Download Proliferation-Independent Control of Tumor Glycolysis by PDGFR

Published OnlineFirst January 15, 2013; DOI: 10.1158/0008-5472.CAN-12-2460
Cancer
Research
Molecular and Cellular Pathobiology
Proliferation-Independent Control of Tumor Glycolysis by
PDGFR-Mediated AKT Activation
Cong Ran1,2, Huan Liu1,2, Yasuyuki Hitoshi1,2,3, and Mark A. Israel1,2
Abstract
The differences in glucose metabolism that distinguish most malignant and normal tissues have called
attention to the importance of understanding the molecular mechanisms by which tumor energy metabolism
is regulated. Receptor tyrosine kinase (RTK) pathways that are implicated in proliferation and transformation
have been linked to several aspects of tumor glucose metabolism. However, the regulation of glycolysis has
invariably been examined under conditions in which proliferation is concomitantly altered. To determine
whether RTKs directly regulate glycolysis without prerequisite growth modulation, we first identified a specific
RTK signaling pathway, platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) that regulates
glycolysis in glioma-derived tumor stem-like cells from a novel mouse model. We determined that PDGFregulated glycolysis occurs independent of PDGF-regulated proliferation but requires the activation of AKT, a
known metabolic regulator in tumor. Our findings identifying a key characteristic of brain tumors, aerobic
glycolysis, mediated by a pathway with multiple therapeutic targets suggests the possibility of inhibiting tumor
energy metabolism while also treating with agents that target other pathways of pathologic significance.
Cancer Res; 73(6); 1–13. 2013 AACR.
Introduction
Recent studies of glucose metabolism in malignant tissues
have suggested that metabolic changes are key alterations
contributing to tumorigenesis and tumor progression. Ongoing work seeks to link these changes to the genetic alterations
underlying oncogenesis (1–4). Normal tissues use aerobic
respiration to metabolize glucose in the presence of physiologic oxygen and glycolysis to metabolize glucose at times of
oxygen deprivation. The Warburg effect refers to the finding
that most malignant tissues, in contrast to normal tissues, used
glycolysis as the main pathway to metabolize glucose even in
the presence of sufficient oxygen (5).
A hallmark of altered metabolism in cancer is increased
glucose uptake. Virtually all high-grade glioma can be shown by
clinical positron emission tomography (PET) to have enhanced
glucose uptake, an aspect of aerobic glycolysis that has been
widely studied in these tumors (6, 7). Growth factor receptor
tyrosine kinases (RTK) implicated in tumorigenesis have been
investigated for their role in tumor glucose metabolism due to
Authors' Affiliations: 1Departments of Pediatrics and Genetics, Norris
Cotton Cancer Center; 2Geisel School of Medicine at Dartmouth, Hanover,
New Hampshire; and 3Department of Neurosurgery Rosai Hospital, Yatsushiro, Kumamoto, Japan
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Author: Mark A. Israel, Dartmouth Medical School, One
Medical Center Drive, Lebanon, NH 03756. Phone: 603-653-3611; Fax:
603-653-9003; E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-12-2460
2013 American Association for Cancer Research.
their structural similarities to the insulin receptor, another
RTK that has a well-defined role in regulating glucose homeostasis (8, 9). The use of common effector molecules by the
insulin receptor and other RTKs suggests that RTKs may
regulate tumor metabolism and thereby contribute to oncogenesis (4, 10). Although the EGF receptor (EGFR) activating a
rarely expressed glucose receptor has been identified in prostate cancer cells and phosphorylation of pyruvate kinase M2 by
the fibroblast growth factor receptor (FGFR) has been reported
(11, 12), it is not known whether any RTKs directly regulate
aerobic glycolysis in tumors. The typically synchronous
changes in proliferation and metabolism that occur in tumors
raises the possibility that RTK-mediated alterations in proliferation results in changes in glucose metabolism. Understanding whether regulation of proliferation and glycolysis by RTKs
are independently mediated by RTK signaling cascades is
essential for deciphering the mechanisms underlying the
reprogramming of glucose metabolism that occurs so frequently in most tumor types.
