ABT-263 and GDC-0941 showed an enhanced
effect, with a further decrease in cellular viability. Furthermore, combination treatment abrogated the ability of stem
cell–like glioma cells to form neurospheres. ABT-263 and GDC-0941, in combination, resulted in a consistent and
significant increase of Annexin V positive cells and loss of mitochondrial membrane potential compared with either
monotherapy. The combination treatment led to enhanced cleavage of both initiator and effector caspases.
Mechanistically, GDC-0941 depleted pAKT (Serine 473) levels and suppressed Mcl-1 protein levels, lowering the
threshold for the cytotoxic actions of ABT-263. GDC-0941 decreased Mcl-1 in a posttranslational manner and
significantly decreased the half-life of Mcl-1 protein. Ectopic expression of human Mcl-1 mitigated apoptotic cell
death induced by the drug combination. Furthermore, GDC-0941 modulated the phosphorylation status of BAD,
thereby further enhancing ABT-263–mediated cell death.
Implications: Combination therapy with ABT-263 and GDC-0941 has novel therapeutic potential by specifically
targeting aberrantly active, deregulated pathways in GBM, overcoming endogenous resistance to apoptosis. Mol
Cancer Res; 12(7); 987–1001. 2014 AACR.
Introduction
Glioblastoma, the most common primary malignant brain
tumor, displays a remarkable resistance toward therapy. The
current mainstay of treatment is surgery followed by radiation/chemotherapy (1). In the last decade, the most promising therapeutic regimens include the oral chemotherapeutic drug, temozolomide (Temodar). This DNA modifying
Authors' Affiliations: 1Department of Pathology & Cell Biology, Columbia
University Medical Center, New York, New York; and 2Department of
Biochemistry and Molecular Pharmacology, University of Massachusetts
Medical School, Worcester, Massachusetts
Note: Supplementary data for this article are available at Molecular Cancer
Research Online (http://mcr.aacrjournals.org/).
Corresponding Author: Markus D. Siegelin, Department of Pathology and
Cell Biology, Columbia University Medical Center, 630 West 168th Street,
P&S 15-401, New York, NY 10032. Phone: 212-305-1993; Fax: 212305-6596; E-mail: [email protected], [email protected],
[email protected]
doi: 10.1158/1541-7786.MCR-13-0650
2014 American Association for Cancer Research.
compound shows efficacy particularly in patients who have
silenced expression of the DNA—repair enzyme O6methylguanine DNA methyltransferase (MGMT). Testing
patient samples for MGMT promoter methylation status
predicts responses to temozolomide treatment. Certain
therapeutic regimens, involving temozolomide, offer slight
survival benefits in the range of 3 months. Thus, there is an
urgent need for novel pathway-related rational therapies.
One of the mechanisms as to why glioblastoma escapes
various treatment modalities is the fact that they exhibit high
resistant to apoptosis (2–4). Multiple factors mediating this
resistance have been described, including the aberrant
expression of the bcl-2 family of proteins. Glioblastomas
display high levels of bcl-2 (5) and bcl-xL (6). Therefore,
these tumors are potential candidates for treatment with
BH3 mimetics, such as ABT-263 (Navitoclax; ref. 7), an
orally available inhibitor of bcl-2 and bcl-xL. From a mechanistic point of view, BH3 mimetics bind to the inhibitory
bcl-2 family of proteins and prevent their interaction with
proapoptotic Bcl-2 family members, such as Bad, Bid, and
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Bim. This allows them to drive intrinsic apoptosis with
translocation of cytochrome c to the cytosol, formation of the
apoptosome, cleavage of caspase-9, and subsequent activation of effector caspase-3 to complete programmed cell
death. This class of molecules is more effective in tumors
with high levels of bcl-2/bcl-xL. ABT-263 is currently under
investigation in various malignancies, either as a single drug
compound, or in combination with other antitumor
reagents (8, 9) and has already reached clinical trials (10).
Specifically, preclinical studies, involving ABT-263, have
shown that it has activity against small cell lung cancer
(SCLC) as well as acute lymphoblastic leukemia (7). Especially, the efficacy in SCLC, which despite treatment nowadays still have a poor prognosis, is of significant relevance
because this type of lung cancer is not amenable to the
modern approaches of personalized medicine so far as
compared with non–small cell lung cancer (NSCLC) in
which certain mutations of the EGFR gene (deletions in exon
19 and point mutations in exon 21) render these tumors
sensitive to the tyrosine kinase inhibitors, gefitinib or erlotinib. The toxicity profile of ABT-263 is favorable compared
with standard chemotherapies. However, one caveat of
ABT-263 and related molecules is that tumors with high
protein expression levels of Mcl-1, another member of the
bcl-2 family of proteins with antiapoptotic properties, exhibit marked resistance against them. Glioblastomas display
high levels of Mcl-1 expression, which is driven by several
upstream cascades, such as NOTCH (11) or CREB3L2ATF5 (12) signaling pathways. To overcome this resistance,
several strategies have been devised, such as the targeting of a
second molecular pathway that allows the depletion of high
levels of endogenous Mcl-1 in cancer cells.
Along these lines, we herein show that GDC-0941, a
recently developed and orally available thienopyrimidine
derivative that inhibits PI3K in the nanomolar range
(13), lowers Mcl-1 levels in glioblastoma cells, rendering
them more sensitive to the apoptotic properties of ABT-263.
In addition, GDC-0941 affects the phosphorylation status
of BAD, thereby modulating the sensitivity of glioblastoma
cells to BH3 mimetics.
Materials and Methods
Cell lines and reagents
Human glioblastoma cell lines, LN229 (p53 mutated,
PTEN wild-type), U87 (p53 wild-type, PTEN mutated),
and U373 (p53 mutated, PTEN mutated) with defined
mutation status were cultured as described previously (14)
and were obtained from the American Type Culture Collection (ATCC). The ATCC confirmed the identities of the
glioblastoma cells (except for U373, which is no longer
verified by the ATCC). The stem cell-like glioma (neurosphere) NCH421K, NCH644, and NCH690 cells were
verified and obtained from Cell Line Services (Heidelberg,
Germany) and were cultured in neural stem cell medium
containing DMEM/F12 (Gibco-Invitrogen), recombinant
human epidermal growth factor (rhEGF, 20 ng/mL; Sigma),
basic fibroblast growth factor (bFGF, 20 ng/mL; Upstate),
BIT: bovine serum albumin, transferrin, insulin, and pen-
988
Mol Cancer Res; 12(7) July 2014
icillin G and streptomycin (Gibco-Invitrogen) in the absence
of serum as previously described (15, 16). Primary neurosphere GS9-6 stem-like glioma cells (12) are p53 wild type
and were cultured in the same media as NCH421K cells.
Cycloheximide and MG132 were purchased from Sigma
Aldrich. GDC-0941 and ABT-263 were purchased from LC
Laboratories.
Neurosphere formation assay
NCH421K cells were mechanically dissociated and plated
at a density of 500 cells per well in 24-well plates. Twentyfour hours later, cells were treated with GDC-0941 and
ABT-263, singly or in combination. Following 10 days,
neurosphere formation was assessed by counting the number
of neurospheres that harbor at least 25 cells per sphere as
described in refs. 17 and 18). Experiments were performed at
least in duplicates and statistical analysis was performed.
Antibodies and Western blotting/immunoblotting
Antibodies to MCl-1 (1:500; CST), phospho-Akt [Serine
473 (Ser 473); 1:500; CST], pan Akt (1:1,000; CST),
cleaved caspase-3 (1:250; CST), human caspase-9
(1:1,000; CST), 14-3-3 (1:1,000; Santa Cruz Biotech),
BAD (1:200; CST), phospho-BAD 112 (1:200; CST),
phospho-BAD 136 (1:200; CST), and actin (1:2,000, clone
AC15; Sigma Aldrich) were used. Western blotting was
performed as described previously. Samples were lysed either
in RIPA cell lysis buffer (CST) supplemented with protease
inhibitor cocktail and phosphatase inhibitors or directly
lysed in Laemmli-sample buffer. Lysates were boiled and
equal amounts of protein were loaded onto a gradient (4%–
12%) poly-acrylamide precast-gel (Invitrogen). Samples
were resolved at 120 V (constant voltage) for 15 minutes
and subsequently for 45 minutes at 180 V (constant voltage).
