Download A Dual PI3K/mTOR Inhibitor, PI-103, Cooperates

Published OnlineFirst November 17, 2010; DOI: 10.1158/0008-5472.CAN-10-1601
Cancer
Research
Therapeutics, Targets, and Chemical Biology
A Dual PI3K/mTOR Inhibitor, PI-103, Cooperates with Stem
Cell–Delivered TRAIL in Experimental Glioma Models
Tugba Bagci-Onder1,2, Hiroaki Wakimoto3, Maarten Anderegg1,2, Cody Cameron1,2,
and Khalid Shah1,2,4
Abstract
The resistance of glioma cells to a number of antitumor agents and the highly invasive nature of glioma cells
that escape the primary tumor mass are key impediments to the eradication of tumors in glioma patients. In this
study, we evaluated the therapeutic efficacy of a novel PI3-kinase/mTOR inhibitor, PI-103, in established glioma
lines and primary CD133þ glioma-initiating cells and explored the potential of combining PI-103 with stem cell–
delivered secretable tumor necrosis factor apoptosis-inducing ligand (S-TRAIL) both in vitro and in orthotopic
mouse models of gliomas. We show that PI-103 inhibits proliferation and invasion, causes G0–G1 arrest in cell
cycle, and results in significant attenuation of orthotopic tumor growth in vivo. Establishing cocultures of neural
stem cells (NSC) and glioma cells, we show that PI-103 augments the response of glioma cells to stem cell–
delivered S-TRAIL. Using bimodal optical imaging, we show that when different regimens of systemic PI-103
delivery are combined with NSC-derived S-TRAIL, a significant reduction in tumor volumes is observed
compared with PI-103 treatment alone. To our knowledge, this is the first study that reveals the antitumor
effect of PI-103 in intracranial gliomas. Our findings offer a preclinical rationale for application of mechanismbased systemically delivered antiproliferative agents and novel stem cell–based proapoptotic therapies to
improve treatment of malignant gliomas. Cancer Res; 71(1); 154–63. 2010 AACR.
Introduction
Glioblastoma multiforme (GBM) is the most common and
aggressive form of malignant brain tumors. Despite the
advances in the understanding of glioma biology, GBMs remain
very difficult to eradicate and the median survival after diagnosis is still less than 12 months (1). The inherent or acquired
resistance of tumor cells to a number of antitumor agents and
the highly invasive nature of tumor cells that escape the
primary tumor mass and subsequently cause recurrence (2,
3) are among the major obstacles in finding an optimal cure for
GBMs. Furthermore, the lack of efficient delivery of promising
anti-GBM agents contribute to therapeutic failure (4).
A number of genetic alterations are implicated in GBM
progression, contributing to high proliferation, resistance to
apoptosis, and extreme invasion capabilities of GBM tumors
(5). One pathway that is commonly deregulated and a promis-
Authors' Affiliations: 1Molecular Neurotherapy and Imaging Laboratory,
and Departments of 2Radiology, 3Neurosurgery, and 4Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Author: Khalid Shah, Massachusetts General Hospital,
Harvard Medical School, Charlestown, MA 02129. E-mail: kshah@helix.
mgh.harvard.edu
doi: 10.1158/0008-5472.CAN-10-1601
2010 American Association for Cancer Research.
154
ing target for therapies is the PI3 kinase (PI3K)-Akt pathway
(6). Although PI3K exerts most of its effects through Akt, its
downstream signaling also activates mammalian target of
rapamycin (mTOR). Because of the high frequency of PI3K
pathway alterations in several cancers, including GBMs, there
has been a major interest in discovering novel modulators of
this pathway's components that are eventually compatible
with preclinical models and clinical studies. Recently, a novel
kinase inhibitor, PI-103, which inhibits both PI3K and mTOR
signaling, has been utilized in several preclinical tumor models including subcutaneous models of glioma (7–14). PI-103
has been shown to inhibit cell proliferation and tumor growth
through its direct effects on the inhibition of PI3K and mTOR
(13, 15, 16). Although PI-103 has shown promising results in
mouse models of different tumor types, its efficacy in mouse
models of intracranial GBMs has not been characterized.
Current treatment regimens for GBMs involve targeting one
or the other hallmark of GBM tumors through surgery,
radiation or chemotherapy with systemically delivered drugs
(5). However, combination therapies that aim to simultaneously tackle different components of glioma progression,
such as proliferation, invasion, and apoptosis resistance,
would ultimately reveal more efficient eradication of these
tumors. There has been a major interest in the development of
tumor-specific cytotoxic therapies for cancers including GBM.