Considerable evidence points to an important role for
platelet-derived growth factor (PDGF) signaling through the
PDGF receptor (PDGFR), an RTK in the pathogenesis of glioma,
particularly in mediating tumor cell proliferation (13–15). The
Cancer Genome Atlas Network (TCGA) recently proposed a
molecular classification of glioblastoma multiforme (GBM),
the highest pathologic grade of glioma, and the most common
brain tumor of adults (16, 17). While alterations in the PDGF/
PDGFR-mediated growth pathway were observed in all 4
TCGA-described subtypes of glioma, such alterations were
identified at a much higher rate in the "proneural" subtype.
Amplification of DNA encoding the PDGFRa and high levels
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of PDGFRA gene expression were seen almost exclusively in
this proneural subtype (17). A second signature genetic alteration of the proneural subtype of glioma was mutation of the
IDH1 gene, which encodes isocitrate dehydrogenase 1, a mitochondrial enzyme of the Krebs cycle (17). Importantly, while
mutation of IDH1 is found almost exclusively in GBM that can
be classified as proneural, very few such GBMs had alterations
of the PDGF/PDGFR pathway and most proneural glioma that
had evidence of altered PDGF/PDGFR signaling did not bear
mutated IDH1 (17). This dichotomy suggested to us that
studying the role of an RTK, namely PDGFR, in regulating
glioma glucose metabolism might be productively pursued in
proneural glioma with normal IDH1 and activation of the
PDGF/PDGFR growth-stimulatory pathway.
Our laboratory has described a mouse model of human
glioma based on overexpression, human PDGFB (hPDGFB) in
tissues such as neural stem cells (NSC) that express glial
fibrillary acidic protein (GFAP; ref. 18). In this mouse model,
GFAP-mediated expression of hPDGFB is required for gliomagenesis and tumor progression and can be regulated by the
administration of tetracycline, which inhibits hPDGFB expression. Using this unique mouse model, we examined the role of
PDGF in regulating glycolysis in cultures enriched for gliomaderived tumor stem-like cells (TSC; ref. 19), a cellular subpopulation thought to be critical for tumor growth and progression. We found that in glioma-derived TSCs, PDGF signaling
regulated the Warburg effect, aerobic glycolysis. We also
observed that such regulation was independent of the effect
of PDGF signaling on cellular proliferation. The phosphoinositide 3-kinase (PI3K)/AKT complex, a PDGFR downstream
effector that has been recognized to mediate multiple activities
of RTKs was found to be a key mediator of PDGF-regulated
glycolysis in these glioma-derived TSCs.
Materials and Methods
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lentivirus encoding short hairpin RNAs (shRNAs) complementary to PDGFRA (Santa Cruz Biotechnology) and Sh-scramble
(ShScram), a lentivirus encoding a random set of 20 nucleotides, were used to generate cultures stably expressing these
transgenes. Recombinant plasmids encoding hemaglutinnin
(HA)-tagged AKT-DN (pCMV-HA-AKT-DN), myristolated
AKT1 (HA-myr-AKT1), and control constructs (pCMV and
PCDNA3; Addgene) were used to establish TSCs expressing
these transgenes (20).
Cell growth and viability
TSCs (100,000 cells/6-well dish) were seeded, harvested, and
counted by trypan blue exclusion. Cell viability was monitored
by fluorescence-activated cell-sorting (FACS) analysis of propidium iodide (PI).
Lactate production, glucose consumption, and glucose
uptake measurement
To examine lactate production and glucose consumption,
cells were seeded in Dulbecco's' Modified Eagle's Media
(DMEM):F12 without glutamine and 1B27 without insulin
supplementation (5,000 cells/24-well dish). Total lactate
secreted and glucose remaining in the conditioned media was
assayed (Point Scientific, L7596 and G7521) on a plate reader
(Molecular Devices Emax). Glucose consumption was determined by subtracting glucose in conditioned media from
glucose in the plating media. Lactate and glucose measurements were normalized to cell numbers. To measure 2-NBDG
uptake, TSCs were treated with doxycycline at 100 pg/mL for 18
hours, incubated DMEM containing 1 mmol/L 2-NBDG and
1 mmol/L of glucose for 1 hours, and evaluated by FACS (21)
Glycolytic rate measurement
TSCs (1.5 106 cells/10-cm dish) were incubated for 48
hours and the glycolytic rate measured (22).