The gel was transferred (wet-transfer) to a PDVF membrane
(Bio-Rad) at 45 V (constant) for 90 minutes. Protein transfer
was confirmed by Ponceau S staining. Membranes were
washed 3 times in TBS-T and blocked with 5% milk in TBST. After blocking, membranes were washed once and incubated with the primary antibody according to the vendor's
instructions. Secondary antimouse or anti-rabbit antibodies
conjugated with horseradish-peroxidase were used at dilutions ranging from 1:2,000 to 1:10,000. For immunodetection, the Pierce ECL Western blotting substrate was utilized.
Membranes were developed either by exposure to a film or by
using the C-DiGit Blot Scanner (LI-COR).
Real-time PCR and cDNA synthesis
Glioblastoma cells were harvested after indicated treatments and RNA was isolated with a Column-Based Isolation
Kit (RNeasy Mini Kit; Qiagen), as described in the manual
of instructions. Subsequently, 500 ng of cDNA was reverse
transcribed, using the "First Strand cDNA Synthesis Kit"
(Origene). Real-time PCR was performed, utilizing a
"Omnimix HS Kit" (Cepheid). The following primers were
used: 18S forward: AGT CCC TGC CCT TTG TAC ACA;
18S reverse: GAT CCG AGG GCC TCA CTA AAC; Mcl-1
forward: CCA AGA AAG CTG CAT CGA ACC AT, and
Molecular Cancer Research
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Published OnlineFirst April 22, 2014; DOI: 10.1158/1541-7786.MCR-13-0650
GDC-0941 Overcomes Intrinsic Apoptotic Resistance in Glioblastoma
Mcl-1 reverse: CAG CAC ATT CCT GAT GCC ACC T.
The PCR cycle parameters are available upon request. Data
analysis were performed by the "Delta-Delta CT METHOD" and data are provided as percentages relative to the
control.
Analysis of cellular viability, apoptosis, and
mitochondrial membrane potential
3(4,5-Dimethyl-thyazoyl-2-yl)2,5 diphenyltetrazolium
bromide (MTT) colorimetric assay was conducted as previously described. In brief, 2,000 cells per well were seeded in
96-well plates at least in duplicate 24 hours before treatment.
After treatment cells were incubated with MTT for 2 hours
at 37 C and assayed for absorbance at 570 nm. For apoptosis
determination, cells were stained with Annexin V (FITC)/
propidium iodide as described in the manufacturer's instructions (BD Bioscience). Following staining, cells were run on
a FACS Calibur machine (Becton Dickinson). Flow cytometric plots were generated by FlowJo software. To analyze
mitochondrial membrane potential (MMP) in response to
treatment, cells were stained with JC-1 (Molecular Probes)
according to the manufacturer's instructions. Cells were
analyzed on a FACS Calibur machine (Becton Dickinson)
on FL1 (green channel) and FL2 (red channel) and the
number/percentage of cells with loss of MMP was determined as described in ref. 19.
Transfections, plasmids, and siRNAs
pcDNA-3, pcDNA3-hMcl1 (Addgene ID: 25375),
pcDNA3 Bad S112A S136A (phosphorylation deficient;
Addgene ID: 8799), and pcDNA3 Bad (Addgene ID: 8778)
were used. For transient transfections, glioblastoma cells
were seeded in 12-well dishes at a density of 50,000 cells per
well. Twenty-four hours later, cells were transfected with the
designated plasmids, using Lipofectamine 2000 or Fugene
HD according to the manufacturer's instructions. Cells were
assayed for protein expression 48 to 72 hours after transfection, and following confirmed ectopic expression of the
protein of interest experiments were performed. Nontargeting human siRNA pools and human siRNA Mcl-1 pools
were obtained from Thermo Fisher Scientific.
Immunohistochemistry of glioblastoma specimens
Tissue microarrays (TMA), containing 34 de-identified
glioblastoma specimens (in triplicate), were provided by the
Division of Neuropathology at the Columbia University
Medical Center. TMAs were created by removing 3 onemillimeter cores of glioblastoma tumors. Four-micrometerthick sections were cut and deparaffinized in xylene. For
antigen, retrieval sections were pretreated by boiling samples
in 0.01 M citrate buffer (pH 6.0) for 5 minutes. TMAs were
stained with a dilution of 1:50 of phosphorylated Akt (phAkt; Ser 473; clone D9E; CST Inc.) 1 and with a dilution of
1:100 of phosphorylated BAD (ph-BAD; Ser 112; clone
40A9; CST, Inc.), respectively. The secondary antibodies
included anti-Rabbit IgG (1:200; purchased from Vector
Laboratories), and antimouse polymeric antibodies (purchased from Dako) and diaminobenzidine was used as
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chromogene. TMAs were scored by 3 board-certified pathologists (J.F. Crary, P.D. Canoll, and M.D. Siegelin), using a
3-tier scoring system (0: no expression, 1: weak expression,
2: strong expression). When there was a disagreement, the
majority of scores ( 2 of 3 wins) was taken. In the rare case,
where the pathologists span the full range (0, 1, 2), then a
score of 1 was assigned. A final score for each tumor was
reached based on the highest scoring core (out of 3 cores per
glioblastoma specimen). Statistical analysis was performed
by Spearman correlation analysis. Representative photographs of the TMAs were taken.
Statistical analysis
Data were analyzed by 2-sided unpaired t tests using a
GraphPad Prism or one-way analysis of variance followed by
Tukey Multiple Comparison Test. Values are provided as
mean SD or mean SEM of replicates of a representative
experiment out of at least 2 independent determinations. A P
value of less than 0.05 (P < 0.05) was accepted as statistical
significant.
Results
Glioblastoma cells display high expression of Mcl-1, phAkt, and ph-BAD
To demonstrate that glioblastoma cells display high levels
of the antiapoptotic Bcl-2 family protein, Mcl-1, as well as a
deregulated PI3K pathway, we determined the expression
levels of ph-Akt (Ser 473) and Mcl-1 in 3 established and 3
glioblastoma neurosphere cultures [NCH644, NCH690,
and GS9-6 (a low passage ex vivo neurosphere cell line),
respectively (Supplementary Fig. S1A)]. Consistent with
their known PTEN mutation status, U87 and U373 glioblastoma cells displayed higher levels of ph-Akt as compared
with the wild-type LN229 glioblastoma cells. About the
expression status of Mcl-1, U373 revealed the lowest expression, whereas U87 showed the highest levels (Supplementary
Fig. S1A). The glioma neurosphere cells showed similar
protein levels of Mcl-1 (Supplementary Fig. S1A). BAD is an
important sensitizer for the intrinsic pathway of apoptosis
and multiple phosphorylation sites were identified, for
example at amino acid 112 and 136, that when phosphorylated render cells more resistant to apoptosis. Our glioblastoma cell line panel showed detectable levels of ph-BAD at
S136 and S112 (Supplementary Fig. S1A), suggesting that
BAD is in a rather inactive state in malignant glioma and that
targeting this molecule might counteract the intrinsic apoptotic resistance associated with high-grade gliomas.
The combination of GDC-0941 and ABT-263 elicits
enhanced loss in cellular viability in glioblastoma cells
First, we determined the effect of the single reagents,
GDC-0941 and ABT-263, on the viability of U373 and
LN229 cells, respectively. Treatment of LN229 glioma cells
with increasing concentrations of GDC-0941 did not result
in a significant reduction in cellular viability after 24 hours
(Fig. 1B). In contrast, increasing concentrations of ABT-263
(1 mmol/L: 63.51% 1.893, P ¼ 0.0064; 2 mmol/L:
69.61% 0.5927, P ¼ 0.0078; 4 mmol/L: 61.32% Mol Cancer Res; 12(7) July 2014
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Pareja et al.
Figure 1. Effects of the combination
treatment of ABT-263 and GDC0941 on cellular viability (A–F) and
neurosphere formation (G–H). A–C,
LN229 cells were treated with
increasing concentrations of ABT263 (1, 2, and 4 mmol/L), GDC-0941
(1, 2, and 4 mmol/L), or a
combination of both (2 mmol/L
ABT-263 and 2 mmol/L GDC-0941),
for 24 hours. Thereafter, MTT
assays were performed to
determine cellular viability. Treated
cells were compared with control
cells (DMSO) and statistical
analysis is provided ( , P < 0.05;
, P < 0.01; , P < 0.001; "ns,"
nonsignificant). D–F, U373 cells
were treated with increasing
concentrations of ABT-263 (1, 2,
and 4 mmol/L), GDC-0941 (1, 2, and
4 mmol/L), or a combination of both
for 48 hours (4 mmol/L ABT-263
and 4 mmol/L GDC-0941).