As such, TRAIL (tumor necrosis factor–related apoptosisinducing ligand), is a promising protein due to its tumorspecific induction of apoptosis in a death receptor–dependent
manner. Although systemically delivered TRAIL has short
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Targeting Gliomas with PI-103 and TRAIL
half-life and requires repeated administration (17), an on-site
TRAIL delivery approach would result in a significantly better
outcome. To this end, we previously established that a
secreted form of TRAIL (S-TRAIL) reduces glioma growth
when delivered by various stem cell types, such as neural
stem cells (NSC) or mesenchymal stem cells (MSC), in orthotopic glioma models (18–21). We also showed that stem cell–
mediated delivery of TRAIL is a very effective method of
delivering the tumor-specific agent on-site of the gliomas
through utilizing the tumor-tracking properties of stem cells.
However, certain genetic alterations in gliomas might counteract the response to TRAIL (22-24). Indeed, over activation
of PI3K/Akt pathway has been reported to antagonize TRAILmediated apoptosis (25) by modulating the TRAIL downstream effectors. Therefore, targeting cell proliferation
through the inhibition of PI3K signaling might not only slow
down the tumor growth and cause a cytostatic response but
also enhance the tumor cytotoxicity in response to TRAIL.
In this study, we first investigated the potency of PI-103 in
intracranial models of established and invasive gliomas.
Furthermore, we addressed the effect of PI-103 in combination
with primary NSC-delivered S-TRAIL in NSC–tumor cell
cocultures in vitro and in mouse models of intracranial tumors
in vivo.
Materials and Methods
Cell lines
Established human glioma lines (Gli36, U87MG, U251, Gli79,
LN229, A172), Gli36 expressing a constitutively active variant
of EGFR (EGFRvIII), herein referred to as Gli36-EvIII, and
Gli36-EvIII engineered to express Fluc-mCherry (Gli36-EvIIIFmC) were grown in DMEM supplemented with 10% FBS and
penicillin/streptomycin. CD133þ GBM8 glioma cells were
cultured in neurobasal medium (Invitrogen/GIBCO) supplemented with 3mmol/L of L-Glutamine (Mediatech), B27 (Invitrogen/GIBCO), 2 mg/mL of heparin (Sigma), 20 ng/mL of
human EGF (R&D Systems), and 20 ng/mL of human FGF-2
(fibroblast growth factor; PeproTech) as described (26). GBM6
and GBM12 primary GBM cells were kind gifts of Dr. Paul Dent
(Virginia Commonwealth University) and cultured in DMEM
supplemented with 5% FBS. mNSCs, which are kindly provided by Dr. Angelo Vescovi (Raffaele Scientific Institute), were
grown in NeuroCult mouse neural stem cell basal medium
(Stem Cell Technologies) supplemented with NeuroCult proliferation supplements, 2 mg/mL of heparin (Sigma), 20 ng/mL
of human EGF (R&D Systems), and 20 ng/mL of human FGF-2
(PeproTech) and penicillin/streptomycin.
Viral vectors, lentiviral packaging, and transduction of
cells
Three lentiviral vectors were used: (a) Pico2-Fluc.mCherry,
a kind gift from Dr. Andrew Kung (Dana Farber Cancer
Institute); (b) LV-S-TRAIL that bears S-TRAIL driven by the
CMV (cytomegalovirus) promoter and also contains an IRESGFP cassette (27); and (c) control GFP, that is driven by a CMV
promoter (27). Lentiviral packaging was performed by transfection of 293T cells as previously described (28). Glioma cells
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(Gli36-EvIII) were transduced with LV-Pico2-Fluc.mCherry at
a multiplicity of infection (MOI) of 2 in medium containing
protamine sulfate (4 mg/mL) and selected with puromycin.
mNSCs were dissociated and cultured as monolayers on
laminin-coated (5 mg/mL) 6-well plates for 24 hours and
transduced with LV-GFP or LV-S-TRAIL at an MOI of 5. All
cells were visualized by fluorescence microscopy for mCherry
or GFP expression 36 hours posttransduction.
Viability and caspase assays
To determine the effects of PI-103 (Cayman Biochemicals)
and/or S-TRAIL on glioma viability, glioma cells were seeded
on 96-well plates (0.5 104 per well) and treated with different
doses of PI-103 (0–5 mmol/L) and/or S-TRAIL (0–1,000 ng/mL)
24 hours after plating. The effect of PI-103 on viability was
assessed at 24, 48, and 72 hours post–PI-103 treatment. The
effect of S-TRAIL was assessed at 24 hours posttreatment. For
combination treatments, glioma cells were treated with PI-103
for 24 hours followed by S-TRAIL treatment. Cell viability was
measured using an ATP-dependent luminescent reagent (CellTiterGlo; Promega); and caspase activity was determined
using DEVD-aminoluciferin (CaspaseGlo 3/7; Promega)
according to manufacturer's instructions. All experiments
were performed in triplicates.
Cell-cycle analysis
Following treatment of glioma cells with PI-103 (1 mmol/
L) for 24 hours, control and treated cells were pulsed for 30
minutes with 10 mmol/L of bromodeoxyuridine (BrdU;
Amersham). The cells were trypsinized and fixed with icecold EtOH and denatured with 2N HCL/0.5% TritonX-100.