Cell cultures
TSCs were isolated as previously described (18). Plasmid
DNA with LipoD293 (SignaGen) was used for transfection. 0.4
mg/mL puromycin (Sigma) or 250 mg/mL G418 (Invitrogen)
was added for selection after transfection and for a minimum
of 10 days. Infection with lentiviruses and selection in 0.4 mg/
mL puromycin for a minimum of 7 days was used to prepared
polyclonal shPDGFRA-expressing cultures.
TSC-R cultures were established recurrent gliomas after 2
months of continuous subcutaneous doxycycline treatment.
To establish doxycycline-resistant tumors from doxycyclinesensitive TSC xenografts, we injected 10,000 doxycycline-sensitive TSCs into nu/nu or syngeneic mice. Following tumor
formation, animals were treated with oral doxycycline and
tumors regressed. After 3 months of treatment, recurrent flank
tumors were excised and cultured. All animal usage was in
accordance with IACUC-approved protocols.
Mitochondrial potential, intracellular ATP assay, and
ADP/ATP ratio
To determine mitochondrial membrane potential, 500,000
cells were collected, washed with PBS, and incubated in PBS
[1% bovine serum albumin (BSA)/150 nmol/L tetramethylrhodamine, methyl ester (TMRM)] for 10 minutes before FACS
analysis (23). To examine ATP, TSCs (25,000 cells/24-well dish)
were seeded and incubated for 12 to 18 hours. Bioluminescence
was used to determine ATP (PerkinElmer) and ADP/ATP ratio
(Abcam, Ab65313).
Materials
PDGFR inhibitor AG1295 (Sigma), MEK1/2 inhibitor UO126
(Qiagen), and PI3K inhibitor LY294002 (Sigma) were diluted in
dimethyl sulfoxide (DMSO) as indicated. Pooled recombinant
Immunoblotting
Cells lysed by radioimmunoprecipitation assay (RIPA)
buffer were fractionated by 8% SDS-PAGE, transferred to
polyvinylidene fluoride (PVDF; Millipore), and evaluated by
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Real-time PCR
RNA was isolated (RNeasy Kit; Qiagen) and reverse transcribed using iScript (Bio-Rad). RNA was examined using iQ
SYBR Green Supermix (Bio-Rad) and the MyIQ Real-Time PCR
system (Bio-Rad). Expression was quantified after normalization using cyclophilin and 18S as controls.
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PDGFR-Mediated Regulation of Glioma Glycolysis
Immunoblots using antibodies: phospho-PATHSCAN at 1:200
and anti-PDGFR, phospho-PDGFRa, AKT, phospho-AKT,
ERK1/2, and phospho-ERK1/2 at 1:2,500 in 5% BSA/TBST (Cell
Signaling); anti-Glut1 antibody at 1:200 in 5% milk/TBST
(Abcam); anti-b-actin at 1:2,000 in 5% milk/TBST (Sigma).
Densitometric measurement of band density was completed
using ImagePro software on scanned blots.
Statistics
Data are representative of 3 or more independent experiments, presented as mean SEM. We used the 2-tailed Student
t test to evaluate differences between 2 groups. P < 0.05 was
designated statistically significant. For experiments involving
multiple comparisons, we conducted one-way ANOVA and
Tukey post hoc test to evaluate differences. Statistical analyses
were completed using GraphPad Prism software.
Results
Modulation of PDGF signaling in glioma-derived TSCs
To model human glioma, we described a GFAP-tTA:TREhPDGFB mouse model overexpressing hPDGFB that can be
regulated by doxycycline (18). To extend these studies, we
established cultures of glioma-derived TSCs from central
nervous system (CNS) glioma arising in these mice (14, 18).