Thereafter, MTT assays were
performed to determine cellular
viability. Treated cells were
compared with control cells
(DMSO) and statistical analysis is
provided ( , P < 0.05; , P < 0.01;
, P < 0.001). G, NCH421K stem
cell–like glioma cells were treated
with ABT-263 (2 mmol/L), GDC0941 (2 mmol/L), or the combination
of both reagents (2 mmol/L of each
compound) and assessed for
neurosphere formation 2 weeks
after plating (G). The number of
neurospheres was counted and is
represented in H. Values are given
as mean SEM of representative
experiments. ABT—ABT-263;
GDC—GDC-0941; ABT þ GDC—
combined treatment of ABT-263
and GDC-0941. Numbers
represent concentrations of the
indicated compounds in mmol/L.
A P value of less than 0.05 is
indicated by one star " ", whereas a
P value of less than 0.01 is
highlighted by 2 stars " ".
A P-value less than 0.001 is
indicated by a star triplet ( ).
Numbers represent concentrations
of the indicated compounds in
mmol/L.
1.528, P ¼ 0.0046) statistically significantly reduced cellular
viability in LN229 cells (Fig. 1A). Next, we aimed to
determine the effect of GDC-0941 and ABT-263 combination therapy on cellular viability. Treatment of LN229
cells with the combination of these drugs exerted a statistically significant decrease of cellular viability (38.76% 0.3905) compared with single treatments with either ABT263 (P < 0.0001) or GDC-0941 (P < 0.0001; Fig. 1C).
Contrasting the results in LN229 cells, treatment of U373
glioma cells with increasing dosages of GDC-0941 (1 mmol/
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Mol Cancer Res; 12(7) July 2014
L: 86.62 0.9061, P ¼ 0.0279; 2 mmol/L: 83.66 2.039,
P ¼ 0.0210; 4 mmol/L: 75.74 3.256, P ¼ 0.0085) elicited
a mild, but statistically significant reduction in cellular
viability with respect to the vehicle-treated cells (Fig. 1E).
Increasing concentrations of ABT-263 (1 mmol/L: 87.40 1.512, P ¼ 0.0028; 2 mmol/L: 101.3 2.867, P ¼ 0.4285;
4 mmol/L: 75.57 1.187, P ¼ 0.0001) resulted in a
minimal reduction of cellular viability in U373 that was
not as pronounced as in LN229 glioblastoma cells (Fig. 1D).
The combination regimen revealed a statistical significant
Molecular Cancer Research
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GDC-0941 Overcomes Intrinsic Apoptotic Resistance in Glioblastoma
decrease of cellular viability (56.49 2.861) compared with
single treatments with either ABT-263 (P ¼ 0.0108) or
GDC-0941 (P ¼ 0.0113; Fig. 1F).
The combination of GDC-0941 and ABT-263 is effective
in ex vivo glioblastoma neurosphere cultures
Glioblastomas are bona-fide examples of heterogeneous
tumors, harboring different cell populations. Among these,
the so-called "cancer stem cells" are of particular interest as
they are implicated in the rapid recurrence of glioblastomas
in patients, and are highly resistant to treatment. To this end,
we asked the question if stem cell–like glioblastoma cells are
susceptible to the ABT-263 and GDC-0941 combination
regimen. With this aim, we treated the neurosphere forming
NCH421K glioblastoma cells with ABT-263 and GDC0941, singly or in combination, and conducted nerosphere
formation assays. Although single reagents had a modest
effect on the formation of NCH421K cell neurospheres, the
combination regimen had a marked inhibitory effect on
neurosphere formation (Fig. 1G and H). This finding
suggests that combination treatment may influence tumorigenicity of these glioblastoma cells and, more importantly,
that it not only affects the bulk of the tumor, but also targets
the potential stem cell–like cellular fraction within malignant gliomas.
The combination of GDC-0941 and ABT-263 as well as
ABT-199 causes enhanced apoptotic cell death with
rapid loss of MMP and increased activation of initiator
and effector caspases
To test the hypothesis that combination of GDC-0941
and ABT-263 works through enhanced apoptosis, we performed Annexin V staining with subsequent flow cytometry
in one low-passage ex vivo glioblastoma neurosphere cell line,
GS9-6, 3 glioblastoma neurosphere cell lines, NCH421K,
NCH690, and NCH644, and in 1 established glioblastoma
cell line, LN229. Glioblastoma cells were treated with 2
mmol/L of GDC-0941, ABT-263, or the combination of
both reagents for 24 hours. Although single treatments with
GDC-0941 and ABT-263 revealed modest induction of
apoptosis, the combination treatment induced a significantly
higher proportion of Annexin V positive cells as compared
with the single treatments in all cell lines tested (Fig. 2A and
B). Furthermore we also tested as to whether another orally
available Bcl-2 inhibitor, ABT-199, which causes fewer
side effects (thrombocytopenia) will induce enhanced cell
death in the presence of GDC-0941. To test this hypothesis,
2 glioblastoma neurosphere cell lines, NCH644 and
NCH690 as well as the established glioblastoma LN229
cells were treated with suboptimal dosages of ABT-199 in
combination with GDC-0941. Akin to ABT-263, ABT-199
cooperates with GDC-0941 to enhance cell death (Fig. 2C
and D). Our findings again suggest that our proposed
treatment combination exerts activity against the stem-like
cells fraction of glioblastoma. Apoptosis is accompanied by
loss of mitochondrial integrity, and because ABT-263 affects
intrinsic apoptotic signaling, we hypothesized that the
enhanced apoptotic cell death induced by GDC-0941 and
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ABT-263 could be accompanied by a concomitant loss of
MMP. LN229 cells were treated under the same conditions
as above, stained with JC-1 and subjected to flow cytometric
analysis. Although LN229 cells treated with ABT-263
(mean: 46.33 1.667) and GDC-0941 (mean: 24.67 0.8819) showed a modest loss in MMP, the combined
treatment led to a significant higher proportion of cells with
loss in MMP (mean: 80.33 0.8819; <0.0001; n ¼ 3; Fig.
2E and F). Because the initiator and effectors of apoptosis are
caspases, we wished to determine if caspase-9 and caspase-3
show activation in the various treatment settings. We theorized that given that the combined treatment of GDC0941 and ABT-263 caused more apoptosis, and more cells
with loss of MMP, cells treated with the drug combination
would exhibit a stronger activation of initiator as well as
effector caspases. To this end, LN229 and U373 glioblastoma cells were treated with ABT-263, GDC-0941, or the
combination of both reagents for 7 hours and subjected to
immunoblot analysis for cleavage/activation of caspases. We
found a slight activation of both initiator caspase-9 and
effector caspase-3 in ABT-263–treated cells. However, the
combination of ABT-263 and GDC-0941 exerted further
activation of caspase-9 and caspase-3 compared with the
single treatments in both LN229 and U373 glioblastoma
cells (Fig. 3C and D), in keeping with the enhanced
apoptosis and loss of MMP observed.
GDC-0941 inhibits PI3K signaling in glioblastoma cells
To elucidate the mechanism as to why ABT-263 and
GDC-0941 demonstrate enhanced apoptotic cell death, loss
of MMP and activation of caspases when compared with the
single treatments, we investigated if GDC-0941 could lower
the apoptotic threshold by suppression of Akt signaling. All,
U373, LN229 (Fig. 3A and B), and U87 (Fig. 4C) cells
showed complete inhibition of Akt phosphorylation at Ser
473 upon GDC-0941 treatment, in the presence or absence
of ABT-263, showing that GDC-0941 is a potent and
inhibitor of the PI3K signaling cascade in malignant glioma
cells.
GDC-0941 depletes Mcl-1 protein levels in a
posttranscriptional manner in glioblastoma cells
ABT-263 binds strongly to Bcl-2 and Bcl-xL, but weakly
to Mcl-1 (7). By interacting with these antiapoptotic molecules, proapoptotic bcl-2 family members, such as Bim and
Bax, are released and can initiate and facilitate intrinsic
apoptosis. Because of the inefficient interaction of ABT263 with Mcl-1, the latter is likely to mediate resistance
against ABT-263 therapy. Thereafter, we hypothesized that
GDC-0941 could cause a reduction in Mcl-1 levels and
thereby primes glioblastoma cells to the cytotoxic actions of
ABT-263. To clarify this, we treated stem cell–like glioma
cells (grown as neurospheres), GS9-6 and NCH421K cells as
well as established glioblastoma cell lines LN229, U373, and
U87 glioblastoma cells with increasing concentrations of
GDC-0941 and subjected them to Western blot analysis.