BrdU incorporation was detected by incubation of cells with
anti-BrdU antibody (Becton Dickinson) followed by FITC
(fluorescein isothiocyanate)-conjugated anti-mouse antibody (Vector Labs) for 30 minutes at room temperature
(RT). Cells were washed and incubated with propidium
iodide (10 mg/mL; Sigma) and RNase A (250 mg/mL) and
analyzed using flow cytometry (Becton Dickinson) and
CellQuest software.
Invasion assays
The invasive capacity of primary human GBM cells was
tested using in vitro invasion assays (Becton Dickinson BioCoat Matrigel Invasion Chamber). GBM6, GBM8, and GBM12
lines were collected with Versene and 1 105 cells were
seeded on each well of a 24-well, matrigel-coated invasion
plate in serum-free and phenol red–free DMEM. The cells were
induced to invade toward a chemoattractant placed in the
lower chambers (5% FBS in phenol red–free DMEM). After
incubation for 24 hours, the noninvading cells were removed
from the upper surface of the invasion membrane and the cells
on the lower surface were stained with Diff-Quick staining kit
(IMEB Inc.). The average number of cells per field was
determined by counting the cells in 6 random fields per well
in 10 images of each well captured using (Olympus IX51).
The effect of PI-103 on invasion was tested by adding PI-103 at
the time of seeding cells in invasion chambers. All experiments
were performed in triplicates.
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Bagci-Onder et al.
Western blotting
Following sequential treatment with PI-103 and S-TRAIL
(each for 24 hours) glioma cells were lysed with NP-40 buffer
supplemented with protease (Roche) and phosphatase inhibitors (Sigma). Thirty micrograms of harvested proteins from
each lysate was resolved on 10% SDS-PAGE, immunoblotted
with antibodies against p-Akt (Ser473), total Akt, p-S6 (Ser235/
236), total S6, poly(ADP-ribose) polymerase (PARP; Cell Signaling) or a-tubulin (Sigma), and detected by chemiluminescence after incubation with HRP (horse radish peroxidase)conjugated secondary antibodies.
Coculture experiments
To establish cocultures of glioma cells and mNSCs, 0.5 105
Gli36-EvIII-FmC cells were seeded on 24-well plates and
grown to 80% confluence in DMEM supplemented with 5%
FBS for 24 hours. mNSC neurospheres expressing S-TRAIL or
GFP (consisting of 0.5 105 cells per neurospheres) were
placed on top of untreated or PI-103–treated glioma monolayers and cultured in mNSC medium. Cells were visualized 24
hours post–mNSC addition using fluorescence microscope
(Olympus IX51). Representative images were processed with
DP2-BSW Software (Olympus). Glioma cell viability was measured by measuring the Fluc activity of cells with D-luciferin as
described previously (20).
In vivo experiments
SCID mice (3 weeks of age; Charles River Laboratories) were
implanted with Gli36-EvIII-FmC cells (5 106 per mouse; n ¼
20) subcutaneously. To test the effect of PI-103 in vivo, each
mouse was administered 25 mg/kg of PI-103 in a mixture of
saline (prepared from stocks 50 mmol/L in DMSO) or control
solution intraperitoneally (i.p.) every day. Mice were imaged
for Fluc activity (tumor volumes) by bioluminescence imaging
(BLI) on days 0 (the first day of drug administration), 7, 11, 14,
and 17 as described previously (28). To establish intracranial
gliomas, 0.5 105 Gli36-EvIII-FmC cells were implanted
stereotactically [from bregma, AP: 2 mm, ML: 1.5 mm V
(from dura): 2 mm; n ¼ 24]. Three days postimplantation, PI103 was administered to mice daily for a period of 17 days (for
PI-103 alone) or shorter (3 or 7 days for combination therapies). For combination therapy, mice were implanted with
mNSC-S-TRAIL (n ¼ 6) or control mNSC-GFP (n ¼ 6) on the
last day of PI-103 administration. The effects on intracranial
tumor growth were monitored by bioluminescence imaging as
described above. On day 15, mice were perfused with 4% PFA
(paraformaldehyde) and tissue was processed for histopathologic analysis. All in vivo procedures were approved by the
Subcommittee on Research Animal Care at Massachusetts
General Hospital.