To characterize the in vitro regulation of the hPDGFB transgene by doxycycline, we incubated cultures from 3 individual
tumors and found that hPDGFB mRNA levels decreased in a
dose-dependent manner (Fig. 1A). Because PDGFR is activated
by phosphorylation, we examined whether inhibition of
hPDGFB expression by doxycycline was associated with a
decrease in PDGFR phosphorylation (Fig. 1B). We found
inhibition of PDGFR phosphorylation and examined the effect
of doxycycline on activation of other kinases downstream of
PDGFR. Levels of activated, phosphorylated SHP2, AKT, and
ERK1/2, and total ERK1/2 expression were decreased when
hPDGFB expression was inhibited (Fig. 1C). To alternatively
modulate PDGFR, we treated these TSCs with AG1295, a
pharmacologic inhibitor of PDGFR (24). We found decreased
PDGFR phosphorylation and decreased activation of AKT and
ERK1/2 (Fig. 1D).
As predicted from our previously published work examining
doxycycline treatment of GFAP-tTA:TRE-hPDGFB mice (18),
we observed growth inhibition with doxycycline at concentrations that did not significantly alter cell survival (Fig. 1E,
Supplementary Fig. S1). Also, proliferation of TSCs was inhibited by AG1295 (Fig. 1F and data not shown). We named these
TSCs as doxycycline-sensitive: TSC-S1, TSC-S2, and TSC-S3.
GFAP-tTA:TRE-hPDGFB TSCs exhibit altered glucose
metabolism
Amongst glioma of the proneural subtype, alterations of
PDGF signaling and IDH1 mutations affecting glucose metabolism rarely occur in the same tumor (17). Glioma-derived TSCs
did not have alterations in IDH1 function (personal communications, Dr. Hai Yan, Duke University, Durham, NC), and so we
investigated PDGF regulation of glucose metabolism. We found
that in addition to slightly enhanced growth compared with
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NSCs from wild-type animals (WT NSC; Fig. 2A), TSCs exhibited
increased lactate production (Fig. 2B) and glucose consumption
(Fig. 2C). These changes are emblematic in tumor cells exhibiting the Warburg effect. We then used 5-[3H]-glucose to measure
their glycolytic rate (22). We found that the glycolytic rate was
higher in TSCs than in WT NSCs (Fig. 2D).
To exclude the possibility that enhanced glycolysis was
secondary to defective mitochondria, we examined mitochondrial membrane potential in TSCs using TMRM, a cationic
tetramethylrhodamine methyl ether (25). We found that mitochondrial membrane potential was not decreased in TSCs (Fig.
2E), indicating that increased glycolysis was not a result of
defective mitochondria. To determine the contributions of
mitochondrial activity and glycolysis to intracellular ATP, we
inhibited mitochondrial phosphorylation in TSCs and WT
NSCs using carbonyl cyanide m-chlorophenyl hydrozone
(CCCP), an uncoupling agent, as well as, antimycin A, an
electron transport inhibitor (12). We found that ATP in TSCs
following inhibition of mitochondrial respiration remained
significantly above the levels observed in treated NSCs (Fig.
2F), indicating that increased glycolysis contributed to the
increased ATP in TSCs. The observation that decreased ATP
levels resulting from treatment with these agents was similar in
WT NSCs and TSCs suggests a similar contribution of mitochondrial respiration to ATP levels.
We also surveyed mRNA expression of multiple enzymes in
glucose metabolism and found that mRNA levels of hexokinase
(HK) 1 and 2, lactate dehydrogenase A (LDHA), and Glut1 were
elevated in TSCs.
PDGF regulates glucose metabolism in glioma-derived
TSCs
In vascular smooth muscle cells, enhanced glycolytic flux
and increased mitochondrial reserve are associated with
PDGFR activation (26–28). Activation of PDGFR is a key
oncogenic alteration leading to tumorigenesis, and we hypothesized that PDGFR activation contributed to the alterated
glucose metabolism in TSCs (Fig. 2). To examine this, we first
determined whether modulating hPDGFB expression and
PDGFR signaling altered glucose metabolism. Doxycycline
treatment diminished hPDGFB expression and decreased lactate production and glucose consumption (Fig. 3A and B).