Although GDC-0941 did not have an effect on Mcl-1
protein levels in U373 glioma cells (Supplementary
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NCH690
A
Control
B
GDC-0941
6
0
0
8
###
***
89
5
ABT-263
0
8
85
7
% Annexin V–positive cells
Propidium iodide
100
84
8
ABT263+GDC-0941
0
53
4
43
###
**
80
Control
GDC-0941 2 μmol/L
ABT-263 2 μmol/L
ABT+GDC 2 μmol/L+2 μmol/L
###
###
***
***
###
*
60
40
20
0
NCH421K GS9-6
Annexin V
NCH690 NCH644
LN229
NCH644
C
Control
GDC-0941
11
2
1
28
D
2
85
17
78
2
ABT-199+GDC-0941
ABT-199
3
69
% Annexin V–positive cells
Propidium iodide
80
2
3
64
32
***
60
###
***
###
40
***
20
0
1
Control
GDC-0941 2 μmol/L
ABT-199 2 μmol/L
ABT+GDC 2 μmol/L+2 μmol/L
###
NCH644
NCH690
LN229
Annexin V
GDC-0941
Control
Red channel
0
85
0
15
ABT-263
0
0
57
43
0
0
77
23
ABT-263+GDC-0941
0
26
0
F
% of cells with loss in MMP
E
***
LN229
100
2 μmol/L
2 μmol/L
80
2 μmol/L
60
40
2 μmol/L
20
0
Control
GDC
ABT
ABT+GDC
74
Green channel
Figure 2. Effects of the combination treatment of ABT-263/ABT-199 and GDC-0941 on apoptosis induction and MMP in LN229 glioblastoma cells. A and B,
NCH644, NCH690, NCH421k, GS9-6, and LN229 cells were treated with 2 mmol/L ABT-263, 2 mmol/L GDC-0941, and the combination of both for 24 hours.
Subsequently, cells were stained with Annexin V/propidium iodide (PI) and analyzed by flow cytometry. FL1 (Annexin V) and FL3 (propidium iodide) are
represented on the x-axis and y-axis, respectively. One representative plot is shown in A. (Continued on the following page.)
992
Mol Cancer Res; 12(7) July 2014
Molecular Cancer Research
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Published OnlineFirst April 22, 2014; DOI: 10.1158/1541-7786.MCR-13-0650
GDC-0941 Overcomes Intrinsic Apoptotic Resistance in Glioblastoma
A
B
LN229
Figure 3. Inhibition of PI3K
signaling by GDC-0941 in
malignant glioma cells and
enhanced activation of initiator
and effector caspases by the
combined treatment of ABT-263
and GDC-0941 A–D) LN229 (A and
C) and U373 (B and D) were treated
with 2 mmol/L GDC-0941, 2 mmol/L
ABT-263 or the combination of
both reagents for 7 hours and
subjected to immunoblotting.
Shown are the results (A and B) for
ph-Akt (Ser 473; ph-Akt) and total
Akt. Whole cell lysates were also
resolved by Western blot analysis
and probed for initiator caspase-9
(CP9) and effector caspase-3
(cCP3) in both LN229 (C) and U373
(D) cells. ABT—ABT-263; GDC—
GDC-0941; ABT þ GDC—
combined treatment of ABT-263
and GDC-0941.
U373
GDC (μmol/L)
0
0
2
2
GDC (μmol/L)
0
2
0
2
ABT263 (μmol/L)
0
2
0
2
ABT263 (μmol/L)
0
0
2
2
ph-Akt
60 kDa
ph-Akt
60 kDa
Akt
60 kDa
Akt
60 kDa
C
D
LN229
U373
GDC (μmol/L)
0
2
0
2
GDC (μmol/L)
0
2
0
2
ABT263 (μmol/L)
0
0
2
2
ABT263 (μmol/L)
0
0
2
2
CP9
47
35/37
kDa
CP9
47
35/37
kDa
cCP3
17/19
kDa
cCP3
17/19
kDa
Actin
45 kDa
Actin
45 kDa
Fig. S1B), treatment of LN229 and U87 glioblastoma cells as
well as the neurosphere forming NCH421K and GS9-6 cells
(18), caused a marked reduction of Mcl-1 protein levels,
which was observed to be dose dependent (Fig. 4A–D). We
then asked the question as to whether the combination
treatment of ABT-263 and GDC-0941 could also reduce
Mcl-1 levels. Confirming this notion, the combination
therapy led to a further decrease of Mcl-1 levels in LN229
cells in a time course experiment as early as 4 hours after
treatment (Fig. 4E). The timeframe of the ABT-263/GDC0941–mediated reduction of Mcl-1 protein levels coincided
with the peak cleavage of both initiator caspase-9 and effector
caspase-3 (Fig. 4E). Of note, phosphorylation of Akt at Ser
473 was undetectable in the presence of GDC-0941 regard-
less of it being administered as a single reagent or in
combination with ABT-263 (Fig. 4E). We then evaluated
the effect of ABT-263 on Mcl-1 levels. We did not detect any
changes of expression in Mcl-1 levels in LN229 upon ABT263 treatment (Supplementary Fig. S1D), whereas in U373
cells ABT-263 led to a marked increase in Mcl-1 protein
levels (Supplementary Fig. S1C), the significance of which
remains to the elucidated. Phosphorylation of Akt was
minimal or not affected by ABT-263 in both LN229 and
U373 glioblastoma cells 7 hours after treatment (Fig. 4A and
Supplementary Fig. S1C). To determine if Mcl-1 is regulated at the transcriptional level by GDC-0941, LN229 cells
were exposed to increasing concentrations of GDC-0941 for
7 hours. Despite a tendency to decrease, no statistically
(Continued.) B, results are quantified and statistically analyzed. The number of Annexin V positive cells following combination treatment (2 mmol/L ABT-263
and 2 mmol/L GDC-941) is different in a statistically significant manner from ABT-263, GDC-0941, and the control treatment. Two-way analysis of variance was
conducted followed by Bonferroni posttests. Stars indicate statistical significance between ABT-263 vs. ABT-263 þ GDC-0941 ( , P < 0.05; , P < 0.01;
#
##
###
, P < 0.001), whereas "#" highlights statistical significance between GDC-0941 vs. ABT-263þGDC-0941 ( , P < 0.05; , P < 0.01; , P < 0.001). C and D,
NCH644, NCH690, and LN229 glioblastomas cells were treated with 2 mmol/L ABT-199, 2 mmol/L GDC-0941, and the combination of both for 24 hours.
Subsequently, cells were stained with Annexin V/PI and analyzed by flow cytometry. FL1 (Annexin V) and FL3 (PI) are represented on the x-axis and
y-axis, respectively. One representative plot is shown in C. D, results are quantified and statistically analyzed. The number of Annexin V positive cells following
combination treatment (ABT-263 and GDC-941) is different in a statistically significant manner from ABT-263, GDC-0941, and the control treatment (two-way
analysis of variance was conducted followed by Bonferroni posttests). Stars indicate statistical significance between ABT-199 vs. ABT-199 þ GDC-0941
#
( , P < 0.05; , P < 0.01; , P < 0.001), whereas "#" highlights statistical significance between GDC-0941 vs. ABT-199 þ GDC-0941 ( , P < 0.05;
##
###
, P < 0.01; , P < 0.001). E and F, LN229 glioblastomas cells were treated with 2 mmol/L ABT-263, 2 mmol/L GDC-0941, and the combination of both for
24 hours. Subsequently, LN229 cells were stained with the mitochondrial dye JC-1, and analyzed by flow cytometry. FL1 (green channel) and FL2 (red channel)
are represented on the x-axis and y-axis, respectively. F, results are quantified and statistically analyzed. The percentage of cells, demonstrating loss of
MMP, following the combination treatment (ABT-263 and GDC-941) is different, in an statistically significant manner, from ABT-263, GDC-0941, and the
control treatment (one-way analysis of variance was performed followed by Tukey Multiple Comparison Test). Values are given as mean SEM of
representative experiments. ABT—ABT-263 (Fig. 2B and E); ABT—ABT-199 (Fig. 2D); GDC—GDC-0941; ABT þ GDC—combined treatment of ABT-263
and GDC-0941, MMP. A P value of less than 0.05 is indicated by 1 star " ", whereas a P value of less than 0.01 is highlighted by 2 stars " ". A P value
less than 0.001 is indicated by a star triplet ( ).