Pharmacokinetic/pharmacodynamic assessment of
PI-103
Mice bearing intracranial or subcutaneous gliomas were
dosed i.p. with 25 mg/kg of PI-103 in 20% hydroxypropyl
b-cyclodextrin daily for 5 days. Two or 5 hours after the last
dose, blood was collected from vehicle- or PI-103–treated
mice, centrifuged, and plasma was kept at 80 C. Subcuta-
156
Cancer Res; 71(1) January 1, 2011
neous and intracranial tumors were isolated, snap frozen in
liquid nitrogen, and kept at 80 C until analysis. Quantitative
analysis of PI-103 was achieved by liquid chromatography
tandem mass spectrometry (LC-MS/MS). Specifically, tumor
tissues were homogenized with Tissuelyzer (Qiagen) and all
samples were precipitated in acetonitrile containing 250 ng/
mL of cerbutamide (internal standard) and diluted in mouse
serum. Analysis was performed using multiple reaction monitoring on API 4000 triple quadrupole system that is equipped
with electrospray ionization source (Applied Biosystems).
Chromatographic separation was done on LC-10A DvP pump
(Shimadzu) and Polar-RP column (50 2.0 mm, 4 mm;
Phenomenex) in 0.1% formic acid at 0.5 mL/min for 3 minutes.
Quantitation was done against the mouse serum standard
curves. For pharmacodynamic assessment of PI-103, tissue
proteins were extracted and analyzed for p-Akt by Western
blotting as described above.
Immunohistochemistry
Mice were perfused and tumors were processed for paraffin
embedding. For p-Akt staining, paraffin sections were subject
to de-paraffinization and antigen retrieval in citrate buffer
(pH ¼ 6.0), blocked with 5% normal goat serum in PBSTritonX and incubated with primary antibody at 4 C overnight. Sections were washed 3 times with PBS, incubated with
biotinylated secondary antibody for 30 minutes at RT and with
ABC reagent (Vectorlabs), followed by development with
diaminobenzidine. After hematoxylin counterstaining, sections were visualized with light microscopy (Olympus IX51)
Statistical analysis
Data were analyzed by Student's t test when comparing 2
groups. Data were expressed as mean SEM and differences
were considered significant at P < 0.05.
Results
PI-103 inhibits glioma cell proliferation and attenuates
tumor growth in vivo
To test the effect of PI-103 on glioma cell proliferation, we
chose 7 different established glioma lines, Gli36, Gli36-EvIII (as
previously characterized; ref. 29; fast-growing Gli36 line that
overexpresses a constitutively active form of EGFR), Gli79,
U87MG, U251, LN229, and A172. Among these cell lines,
U87MG, U251, and A172 harbor PTEN mutations with a
comparably overactive PI3K-Akt pathway. PI-103 caused inhibition of Akt phosphorylation as well as S6 phosphorylation as
assessed by immunoblotting (Fig. 1A). PI-103 also resulted in a
dose-dependent (Fig. 1B) and time-dependent (Supplementary Fig. 1) inhibition of cell proliferation in all tested glioma
lines. We further analyzed the effects of PI-103 on a selected
glioma line, Gli36-EvIII, and showed that PI-103 resulted in a
proliferative arrest in the cell cycle with a proportional
increase in G0–G1, a decrease in the S phase and no change
in the G2 phases of cell cycle (Fig. 1C).
To assess the effect of PI-103 in both subcutaneous and
orthotopic models of glioma, we used the highly malignant
Gli36-EvIII glioma line, engineered to express Fluc-mCherry,
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Targeting Gliomas with PI-103 and TRAIL
C
A
B
F
D
E
Figure 1. PI-103 inhibits glioma cell proliferation and attenuates tumor growth in vivo. A, Western blot analysis of p-Akt, p-S6, and tubulin levels in
established glioma lines. B, viability of glioma lines in response to 48-hour treatment of different doses of PI-103 as measured by CellTiterGlo assay.
*, P < 0.05 in the comparison of each treatment with controls, Student's t test. C, cell-cycle analysis of a selected glioma line, Gli36-EvIII, in response to
24-hour treatment of PI-103. D, experimental approach to test the effect of PI-103 on glioma growth in vivo. E, plot showing the Fluc bioluminescence intensity
of mice implanted with subcutaneous tumors and treated intraperitoneally with daily doses of 25 mg/kg of PI-103 or control (DMSO) for 17 days
(n ¼ 8 per group). Representative pseudocolor BLI images on days 0, 7, 14, and 17 are shown. F, plot showing the Fluc bioluminescence intensity of mice
implanted with intracranial tumors and treated intraperitoneally with daily doses of 25 mg/kg of PI-103 or control (DMSO) for 17 days (n ¼ 4 per group).
Representative pseudocolor BLI images on days 0, 11, 14, and 17 are shown. G and H, representative 100 images from p-Akt immunohistochemistry in
tumors dissected from control and PI-103–treated mice at day 17.