AG1295 inhibition of PDGFR caused a decrease in lactate
production and glucose consumption (Fig. 3C and D). Findings
in all 3 TSC cultures examined gave similar results (data not
shown).
We examined the glycolytic rate of TSCs following PDGF/
PDGFR inhibition. We observed approximately a 40% decrease
in glycolytic rate in 2 independent TSC cultures following
inhibition of PDGF/PDGFR by doxycycline (Fig. 3E), indicating
PDGF-induced glucose uptake contributed to enhanced
glycolysis. We then evaluated glucose uptake using the fluorescent glucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol4-yl)amino]-2-deoxyglucose (2-NBDG), in TSCs (21). A
decrease in glucose uptake was detectable by FACS following
inhibition of PDGF/PDGFR (Fig. 3F). Also, the expression of
HK1, HK2, and Glut1, molecules contributing glucose uptake
(29), was enhanced by PDGF (Fig. 3G, Supplementary Fig. S2A
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Figure 1. Doxycycline and PDGFR inhibitor, AG1295, effectively inhibit PDGF signaling in primary glioma-derived TSCs. TSC cultures, TSC-S1, S2, and
S3, were derived from 3 independent mouse CNS gliomas (18). A, steady-state hPDGFB mRNA levels in TSCs following 36 hours of doxycycline treatments.
B, immunoblot analysis of PDGFR phosphorylation in TSCs following 48 hours of doxycycline treatments. C, immunoblot analysis of PDGFRa, PDGFRb,
SHP2, AKT, and ERK1/2 phosphorylation and AKT and ERK1/2 total expression in TSCs following 48 hours of doxycycline treatments. D, left,
immunoblot analysis of PDGFR, AKT, and ERK1/2 activation in lysates of TSC-S1 following 48 hours of AG1295 treatments. Right, bar graph of immunoblot
band density measured in triplicate normalized to actin. B–D, derived from multiple gels independently blotted and examined with indicated antibodies. E,
growth curves of TSC-S1 following doxycycline treatment. F, growth curves of TSC-S1 following AG1295 treatments. Data in all panels are representative of 3
independent experiments and presented as mean SEM of triplicates ( , P < 0.05; , P < 0.01).
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PDGFR-Mediated Regulation of Glioma Glycolysis
Figure 2. Glioma-derived TSCs
exhibit Warburg-like glucose
metabolism. A, growth curves of WT
NSCs and TSC-S1. Lactate
production (B) and glucose
consumption (C) of WT NSCs and
TSC-S1. D, glycolytic rate of WT
NSCs and TSCs. E, FACS analysis to
evaluate mitochondrial membrane
potential of WT NSCs and TSCs by
TMRM. Left, histogram
representation of TMRM intensity in
WT NSC and TSCs (20,000 cells).
Right, average median fluorescence
of WT NSCs and TSCs. F,
intracellular ATP in WT NSCs and
TSC-S1 following treatment with
CCCP (40 minutes) or antimycin A
(40 minutes). G, steady-state mRNA
levels of enzymes involved in glucose
and lactate metabolism in WT NSCs
and TSC-S1. Data in all panels are
representative of 3 or more
independent experiments,
presented as mean SEM of
triplicates ( , P < 0.05; , P < 0.01;
, P < 0.005).
and S2B). These findings indicate that glucose influx is
enhanced in TSCs by activation of PDGFR. The expression of
LDHA and pyruvate dehydrogenase kinase 1 (PDK1), enzymes
affecting the conversion of pyruvate to lactate, decreased
following hPDGFB inhibition (Fig. 3G, Supplementary Fig.
S2C), suggesting that PDGF signaling is associated with other
changes enhancing glycolysis.