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Mol Cancer Res; 12(7) July 2014
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993
Published OnlineFirst April 22, 2014; DOI: 10.1158/1541-7786.MCR-13-0650
Pareja et al.
A
B
GS9-6
GDC (μmol/L) 0
2
4
8
LN229
GDC (μmol/L) 0
2
4
8
ph-Akt
60 kDa
ph-Akt
60 kDa
Akt
60 kDa
Akt
60 kDa
Mcl-1
40 kDa
Mcl-1
40 kDa
Actin
45 kDa
14-3-3β
30 kDa
C
E
LN229
U87
GDC (μmol/L) 0
2
5
ph-Akt
60 kDa
Akt
60 kDa
rel. exp. 1.0
0.4
0
7
7
.5 1.5 2
3
5
7
GDC
−
−
+
+
+
+
+
+
+
ABT
−
+
−
+
+
+
+
+
+
47 kDa
CP9
0.25
Mcl-1
40 kDa
14-3-3β
30 kDa
D
Time (h)
cCP3
17 kDa
14-3-3β
30 kDa
ph-Akt
60 kDa
Akt
60 kDa
Mcl-1
40 kDa
14-3-3β
30 kDa
NCH421K
GDC −
−
+
ABT −
+
−
rel. exp. 1.0
1.07
0.75
Mcl-1
40 kDa
Actin
45 kDa
significant alterations of Mcl-1 mRNA levels were detected
at 2, 4, and 8 mmol/L of GDC-0941 (P ¼ 0.1441; Fig. 5A),
which suggests that a posttranscriptional mechanism may
primarily contribute to GDC-0941–mediated suppression
of Mcl-1 levels. Recent studies show that Mcl-1 is a protein
with a short half-life, prone to degradation by the proteasome and caspases (20–22). Hence, we postulated that a
potential mechanism for GDC-0941–driven depletion of
Mcl-1 protein levels could be enhanced proteasomal degradation. To test this, we treated LN229 cells for 7 hours with
different concentrations of GDC-0941 in the presence or
absence of MG132, a proteasomal inhibitor. Treatment with
MG132 partially rescued GDC-0941–mediated Mcl-1
depletion (Fig. 5B), in keeping with the hypothesis that
GDC-0941 enhances Mcl-1 degradation through the proteasome. We demonstrated that the combined treatment of
ABT-263 and GDC-0941 induces apoptosis to a greater
extent than single treatments, it would be thus conceivable
that the combination treatment as well as GDC-0941
994
35 kDa
Mol Cancer Res; 12(7) July 2014
Figure 4. Effects of ABT-263 and
GDC-0941 on phosphorylation of
Akt, Mcl-1 levels, and cleavage of
caspases 9 and 3. A, primary GS96 glioma stem cell–like cells were
treated with increasing
concentrations of GDC-0941
(mmol/L) for 7 hours and subjected
to immunoblotting with antibodies
against ph-Akt (Ser 473), Akt, and
Mcl-1. Actin protein served as
loading control. B and C, LN229,
and U87 were treated with
increasing concentrations of
GDC-0941 (mmol/L) for 7 hours
and subjected to immunoblotting
with antibodies against
phosphorylated Akt (Ser 473), Akt
and Mcl-1. 14-3-3 protein served
as loading control. D, NCH421K
cells were treated with 2 mmol/L
GDC-0941 or 2 mmol/L ABT-263
for 7 hours and subjected to
immunoblotting with antibodies
for Mcl-1. Actin served as loading
control. E, LN229 cells were
treated with suboptimal dosages
of GDC-0941 (1 mmol/L), ABT-263
(2 mmol/L) or the combination of
both for the depicted times and
subjected to immunoblotting for
antibodies against Caspase-9,
cleaved Caspase 3,
phosphorylated Akt (Ser 473), Akt
and Mcl-1. The antibody against
caspase-9 detects its total form
(47 kDa), as well as its cleaved
(active) form at 35–37 kDa. 14-3-3
served as a loading control. ABT –
ABT-263; GDC – GDC-0941;
ABTþGDC – combined treatment
of ABT-263 and GDC-0941.
administered alone would impact the protein half-life of
Mcl-1. To examine this postulate, we treated LN229
glioblastoma cells with the protein synthesis inhibitor,
cycloheximide in the presence or absence of ABT-263 and
GDC-0941. Single treatment with GDC-0941, in the
presence of cycloheximide, significantly reduced the protein half-life of Mcl-1, with a marked decrease of Mcl-1
protein levels already after 1.5 hours of treatment (Fig.
5C). Similarly, combination treatment of ABT-263 and
GDC-0941 significantly altered protein half-life of Mcl-1,
causing a decline of Mcl-1 levels starting 45 minutes
following treatment, with a substantial depletion 3 hours
of therapy (Fig. 5D). These results indicate the existence
of a posttranslational mechanism by which GDC-0941
alone and in combination with ABT-263 depletes Mcl-1
protein levels. Mcl-1 reduction by GDC-0941 was rescued by the proteasome inhibitor MG132, indicating that
that Mcl-1 protein levels are suppressed by enhanced
proteasomal degradation.
Molecular Cancer Research
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Published OnlineFirst April 22, 2014; DOI: 10.1158/1541-7786.MCR-13-0650
GDC-0941 Overcomes Intrinsic Apoptotic Resistance in Glioblastoma
Mcl-1 mRNA levels (%)
A
B
LN229
LN229
7h
100
GDC (μmol/L)
MG132 (μmol/L)
80
60
0
0
5
0
5
8
2
0
2
2
Mcl1
40 kDa
14-3-3β
30 kDa
40
20
0
Control
GDC2
GDC4
GDC8
(μmol/L) (μmol/L) (μmol/L)
LN229
C
Time (h) 0
0
.75
.75
1.5
1.5
3.0
3.0
Cycloheximide
+
+
+
+
+
+
+
+
GDC
−
+
−
+
−
+
−
+
Mcl-1
40 kDa
Actin
45 kDa
D
LN229
0
.75
.75
1.5
1.5
3.0
3.0
Cycloheximide
Time (h) 0
+
+
+
+
+
+
+
+
GDC+ABT
−
+
−
+
−
+
−
+
Mcl-1
40 kDa
14-3-3β
30 kDa
Figure 5. Regulation of Mcl-1 by GDC-0941. A, LN229 cells were treated with increasing concentrations of GDC-0941 for 7 hours. Subsequently, mRNA was
isolated, transcribed into cDNA, and real-time PCR analysis with primers spanning the Mcl-1 transcript was performed. Data are given as relative percentage
to the Control and were calculated as described in the Materials and Methods section. Data were analyzed by using one-way ANOVA, followed by a Tukey
Multiple Comparison Test. B, LN229 cells were treated with GDC-0941, with or without the additional of the proteasome inhibitor MG132 or the combination of
both reagents for 7 hours and subjected to immunoblot analysis with antibodies against Mcl-1 or 14-3-3b (loading control). The vertical line on the immunoblot
indicates that the first sample was noncontiguous, but run on the same gel simultaneously with the other samples. C, LN229 cells were incubated with the
protein synthesis inhibitor, cycloheximide (10 mg/mL), in the presence or absence of GDC-0941 (2 mmol/L) for the indicated time points and then subjected to
immunoblotting. Membranes were incubated with a Mcl-1–specific antibody as well as with b-actin, serving as a loading control. D, LN229 cells were
incubated with the protein synthesis inhibitor cycloheximide (10 mg/mL), alone or in combination with ABT-263/GDC-0941 (2 mmol/L each) for the indicated
time points and then subjected to immunoblotting. Membranes were incubated with a Mcl-1–specific antibody as well as with 14-3-3b, serving as a loading
control. Values are given as mean SEM of representative experiments. GDC2, GDC4, and GDC8—2, 4, and 8 mmol/L of GDC-0941.