Gli36-EvIII-FmC (Supplementary Fig. 2). A significant attenuation of tumor growth was observed in mice bearing subcutaneous tumors treated with daily PI-103 i.p. injections as
compared with controls (Fig. 1E), revealing that PI-103 has
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the potential to block the growth of this fast-growing glioma in
mice. To test the effect of PI-103 orthotopically, we established
Gli36-EvIII-FmC intracranial gliomas in mice. PI-103 treatment slowed down the growth of intracranial tumors as
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Table 1. PI-103 levels in plasma, intracranial, and subcutaneous tumors
Treatment
Vehicle
PI-103a
PI-103b
Total PI-103 concentration, ng/mL
Relative ratios
Brain
SubQ
Plasma
Brain/plasma
SubQ/plasma
BQL
25.6
16.9
BQL
29.9
24
BQL
2.6
9.8
–
9.9
1.7
–
11.5
2.5
NOTE: Pharmacokinetic detection of PI-103 levels by LC-MS/MS using multiple reaction monitoring in plasma, intracranial tumor, and
subcutaneous tumor tissue collected from mice dosed with 25 mg/kg of PI-103 i.p.
Abbreviation: BQL, below quantization limit of 1 ng/mL.
a
Repeated daily dose of 25 mg/kg PI-103 i.p. (collection at 2 hours after last administration).
b
Single dose of 25 mg/kg PI-103 i.p. (collection at 2 hours after administration).
compared with the controls (Fig. 1F). The presence of PI-103 in
the intracranial tumors was confirmed by the assessment of
PI-103 levels in the brains of tumor-bearing mice (Table 1). To
assess the concentration of PI-103 in the subcutaneous and
intracranial tumor compartments relative to plasma levels, we
performed LC-MS/MS on tumors and plasma collected from
mice treated with (i) a single dose i.p. injection of PI-103 and
(ii) repeated daily i.p. injection of PI-103. Accordingly, the daily
injections of PI-103 resulted in higher levels of the drug in both
compartments possibly due to the establishment of steadystate levels (Table 1). Importantly, we observed that the
intracranial tumor compartment had comparable PI-103 levels
to the subcutaneous tumor compartment relative to plasma
levels in both treatment regimens (Table 1), suggesting that PI103 has the potential to reveal efficacy in orthotopic gliomas.
The growth-limiting effects of PI-103 in the in vivo models were
partly due to a downregulation of Akt activity as evident by the
decreased p-Akt staining in the paraffin sections extracted
A
PI-103 augments the response of glioma cells
to S-TRAIL
After confirming the in vivo potential of PI-103 in gliomas,
we tested whether PI-103 acts in concert with locally delivered
TRAIL through simultaneous inhibition of cell proliferation
and activation of cell death pathways. A combination treatment of PI-103 and S-TRAIL resulted in a significant inhibition
of cell viability compared with control conditions or S-TRAIL
alone in the majority of glioma lines with varying sensitivities
to TRAIL-mediated apoptosis (Fig. 2A). Next, we assessed the
effect of PI-103 on TRAIL response of the highly proliferating
and malignant Gli36-EvIII line. PI-103 treatment prior to STRAIL treatment elevated the TRAIL-induced cell killing as
B
C
158
from the tumor tissue (Fig. 1G and H) and decreased p-Akt
levels in the tumor tissue extracts (Supplementary Fig. 3).
These results demonstrate that PI-103 is a potent inhibitor
of growth in mouse models of malignant gliomas.
D
Cancer Res; 71(1) January 1, 2011
Figure 2. PI-103 augments the
response of glioma cells to STRAIL (A and B). Glioma cell
viability showing the combined
effect of PI-103 and S-TRAIL
treatment on different glioma lines
with PI-103 (5 mmol/L) and STRAIL (10 ng/mL; A) and dosedependent synergistic effect of PI103 and S-TRAIL on Gli36-EvIII
(B). C, caspase 3/7 activity
showing the combined effect of
PI-103 and S-TRAIL treatment on
Gli36-EvIII. *, P < 0.05 in the
comparison of each treatment
with controls, Student's t test.
D, Western blotting showing
changes in PARP cleavage with
S-TRAIL (100 ng/mL) alone or in
combination with PI-103
(1 mmol/L) in Gli36-EvIII.
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Targeting Gliomas with PI-103 and TRAIL
shown with varying doses of PI-103 and S-TRAIL (Fig. 2B). The
augmented TRAIL response with PI-103 treatment was also
evident at the caspase activation level. Specifically, whereas
TRAIL treatment induced caspase 3/7 activity up to 2.5-fold of
basal levels in control cells, cells previously exposed to PI-103
exerted up to 4-fold increase in caspase activity with TRAIL
(Fig. 2C). One of the effector proteins downstream of caspases,
PARP, was not affected by PI-103 alone but its cleavage upon
TRAIL treatment was enhanced by PI-103 (Fig. 2D). The
augmented TRAIL response was also observed in the Gli36
line that does not express EGFRvIII, suggesting that the effect
of PI-103 on TRAIL-induced cell killing is independent of
EGFR variant status in gliomas (Supplementary Fig. 4). These
results reveal that PI-103 works in concert with TRAILinduced apoptosis in glioma cells.