We observed in TSCs intracellular ATP levels above those in
corresponding normal cells (Fig. 2F), suggesting that PDGFB
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may regulate ATP in glioma-derived TSCs. We evaluated ATP
following inhibition of PDGFB and found that following doxycycline treatment intracellular ATP was reduced (Fig. 4A,
Supplementary Fig. S3A). This dramatic decrease in ATP was
also observed in TSCs treated with AG1295 (Fig. 4B, Supplementary Fig. S3B). We then examined the ADP/ATP ratio in
TSCs. Diminished PDGF signaling resulted in an increased
ADP/ATP ratio (Fig. 4C). In addition, we examined activation
of AMP-activated protein kinase (AMPK), an energy sensor
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Figure 3. PDGF pathway regulation of glucose metabolism in glioma-derived TSCs. Lactate production (A) and glucose consumption (B) of TSCs following
96 hours of doxycycline treatments. Lactate production (C) and glucose consumption (D) of TSC-S1 following 96 hours of AG1295 treatments. E, glycolytic
rate of TSCs in the presence (þ) or absence () of doxycycline treatment. F, FACS analysis of glucose uptake in TSC-S1 after 18 hours of doxycycline
treatment (100 pg/mL) using 2-NBDG. Left, histogram representation of 2-NBDG intensity (20,000 cells). Right, quantification of FACS analysis. G,
steady-state mRNA levels of TSC-S1 following 48 hours of doxycycline treatment (100 pg/mL). Data in all panels are representative of 3 or more independent
experiments, presented as mean SEM of triplicates ( , P < 0.05; , P < 0.01; , P < 0.005).
activated by increased intracellular AMP:ATP ratio (30). We
detected increasing levels of phosphorylated, activated AMPK
when hPDGFB expression was inhibited (Fig. 4D) consistent
with the decreased ATP following inhibition of PDGF/PDGFR
(Fig. 4A and B, Supplementary Fig. S3).
Decreased ATP following PDGF/PDGFR inhibition (Fig.
4A and B) could be due to decreased glycolysis or decreased
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mitochondrial function (27, 31). To characterize the contribution of these mechanisms to decreased ATP, we measured
mitochondrial membrane potential before and after hPDGFB
inhibition. No changes in membrane potential were
observed following doxycycline (Fig. 4E), indicating that
PDGF/PDGFR signaling does not inhibit mitochondrial
function.
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PDGFR-Mediated Regulation of Glioma Glycolysis
Figure 4. Intracellular ATP levels in
doxycycline-sensitive TSCs are
maintained by PDGF signaling.
Intracellular ATP in TSCs following 18
hours of doxycycline treatments (A)
or AG1295 treatments (B). C, ADP/
ATP ratio in TSCs following 18 hours
of dox treatment. D, immunoblot
analysis of AMPK a-subunit
phosphorylation in TSCs following
24 hours of doxycycline treatments.
E, FACS analysis of TSC-S1
following 24 hours of doxycycline
treatment to evaluate mitochondrial
membrane potential by TMRM. Left,
histogram representation of TMRM
intensity in TSCs (20,000 cells).
Right, quantification of FACS
analysis. All data are representative
of 3 or more independent
experiments and presented as mean
SEM of triplicate ( , P < 0.05;
, P < 0.01; , P < 0.005).
PDGF regulation of glycolysis in TSCs is independent of
its effect on proliferation
To determine whether PDGF-regulated glycolysis was a
consequence of changes in proliferation, we examined proliferation-arrested cells for changes in glucose metabolism. If
hPDGFB regulates glycolysis, its effects on glucose metabolism
would be independent of proliferation. We induced cell-cycle
arrest of glioma TSCs in M phase with mitomycin C (MMC;
ref. 32) and evaluated glycolysis in these cells (Supplementary
Fig. S4A). We found that doxycycline-mediated decreases in
hPDGFB expression resulted in decreased lactate production
and glucose consumption (Supplementary Fig. S4B and S4C),
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suggesting that PDGFR signaling modulated glucose metabolism independent of cellular proliferation.