Ectopic overexpression of human Mcl-1 attenuated
apoptosis induced by the combination therapy of GDC0941 and ABT-263
To further confirm the notion that Mcl-1 mediates
apoptotic resistance, we tested 2 cell lines with high
(U87) and low levels of Mcl-1 (LN229) protein expression
for the sensitivity of ABT-263. Consistent with the expression pattern, U87 cells were more resistant to the cytotoxic
effects of ABT-263 as compared with LN229 cells (Supplementary Fig. S2A). Next, we investigated if overexpression of
Mcl-1 would attenuate ABT-263/GDC-0941–mediated
www.aacrjournals.org
induction of apoptosis. In these experiments, LN229 cells
were transfected with a plasmid encoding the open reading
frame of human Mcl-1. Forty-eight hours after transfection with either a control empty pcDNA3 plasmid, or a
plasmid encoding human Mcl-1, LN229 cells were harvested, lysed, and analyzed by Western blotting for Mcl-1
expression (Fig. 6A). After selection cells were subjected to
ABT-263 and GDC-0941 combination therapy (Fig. 6B).
Moreover, LN229 cells overexpressing human Mcl-1
displayed a statistically significant protection from
ABT-263/GDC-0941–induced cell death as compared
Mol Cancer Res; 12(7) July 2014
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Published OnlineFirst April 22, 2014; DOI: 10.1158/1541-7786.MCR-13-0650
Pareja et al.
B
LN229
Empty
+
−
pcDNA-hMcl-1
−
+
25
40 kDa
Mcl-1
***
30
% Dead cells
A
20
15
10
5
14-3-3β
30 kDa
0
GDC+ABT
GDC+ABT
2 μmol/L+2 μmol/L 2 μmol/L+2 μmol/L
Empty
Mcl-1
empty
Mcl-1
LN229
C
D
LN229
100
−
−
+
+
n.t.-siRNA
+
−
+
−
Mcl-1 siRNA
−
+
−
+
Mcl-1
40 kDa
Bcl-xL
***
***
80
% PI-positive cells
ABT199
***
60
40
20
30 kDa
0
45 kDa
Actin
Control
Control
ABT-199
2 μmol/L
ABT-199
2 μmol/L
siRNAn.t.
siRNAMcl-1
siRNAn.t.
siRNAMcl-1
LN229
LN229
E
F
LN229
ABT263
−
−
+
+
ABT199
−
−
+
+
n.t.-siRNA
+
−
+
−
n.t.-siRNA
+
−
+
−
Mcl-1 siRNA
−
+
−
+
Mcl-1 siRNA
−
+
−
+
47 kDa
47 kDa
CP9
CP9
35 kDa
35 kDa
cCP3
17/19 kDa
cCP3
17/19 kDa
Actin
45 kDa
Actin
45 kDa
Figure 6. Overexpression of Mcl-1 attenuates cell death induced by the combination treatment of ABT-263 and GDC-0941 and specific suppression of Mcl-1
sensitizes glioma cells to ABT-263/ABT-199–mediated cell death. A, LN229 glioma cells were transfected with a plasmid encoding human Mcl-1 or with
the respective empty control plasmid. Forty-eight hours after transfection cells were harvested and subjected to immunoblotting. Membranes were
incubated with a Mcl-1–specific antibody or an antibody against 14-3-3b as loading control. B, LN229 cells were transfected, selected with Geneticin and were
treated with the combination of ABT-263 (ABT; 2 mmol/L) and GDC-0941 (GDC; 2 mmol/L) for 24 hours. Thereafter, cells were harvested and analyzed for
cellular viability by flow cytometric analysis. LN229 cells, overexpressing human Mcl-1, displayed less dead cells in response to treatment with ABT-263/GDC0941. A P value of less than 0.001 is indicated by 3 stars " ". (Continued on the following page.)
996
Mol Cancer Res; 12(7) July 2014
Molecular Cancer Research
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Published OnlineFirst April 22, 2014; DOI: 10.1158/1541-7786.MCR-13-0650
GDC-0941 Overcomes Intrinsic Apoptotic Resistance in Glioblastoma
with the respective control. These results indicate that
Mcl-1 is implicated in death induced by ABT-263 as a
monotherapeutic as well as by the combination treatment
of ABT-263 and GDC-0941.
Specific suppression of Mcl-1 by siRNA sensitizes
glioblastoma cells to ABT-263/ABT-199
Next we assessed if specific suppression of Mcl-1 can
sensitize glioblastoma cells to the cytotoxic effects of ABT263 and ABT-199, respectively. LN229 cells transfected
with a Mcl-1–specific siRNA revealed a strong reduction of
endogenous Mcl-1 levels as compared with the nontargeting
siRNA (Fig. 6C). Furthermore, the Mcl-1 siRNA was
specific for Mcl-1 as Bcl-xL was not affected (Fig. 6C).
About cell death downregulation of Mcl-1 in LN229
increased the number of dead cells as compared with LN229
cells transfected with a nontargeting siRNA (Fig. 6D).
Furthermore, a suboptimal dosage of ABT-199 had no effect
on nontargeting siRNA-transfected LN229 cells, whereas
LN229 cells with suppressed Mcl-1 protein levels showed a
remarked induction of cell death after ABT-199 treatment
(Fig. 6D). In addition, ABT-199 treatment further
enhanced Mcl-1 siRNA-mediated cell death (Fig. 6D). Next
we tested as to whether ABT-263 and ABT-199 enhance
cleavage of initiator/effector caspases-9/-3 in LN229 cells
transfected with a Mcl-1–specific siRNA. Indeed, both
ABT-199 and ABT-263 revealed a marked cleavage of
caspase-9/-3 under Mcl-1–depleted conditions (Fig. 6E
and F).
GDC-0941 dephosphorylates BAD and a
phosphorylation-deficient BAD mutant enhances ABT263–mediated cell death
Because glioblastoma cells contain high levels of phosphorylated endogenous BAD (Supplementary Fig. S1A),
which is considered to be inefficient to mediate apoptosis, we
seek to determine as to whether GDC-0941 modulates the
phosphorylation status of BAD at Ser 112 and Ser 136. In
addition, GDC-0941 and ABT-263 also cooperated to
induce death of U373 glioma cells, even though there was
no suppression of Mcl-1 protein levels after GDC-0941
treatment. We found that GDC-0941 led to a dephosphorylation of BAD at Ser 112 and Ser 136, respectively (Fig. 7A–
C). Consistently, GDC-0941 mediated a dramatic decrease
in the phosphorylation status of Akt, paralleling the levels of
BAD Ser 112 and Ser 136. Given the dephosphorylation of
BAD we hypothesized that a phosphorylation-deficient
BAD (S112A, S136A) should have stronger cell death
sensitizing/inducing properties. For this purpose we transfected wild-type BAD and a phosphorylation-deficient
mutant BAD (S112A, S136A) into LN229 glioblastoma
cells. Transfection of both wild-type and mutant BAD
increased the percentage of dead cells. However, phosphorylation-deficient BAD had a significant stronger effect on cell
death as compared with wild-type BAD (Fig. 7D). Furthermore, mutated BAD cooperated with ABT-263 in apoptosis
induction (Fig. 7D). These findings support the notion that
GDC-0941-mediated dephosphorylation of BAD is implicated in ABT-263/GDC-0941 enhanced cell death when
compared with each drug alone.
The protein expression levels of ph-Akt (Ser 473) and phBAD (Ser 112) correlate in glioblastoma tissue specimens
TMAs, containing 34 glioblastoma tissue specimens, were
stained for the expression of ph-Akt and ph-BAD (Ser 112),
respectively. Most glioblastomas tumor specimens showed
expression of ph-Akt (Ser 473) and ph-BAD (Ser 112),
whereas a minority (ph-Akt) and small fraction (ph-BAD)
of cases displayed no detectable staining (Fig. 8A–C
and Table 1). Although ph-Akt was both detected in the
cytoplasm and nucleus of glioblastoma cells, the expression
of ph-BAD (Ser 112) was localized to the cytoplasm. Tumor
vasculature seemed to be mostly negative for both ph-Akt
and ph-BAD, respectively. Notably, the expression levels of
ph-Akt (Ser 473) and ph-BAD (Ser 112) revealed a statistically significant positive correlation (n ¼ 34; Spearman
correlation coefficient r ¼ 0.5289; 95% confidence interval,
0.2223–0.7403; P < 0.0013). These data support the notion
that the Akt pathway is active in glioblastoma and affects
BAD phosphorylation, thereby inhibiting its proapoptotic
properties.