Effect of PI-103 and stem cell–derived TRAIL in
cocultures of mNSCs and glioma cells and in vivo
To test the combined effect of PI-103 and stem cell–
delivered TRAIL on glioma viability in vitro, we first engineered primary mouse NSCs (mNSC) to express a secretable and a highly potent variant of TRAIL, S-TRAIL (from
here on referred as mNSC-TRAIL; Supplementary Fig. 5). To
observe the effect of mNSC-TRAIL on glioma cells in vitro,
we established cocultures of glioma cells (Gli36-EvIII-FmC)
and mNSCs (mNSC-TRAIL; and control mNSC-GFP).
mNSCs initially spread and infiltrated into the Gli36EvIII-FmC monolayer (Fig. 3A) and mNSC-TRAIL resulted
in glioma killing as confirmed by significant decrease in
glioma cell viability as compared with the controls
(Fig. 3A). To test the effect of PI-103 in this system, the
monolayer of Gli36-EvIII-FmC cells were treated with PI103 for 24 hours prior to the seeding of mNSCs. A significantly increased glioma cell killing was observed in PI103 and mNSC-TRAIL treatment over 24 and 48 hours as
compared with the controls (Fig. 3B). These results reveal
that PI-103 acts in concert with mNSC-TRAIL and results in
increased cell killing in vitro.
To investigate the combined antiglioma potential of PI-103
and mNSC-TRAIL in vivo, we first demonstrated the functionality of mNSC-TRAIL by admixing glioma cells and mNSCs in
subcutaneous model. Tumors growing in the presence of
mNSC-TRAIL were significantly smaller than controls
(Fig. 3C). To ultimately examine the effect of combined PI103 and mNSC-TRAIL therapy in intracranial gliomas, mice
bearing established Gli36-EvIII-FmC gliomas were treated
with PI-103 over a brief period of 3 or 7 days and then
implanted with mNSC-TRAIL or control mNSC-GFP in the
close vicinity of the tumors (Fig. 3D). A significant decrease in
tumor volumes was observed in mice implanted with mNSCTRAIL after 3 day PI-103 pretreatment (Fig. 3E). To test the
effect of a longer PI-103 administration in this setting, we
administered PI-103 for 7 days prior to mNSC implantation. A
significantly reduced tumor size compared with control mice
was seen after mNSC-TRAIL implantation in PI-103–treated
mice (Fig. 3F). Taken together, these results reveal that
systemic delivery of PI-103 combined with mNSC-TRAIL
results in marked attenuation of intracranial tumor growth.
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PI-103 inhibits the proliferation and invasion of
primary glioma–initiating cells
Although our established glioma lines serve as potential
tools to examine the effect for PI-103 and TRAIL, they still lack
the characteristics that mimic clinical glioma settings. As
such, established glioma lines mainly form solid tumors when
implanted into mice as opposed to the highly infiltrative
nature of human gliomas. To this end, we utilized 3 primary
human GBM lines that are enriched in CD133þ glioma-initiating cells and form highly invasive tumors in mice (26, 30).
The treatment of GBM6, GBM8, and GBM12 lines with PI-103
slowed down their rate of proliferation in a dose- and timedependent manner (Fig. 4A), with GBM8 being the most
responsive cell line. Moreover, PI-103 treatment reduced
the invasive ability of GBM cells in a matrigel-coated invasion
assay to a far more extent than the inhibition of proliferation
(Fig. 4B and C). Accordingly, GBM6 and GBM8 invasion was
most affected by PI-103 after 24 hours (90% inhibition of
invasion in these cells compared with only 40% inhibition in
viability). PI-103 also inhibited PI3K and mTOR pathways as
indicated by reduced Akt and S6 phosphorylation in a dosedependent manner (Fig. 4D). To test whether PI-103 and
TRAIL can work together to inhibit the proliferation of these
clinically relevant GBM cells, we selected GBM8 line, which we
previously characterized for its TRAIL response (18). Treatment of GBM8 cells with PI-103 prior to S-TRAIL augmented
the TRAIL response in these cells (Fig. 4E). These results reveal
that PI-103 inhibits invasive properties of GBMs and that PI103 augments the TRAIL response, thus attenuating the
aggressive characteristic of primary GBMs.
Discussion
In this study, we demonstrate the effect of a dual PI3K/
mTOR inhibitor, PI-103, in a panel of established and primary
invasive glioma cell lines and provide evidence of the antitumor effects of systemically delivered PI-103 in intracranial
glioma models. Furthermore, we show that PI-103 co-operates
with stem cell–delivered S-TRAIL in mouse models of gliomas.