To extend this observation (Supplementary Fig. S4), we
determined whether PDGF-regulated glycolysis in TSCs isolated from GFAP-tTA:TRE-hPDGFB tumors resistant to the
growth-inhibitory effects of decreased PDGF signaling. We
previously observed that a small percentage of GFAP-tTA:
TRE-hPDGFB glioma recurred in vivo despite continuous
doxycycline treatment (Supplementary Fig. S5A and data not
shown). Because in vivo recurrence was rare, we sought to
develop a system in which recurrent tumors occur more
frequently. We therefore established flank xenografts of
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doxycycline-sensitive TSCs in syngeneic or nude mice (Supplementary Fig. S5B). Despite continuous doxycycline treatment, approximately 70% of these animals developed recurrent
tumors. Three in vivo recurrent tumors and 3 xenografts that
recurred after continuous doxycycline treatment, a total of 6
recurrent tumors, which were each resistant to doxycycline
inhibition on tumor growth, were excised and propagated in
sphere culture as TSCs. We found that these TSCs were not
growth inhibited in vitro by doxycycline (Supplementary Fig.
S5C and data not shown). We named these TSCs that are
resistant to the antiproliferative effects of doxycycline TSC-R.
TSC-R1, 2, and 3 were derived from recurrences of primary
tumors and the TSC-R4, 5, and 6 were from the xenograft
model.
We evaluated TSC-R cells from these 6 independent tumors
and found that PDGF expression remained responsive to
doxycycline (data not shown), although the cells were not
growth inhibited by doxycycline. Similarly, AG1295 did not
affect proliferation in any of these cultures (Supplementary Fig.
S5D). As in doxycycline-sensitive TSCs, inactivation of PDGF/
PDGFR signaling in doxycycline-resistant TSCs decreased
lactate production and glucose consumption (Fig. 5A and
B), without detectable changes in proliferation (Supplementary Fig. S5D). Also, we detected a decrease in intracellular ATP
following PDGFR inhibition in these doxycycline-resistant
TSCs (Fig. 5C) comparable with the decreases in doxycycline-sensitive TSCs (Fig. 4B). Finally, we observed a decrease
in the glycolytic rate of doxycycline-resistant TSCs when
PDGFR was inhibited (Fig. 5D), confirming that glycolysis
was regulated by PDGF/PDGFR. To provide further evidence
that these changes in glycolysis were mediated specifically
by PDGFR, we inhibited the expression of surface receptor
PDGFRA using a short hairpin (Sh) to diminish PDGFR.
Two concentrations of recombinant retrovirus encoding
shPDGFRA, 0.5 and 2 multiplicity of infection (moi), were used.
In both instances phosphorylated and total PDGFRA
decreased (Fig. 5E). Also, glioma-derived doxycycline-resistant
TSCs infected with a lentivirus encoding shPDGFRA (0.5 and
2 moi) exhibited reduced lactate production (Fig. 5F),
decreased glucose consumption (Fig. 5G), and decreased
intracellular ATP (Fig. 5H).
AKT is a key mediator of PDGF-regulated tumor
glycolysis
To pursue the mechanism by which PDGF regulates
glycolysis in glioma TSCs, we evaluated downstream pathways including the mitogen-activated protein kinase
(MAPK) and PI3K/AKT pathways that are modulated by
PDGF/PDGFR signaling (10, 33). To evaluate the MAPK
pathway, we used UO126 to inhibit MEK1/2 kinase activity (34). Treatment decreased phosphorylation of the MEK
target, ERK1/2, although other downstream targets of
PDGFR were unaffected (Fig. 6A). While MEK1/2 inhibition
resulted in a decrease in proliferation (Fig. 6B), lactate
production and glucose consumption at multiple time
points after treatment exhibited no appreciable difference
(Fig. 6C and D). We interpreted these data to indicate that
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MEK signaling did not mediate PDGF/PDGFR regulation of
glycolysis.