Discussion
Glioblastoma WHO IV is a treatment-resistant cancer
with a short survival time after diagnosis. Despite a significant increase in our understanding of the biology of these
tumors, effective treatment strategies to overcome this disease are still unavailable. Given the heterogeneous nature of
these neoplasms, it is unlikely for a "smoking-gun" therapy
to be effective in all glioblastomas. A personalized approach
tailored to each individual is more likely to be successful. In
this context, our current understanding of glioblastoma may
allow us to stratify patients into groups according to the
nature of molecular aberrations in their tumors. For example, many glioblastomas harbor aberrantly active PI3K
signaling and high levels of bcl-2/bcl-xL, which suggests
that these tumors depend on these pathways to achieve their
growth and resistance to therapy. In this work, we have
targeted the 2 above-mentioned signaling networks simultaneously by administering 2 orally available drugs that
(Continued.) C, LN229 cells were transfected with a specific siRNA against Mcl-1 or with a nontargeting siRNA, respectively. Forty-eight hours after
transfection cells were harvested and analyzed for protein expression of Mcl-1 and Bcl-xL by immunoblotting, respectively. Actin serves as a loading control.
D, LN229 cells were transfected with a specific siRNA against Mcl-1 or with a nontargeting siRNA, respectively. Forty-eight hours after transfection cells were
either untreated or exposed to a suboptimal dosage of ABT-199 (2 mmol/L) overnight. Flow cytometric analysis was performed to quantify the amount of
Propidium iodide positive cells (% PI positive cells). Statistical analysis was performed by one-way ANOVA analysis. A P value of less than 0.001 is indicated by
3 stars " ". E and F, LN229 were transfected as in C, were left untreated or treated with ABT-263 (E) or ABT-199 (F) and analyzed for CP9 (includes proform at
47 kDa as well as cleavage products at 35 kDa) and caspase-3 cleavage (cCP3).
www.aacrjournals.org
Mol Cancer Res; 12(7) July 2014
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997
Published OnlineFirst April 22, 2014; DOI: 10.1158/1541-7786.MCR-13-0650
Pareja et al.
B
LN229
GDC (μmol/L)
0
2
4
8
ph-Akt
Ser473
60 kDa
Akt
60 kDa
ph-BAD
Ser112
23 kDa
ph-BAD
Ser136
23 kDa
BAD
23 kDa
Actin
45 kDa
C
0
2
GDC (μmol/L) 0
ph-Akt
Ser473
Akt
4
60 kDa
60 kDa
ph-BAD
Ser136
ph-BAD
Ser112
23 kDa
23 kDa
BAD
23 kDa
Actin
45 kDa
**
4
50
ph-Akt
Ser473
60 kDa
Akt
60 kDa
ph-BAD
Ser112
ph-BAD
Ser136
BAD
Actin
23 kDa
23 kDa
23 kDa
45 kDa
Mol Cancer Res; 12(7) July 2014
**
40
30
20
10
0
Empty
+
−
−
+
−
−
BAD
−
+
−
−
+
−
BAD S112A/
S136A
−
−
+
−
−
+
ABT-263
1 μmol/L
−
−
−
+
+
+
specifically inhibit either PI3K, GDC-0941, or Bcl-2/BclxL, ABT-263. ABT-263 binds to a hydrophobic groove in
Bcl-2/Bcl-xL and thereby displaces proapoptotic molecules
of the Bcl-2 family of proteins, such as Bim. In turn, it elicits
a bax/bak-dependent cell death, which corresponds to rapid
apoptosis. Similarly to ABT-737, a nonoral predecessor of
ABT-263, ABT-263 reveals antiglioma activity and may
thus be suitable for the treatment of glioblastoma. Plasmatic
levels of 5.4 to 7.7 mmol/L were achieved in mice and higher
concentrations of the compound were detected in the plasma
of dogs. In this study, we used relevant plasmatic concentrations of ABT-263 to closely resemble the clinical scenario.
Relevant plasma concentrations of ABT-263 had a mild, but
significant growth inhibitory effect on U373 and LN229
glioblastoma cells. The major therapeutic mechanism of
ABT-263 is rapid apoptosis induction, and at the highest
dosage of ABT-263, the reduction of cellular viability was
less than 50%. Hence, we speculated that the addition of a
sensitizing reagent that inhibits an additional prosurvival
pathway may facilitate ABT-263–mediated cell death. To
this end, we used GDC-0941, an orally available novel PI3K
inhibitor. Previous studies indicate that this compound is
998
2
D
U373
GDC (μmol/L)
U87
% Dead cells
A
Figure 7. GDC-0941 modulates the
phosphorylation status of BAD at
Ser 112 and Ser 136 in
glioblastoma cell lines. A–C,
LN229, U87 and U373 cells were
treated with increasing
concentrations of GDC-0941
(concentrations in mmol/L) for 7
hours and subjected to
immunoblotting with antibodies
against phosphorylated Akt (Ser
473), Akt, ph-BAD (Ser 112 and Ser
136), and total BAD. Actin protein
served as loading control. D,
LN229 glioblastoma cells were
transfected with plasmids,
encoding wild-type BAD, a
phosphorylation deficient BAD
(S112A, S136A, Serine ! Alanine)
or the respective control plasmid.
Thereafter, cells were either left
untreated or treated with a
suboptimal concentration of ABT263 (1 mmol/L) and cell death was
quantified by flow cytometry.
Shown are the total percentages of
dead cells. A P value of less than
0.01 is highlighted by 2 stars " ".
predominantly cytostatic and only under certain circumstances induces apoptosis. Combination therapies oftentimes include reagents that induce rapid apoptosis, which
are administered in conjunction with slow acting compounds as sensitizers. This is exemplified by molecules that
sensitize cancer cells to death receptor–mediated apoptosis,
such as TNF-related apoptosis-inducing ligand (TRAIL;
refs. 14, and 23–25). We found that the combination
treatment of GDC-0941 and ABT-263 exerted a stronger
effect on loss of cellular viability in glioblastoma cells as
compared with each compound alone. This effect seemed to
be independent of the TP53 status of the tumor cell, both
both LN229 and U373 harbor mutated TP53 alleles,
whereas GS9-6 harbors wild-type TP53. Moreover, combination treatment not only had an effect in established
glioblastoma cells grown in full serum, which represent the
bulk of tumor cells, but also significantly inhibited neurosphere formation in stem cell–like glioma cells, suggesting
that this treatment regimen may be efficacious in both the
initial reduction of the tumoral, as well as reducing and
delaying tumor recurrence, by targeting the stem cell niche
(26) within high-grade glial tumors. Mechanistically, ABT-
Molecular Cancer Research
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Published OnlineFirst April 22, 2014; DOI: 10.1158/1541-7786.MCR-13-0650
GDC-0941 Overcomes Intrinsic Apoptotic Resistance in Glioblastoma
Figure 8. Glioblastoma TMAs, containing 34 tumor samples, were stained
with antibodies against ph-Akt (Ser 473) or ph-BAD (Ser 112).
Representative photographs were taken. A, 0: absent expression; B, 1:
low expression; and C, 2: high expression. Three representative
glioblastomas (GBM1, GBM2, GBM3) are shown with different
expression levels of ph-Akt (Ser 473) and ph-BAD (Ser 112).