PI3K pathway is one of the most commonly deregulated
signaling networks in gliomas (6); therefore, the components
of this pathway are candidates for targeted therapies. A novel
pyridinylfuranopyrimidine inhibitor, PI-103, has been shown
to dually inhibit PI3K and mTOR and block cell proliferation in
several cancer cell lines including gliomas (7–16, 31–34). In
this study, we show that PI-103 inhibits cell proliferation in a
dose- and time-dependent manner. Although these established glioma lines are the most commonly used models in
vitro, they fail to recapitulate the clinical properties of tumors.
Therefore, recent studies have focused on primary glioma
lines and indicated a role for tumor-initiating cells in these
lines. In an effort to test the effect of PI-103 in such models, we
employed 3 invasive primary glioma lines with high CD133þ
expression (26, 30) and showed that PI-103 alone is effective in
attenuating growth as well as invasiveness of these cells. This
suggests that, once delivered efficiently, PI-103 might block
not only the proliferation but also the invasion of these
tumors. Our results agree with previous reports that PI-103
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Bagci-Onder et al.
A
B
C
d0
d5
D
E
F
Figure 3. Effect of PI-103 and stem cell–derived TRAIL in cocultures of mNSCs and glioma cells and in vivo. A and B, glioma cells, Gli36-EvIII-FmC
(red) either untreated (A) or PI-103 treated (B) and mNSCs (mNSC-TRAIL or mNSC-GFP; green) were cocultured; and 3 days (A) or 1 and 2 days (B) later glioma
cells were assessed for their viability by Fluc activity. Plots revealing glioma cell viability and representative bright-field and fluorescent coculture
photomicrographs are shown. Arrows show the space formed around mNSC-TRAIL as a result of glioma cell killing. *, P < 0.05 in the comparison of each
treatment with controls, Student's t test. C, demonstration of mNSC-TRAIL function in vivo. Plot showing the Fluc bioluminescence intensity of mice
implanted subcutaneously with a mixture of Gli36-EvIII-FmC and mNSCs (1: Gli36-EvIII, 2: Gli36-EvIII þ mNSC-GFP, 3: Gli36-EvIII þ mNSC-TRAIL; n ¼ 3 per
group). Representative pseudocolor BLI images on days 0 and 5 are shown. *, P < 0.05 in the comparison of mNSC-TRAIL group with mNSC-GFP or control
groups, Student's t test. D, experimental approach to test the effect of PI-103 and TRAIL in vivo. Each mouse received intracranial implantation of Gli36-EvIIIFmC cells and imaged 3 days later (day 0) followed by daily i.p. injection of PI-103 for 3 (for E) or 7 days (for F). E, plot showing the Fluc bioluminescence
intensity of mice implanted with intracranial gliomas in response to brief PI-103 (3 days) administration and mNSC-TRAIL. Tumor volume fold increase at day 7
compared with day 0 is plotted (n ¼ 7 per group). Representative pseudocolor BLI images on days 0 and 7 are shown. F, plot showing the Fluc
bioluminescence intensity of mice implanted with intracranial gliomas in response to longer PI-103 (7 days) administration and mNSC-TRAIL. Tumor volumes
at day 14 are plotted (n ¼ 4 per group). Representative pseudocolor BLI images on days 0 and 14 are shown. *, P < 0.05 in the comparison of PI-103 þ mNSCTRAIL groups with other treatment groups, Student's t test.
can serve as a widely effective antiglioma agent (13). Furthermore, we show that PI-103 is effective in reducing the growth
of orthotopic gliomas in vivo by using bioluminescence imaging. Our assessment of PI-103 levels in intracranial tumors in
comparison with subcutaneous tumors by LC-MS/MS demonstrates the availability of PI-103 in both tumor compartments,
suggesting that PI-103 might serve as a good tool compound
160
Cancer Res; 71(1) January 1, 2011
for efficacy studies in orthotopic gliomas as well as subcutaneous models. Also demonstrated by our analysis of p-Akt
levels in the tumor tissues, which can be regarded as a
pharmacodynamic marker, we can now be certain about
the effect of PI-103 in our glioma models. To our knowledge,
this is the first study demonstrating the effect of this dual
PI3K/mTOR inhibitor in an orthotopic glioma and is in line
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Targeting Gliomas with PI-103 and TRAIL
A
B
C
D
Figure 4. PI-103 inhibits the proliferation and invasion of primary glioma-initiating cells. A, PI-103 dose- and time-dependent changes in cell viability of
primary CD133þ glioma cell lines, GBM6, GBM8, and GBM12. B and C, photomicrographs (B) and plots (C) showing the changes in cell invasion upon
PI-103 treatment in 3 different primary glioma lines. D, Western blot analysis of p-Akt, p-S6, and tubulin levels in GBM6, GBM8, and GBM12 primary glioma
lines. E, cell viability showing the combined effect of PI-103 (1 mmol/L) and S-TRAIL (10 ng/mL) on GBM8 cells.
with the findings of a previous report on the pharmacologic
characterization of PI-103 (15).