263 and GDC-0941 in combination induced higher levels of
apoptosis as compared with each reagent on its own. Paralleling the enhanced apoptosis observed was our finding
that combination treatment provoked and increased loss of
MMP. This led us to propose that GDC-0941 primes
glioblastoma cells by acting on mitochondrial antiapoptotic
molecules, such as the Bcl-2 family of proteins. In this
context, it is well known that Mcl-1 is a bona-fide inhibitor
of intrinsic apoptosis playing a major role in resistance to
treatment with BH3 mimetics, such as ABT-737 (27, 28) or
its derivative ABT-263 (7). Mcl-1, localized to the mitochondrial outer membrane, is capable of inhibiting baxmediated intrinsic apoptosis, and its levels in tumors correlate with prognosis. Mcl-1 may, thereafter, serve both as a
therapeutic marker and as a prognostic indicator. Prior
Table 1. Expression levels of ph-Akt (Ser 473)
and ph-BAD (Ser 112) in glioblastoma tissue
specimens
Protein
Score
Tumors (%)
ph-Akt (Ser 473)
0
1
2
0
1
2
2/34 (6%)
15/34 (44%)
17/34 (50%)
7/34 (21%)
21/34 (62%)
6/34 (18%)
ph-BAD (Ser 112)
NOTE: 0, no staining; 1, low expression; 2, high expression.
www.aacrjournals.org
studies showed that interfering with cyclin-dependent kinase
(CDK) with compounds such as roscovitine, lowers Mcl-1
protein levels, and thereby sensitizes cells to the cytotoxic
effects of ABT-737 (7). The effect of roscovitine was
recapitulated by an shRNA-targeting Mcl-1 in human
leukemia cells (7). Suppression of Mcl-1 results in activation
of the proapoptotic Bcl-2 family protein bak through a
conformational change (28), in line with the fact that
Mcl-1 avidly binds to bak. Sorafenib (BAY 43-9006) is a
compound initially identified as a broad kinase inhibitor. In
contrast to other kinase inhibitors, which primarily elicit
cytostasis, sorafenib also induces apoptosis in the low mmol/
L range. Several molecular mechanisms have been proposed
and recent studies in different tumor entities confirm that a
key mechanism for cell death induction by sorafenib is the
downregulation of Mcl-1. Sorafenib-mediated suppression
of Mcl-1 occurs at different levels. Some reports suggest a
posttranscriptional mechanism (20), whereas others favor a
transcriptional one (12, 29). For instance, Yu and colleagues
showed that sorafenib did not modulate Mcl-1 mRNA levels
in breast cancer cells, colon cancer cells, and leukemia cells.
Instead, sorafenib increased proteasomal degradation of
Mcl-1 in these cells (20), in keeping with the fact that
Mcl-1 was described as a protein with a short-half life and
prone to proteasomal degradation. In glioblastoma, sorafenib seems to suppress Mcl-1 at the transcriptional level,
involving ATF5, a transcriptional factor (12). Irrespective of
the mechanism, lowering Mcl-1 levels seems to be an
effective strategy to sensitize tumor cells to therapy. Indeed,
therapeutic strategies, targeting Mcl-1 by sorafenib, have
already reached clinical trials (clinicaltrials.gov). Our findings demonstrate that GDC-0941 lowers Mcl-1 levels in
several established glioblastoma cell lines, as well as in stem
cell–like glioma cells (neurosphere cultures). In addition, we
also showed that the combination of ABT-263/GDC-0941
affected Mcl-1 levels. Our findings suggest that GDC-0941
regulates Mcl-1 protein levels mainly in a posttranslational
manner. This is supported by our experiments, using protein
synthesis inhibitor cycloheximide, which showed that in the
presence of GDC-0941 the protein half-life of Mcl-1 is
significantly reduced. In addition, GDC-0941–mediated
suppression of Mcl-1 protein levels was rescued by the
proteasomal inhibitor, MG-132, corroborating the fact that
GDC-0941 regulates the proteasomal turnover of Mcl-1.
GDC-0941–mediated Mcl-1 depletion occurs within a
couple of hours of treatment, suggesting that the ABT263 and GDC-0941 can be applied together without a
preincubation period with the sensitizing reagent (GDC0941). The concept of combining PI3K inhibitors or "pleiotropic kinase inhibitors" (30) as well as mTOR inhibitors
with BH3 mimetics has also been exploited in other tumor
entities, such as rhabdomyosarcomas (RMS) and non–small
cell lung carcinomas (30–33). For instance, akin to glioblastomas, RMS harbor a deregulated PI3K pathway and
targeting these tumors with AZD8055 proved to be an
effective strategy to sensitize RMS cells to the cytotoxic
action of ABT-737 (31). Mechanistically, the sensitizing
effect of AZD8055 in this malignancy was because of
Mol Cancer Res; 12(7) July 2014
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999
Published OnlineFirst April 22, 2014; DOI: 10.1158/1541-7786.MCR-13-0650
Pareja et al.
suppression of Mcl-1 (31), reminiscent of our findings with
GDC-0941 in malignant glioma cells. Upstream of PI3K
are the membrane bound tyrosine kinase receptors. EGFR
is a classic example, which is often mutated in both lung
adenocarcinomas and glioblastomas. However, the mutation sites differ between these 2 mentioned tumor entities.
Although glioblastomas often harbor the EGFRvIII mutation (34), lung adenocarcinomas have mutations in exons
19 and 21, which in contrast to glioblastomas renders
them remarkably sensitive to Gefitinib/Erlotinib. Upon
Gefitinib treatment lung adenocarcinomas, harboring
EGFR-inhibitor sensitizing mutations, undergo Bimdependent apoptosis, which can be significantly augmented by BH3 mimetics (35). This underscores the fact that
compounds interfering with molecules upstream of the
PI3K signaling pathway, such as membrane bound receptors, may be favorably combined with BH3 mimetic
drugs. The rational for this combination is similar to the
addition of PI3K inhibitors to combinatorial regimens,
because EGF, the natural ligand for EGFR, elevates Mcl-1
protein levels (36).
Currently, there are also small molecule inhibitors
under development that bind to Mcl-1 and inhibit its
antiapoptotic functions, for example Obatoclax (37).
Therefore, it is conceivable that these compounds may
be used in conjunction with BH3 mimetics, such as ABT199 or ABT-263 (this study). The disadvantage of this
strategy compared with the one presented here (the drug
combination of ABT-263 þ GDC-0941) is that Obatoclax acts downstream and will not affect other downstream
pathways. PI3K inhibitors lower Mcl-1 levels and affect
other apoptosis modulating factors, such as BAD (this
study) and caspase-9. Our study nicely showed that
glioblastoma tissue specimens and cell line harbor high
levels of ph-BAD, that GDC-0941 has an effect on BAD
phosphorylation status and that dephosphorylated BAD
enhances apoptosis by BH3 mimetics. Thus, the advantage of the presented combination therapy in this study is
clearly that it not only affects Mcl-1, but also additional
deregulated pathways linked to the PI3K signaling axis.
This becomes particularly relevant in the context of
malignant glioma, in which the PI3K pathway is commonly hyperactive (primary glioblastoma).
Conclusion and Remarks
In summary, we have described a novel potential combinatorial therapy for glioblastoma, which includes the PI3K
inhibitor, GDC-0941, and the BH3-mimetic, ABT-263. At
clinically relevant concentrations when combined, these compounds have a significant anti-glioma effect that is stronger
than each compound on its own in both established and in
stem cell-like glioma cells. Furthermore, these reagents are
orally available, facilitating their administration. Mechanistically, this drug combination is rational because it targets 2
aberrantly active pathways in glioblastomas. In addition,
inhibition of one of these pathways (PI3K) affects the second
one by lowering the expression levels of a key molecule, Mcl-1,
which drives resistance to certain BH3 mimetics.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: F. Pareja, A.H. Ross, M.D. Siegelin
Development of methodology: F. Pareja, J.F. Crary, M.D. Siegelin
Acquisition of data (provided animals, acquired and managed patients, provided
facilities, etc.): C. Shu, J.F. Crary, M.D. Siegelin
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F. Pareja, J.F. Crary, P.D. Canoll, A.H. Ross, M.D. Siegelin
Writing, review, and/or revision of the manuscript: F. Pareja, J.F. Crary, P.D. Canoll,
A.H. Ross, M.D. Siegelin
Administrative, technical, or material support (i.e., reporting or organizing data,
constructing databases): D. Macleod, A.H. Ross, M.D. Siegelin
Study supervision: M.D. Siegelin
Acknowledgments
The authors thank Dr. L. Greene for helpful discussions and providing laboratory
space and reagents. The authors also thank R.P. Moser for supporting isolation of
primary glioma neurosphere cultures.
Grant Support
This study was supported in part by grants from the American Brain Tumor
Association, Translational Grant 2013 (M.D. Siegelin; ABTACU13-0098), from the
2013 AACR-National Brain Tumor Society Career Development Award for Translational Brain Tumor Research, Grant Number 13-20-23-SIEG, and the CTO/CTSA
Pilot Award from the Irving Institute for clinical and translational research (Columbia
University Medical Center, New York, New York; M.D. Siegelin) and NINDS R01
NS021716 (A.H. Ross).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Received December 13, 2013; revised March 31, 2014; accepted April 13, 2014;
published OnlineFirst April 22, 2014.
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