Although targeting the highly proliferative state of gliomas
is a favorable therapeutic approach, it might not be sufficient
in the eradication of tumors that are left behind after surgical
intervention. Therefore, combinatorial approaches that target
tumor cell proliferation and induce tumor-specific cytotoxicity would be very effective in eliminating recurrence and
subsequent therapeutic failure. To this end, apoptosis-inducing reagents, such as TRAIL, have recently gained attention
for the preclinical studies. However, the short half-life and the
off-target toxicity of systemically delivered TRAIL pose a
challenge in translating it into the clinics (17). On the basis
of these limitations, we have previously established that stem
cell–delivered TRAIL is very effective in glioma eradication
(18–20, 27, 35, 36) due to its on-site delivery and long-term
www.aacrjournals.org
expression compared with systemically administered purified
TRAIL. In this study, we report on the combination of PI-103
and mNSC-TRAIL in our glioma models and demonstrate the
in vitro effects of PI-103 and S-TRAIL focusing on the viability
and apoptosis of a panel of established glioma cell lines. Prior
to our study, a few other studies reported the combination
effect of PI-103 with EGFR inhibition (32), radiation (31), and
chemotherapy-induced apoptosis (37). To our knowledge, this
is the first study that examines the combined effect of PI-103
with TRAIL in glioma cells both in vitro and in vivo. Our
findings suggest that PI-103 augments the response of glioma
cells to TRAIL. However, it should be noted that whereas we
did not observe a switch in the response of TRAIL-resistant
gliomas to TRAIL by PI-103 treatment, we noticed an increase
in the TRAIL response of glioma cells that were already
fairly sensitive to TRAIL. Therefore, we do not argue that
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Published OnlineFirst November 17, 2010; DOI: 10.1158/0008-5472.CAN-10-1601
Bagci-Onder et al.
PI-103 is a TRAIL-sensitizing agent, but it has the potential
to significantly augment the TRAIL-induced apoptosis in a
subset of gliomas. Addressing the molecular interactions of
PI-103–mediated PI3k/mTOR inhibition with TRAIL will
further our understanding of their combination as a treatment
strategy.
Our in vivo studies reveal that systemic delivery of PI-103
combined with mNSC-TRAIL results in marked attenuation of
intracranial tumor growth, suggesting that combining systemically delivered PI3K/mTOR inhibitors with stem cell–
delivered agents might lead to marked tumor eradication.
It should be noted that we performed our combination
therapy experiments with caution. We only briefly administered PI-103 prior to mNSC implantation to avoid any possible
collateral damage to mNSCs from PI-103. When we briefly
treated mice for only 3 days with PI-103 prior to mNSC
implantation, we did not observe a difference in tumor growth
due to drug alone. However, upon administration of the drug
for 7 days, we noticed a difference in tumor growth due to PI103, which was further enhanced with mNSC-TRAIL. Therefore, optimal conditions for alternating the systemically delivered drugs with implanted stem cells should be defined for
future translation into clinics.
Our results with intracranial glioma models suggest that the
combination of systemically delivered PI3K/mTOR inhibitors
and stem cell–delivered TRAIL offers promise in gliomas.
Testing this approach with other PI3K/mTOR inhibitors, such
as GDC-0941, which is undergoing phase I clinical trials (38),
would extend our findings and help with the translation of our
combination strategy to the clinics. This could be performed
in brain tumor patients by administering the clinically
approved PI3K/mTOR inhibitor systemically and implanting
stem cell–secreting S-TRAIL in the tumor resection cavity
after surgical resection of the tumor. This combination therapy would enhance the eradication of the residual tumor cells
and prevent tumor recurrence. Taken together, our study
might serve as an excellent foundation for future therapies
combining systemically delivered cytostatic drugs, such as
PI3K/mTOR inhibitors, with stem cell–delivered cytotoxic
agents, such as TRAIL in gliomas.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Dr. Philip Lambert (VivoPath) for help with LC-MS/MS analysis;
Dr. Andrew Kung (Dana Farber Cancer Institute) for providing pico2-FlucmCherry lentiviral vector; and Massachusetts General Hospital Pathology Core
for help with histologic analysis.
Grant Support
This work was supported by American Cancer Society (K. Shah) and Alliance
for Cancer Gene Therapy (K. Shah).
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 May 10, 2010; revised October 13, 2010; accepted November 9, 2010;
published OnlineFirst November 17, 2010.
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Published OnlineFirst November 17, 2010; DOI: 10.1158/0008-5472.CAN-10-1601
A Dual PI3K/mTOR Inhibitor, PI-103, Cooperates with Stem
Cell−Delivered TRAIL in Experimental Glioma Models
Tugba Bagci-Onder, Hiroaki Wakimoto, Maarten Anderegg, et al.
Cancer Res 2011;71:154-163. Published OnlineFirst November 17, 2010.
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