We then examined the possible role of PI3K/AKT signaling
in glycolysis. We observed 2 classes of doxycycline-resistant
TSCs: those in which the AG1295 inhibited AKT phosphorylation (Fig. 6E: TSC-R1, TSC-R5, and data not shown) and those
in which AKT phosphorylation was not inhibited (Fig. 6E: TSCR4 and TSC-R6). Importantly, when AKT phosphorylation
remained responsive to PDGFR activity as in TSC-R1, TSCR2, and TSC-R5, lactate production (Fig. 6F) and glucose
consumption (Fig. 6G) were modulated by PDGFR; however,
when AKT was unaffected by inhibition of PDGFR as it was in
TSC-R4 and TSC-R6, there were no reproducible or significant
changes in lactate production (Fig. 6F) or glucose consumption
(Fig. 6G) following PDGF/PDGFR inhibition.
This apparent association of AKT modulation and glycolysis suggests that AKT may mediate PDGF-regulated
glucose metabolism. We therefore inhibited AKT activation
in both doxycycline-sensitive and -resistant TSCs with a
PI3K inhibitor, LY294002 (Fig. 7A). Following treatment,
we observed significant decreases in glycolysis as determined by lactate production (Fig. 7B), glucose consumption
(Fig. 7C), and the glycolytic rate of (Fig. 7D) This finding was
representative of all other TSC cultures examined (data
not shown).
We then overexpressed in TSC-S1 cells a HA-tagged dominant-negative form of AKT (AKT-DN), which prevents AKT
activation by blocking endogenous AKT from binding to PI3K
(20). Cultures in which approximately 40% of the population
was transfected with AKT-DN as estimated from the cytologic
evaluation of cells transfected with a comparable GFP-expressing control vector exhibited decreased phosphorylation of a
kinase downstream of AKT, S6-kinase (Supplementary Fig.
S6A). In these cells, we observed a 30% to 40% decrease in
lactate production (Supplementary Fig. S6B) and glucose
consumption (Supplementary Fig. S6C). We then expressed
a constitutively activated form of AKT1 in TSC-S3 (Fig. 7E–G)
(35). We transiently expressed HA-tagged myr-AKT1 (HA-myrAKT1) in TSCs. Activated AKT can be evaluated by assaying the
phosphorylation of downstream kinase p70-S6K (Fig. 7E;
ref. 36). In cells transfected with constitutively activated AKT1,
no increase in glycolysis was seen presumably because in the
PDGFB-secreting TSCs, AKT is extensively activated (Fig. 1C).
Adding doxycycline did not inhibit glycolysis, although it
did inhibit glycolysis in cells lacking constitutively activated
AKT1 (Fig. 7F and G). These findings indicate that activated
AKT1 can rescue PDGF inhibition of glycolysis. Consistent with
this interpretation is our finding that modulation of hPDGFB
by doxycycline alters expression of AKT target genes important for glucose metabolism (Fig. 3G, Supplementary Fig. S2;
refs. 21, 37).
Discussion
Altered glucose metabolism is a hallmark of cancer underlying PET imaging, which depends upon quantification of 2deoxy-2(18F)fluoro-D-glucose in tumors (6, 37). Such imaging
distinguishes most tumors from corresponding normal tissues.
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PDGFR-Mediated Regulation of Glioma Glycolysis
Figure 5. The PDGF pathway regulates glucose metabolism independent of proliferation. Lactate production (A) and glucose consumption (B) of TSCs
following 96 hours of AG1295 (þ) or DMSO () treatment. C, intracellular ATP in TSCs after 24 hours of AG1295 (þ) or DMSO () treatment. D, glycolytic rate
following 48 hours of AG1295 (þ) or DMSO () treatment. E, immunoblot analysis of PDGFR activation in TSC-R1 infected with sh-scramble (ShScram)
and sh-PDGFRa (ShPDGFRA) lentiviruses. Lactate production (F) and glucose consumption (G) of TSC-S1 expressing ShScram or ShPDGFRA. H,
intracellular ATP in TSC-R1 expressing shScram or ShPDGFRA. Data in all panels are representative of 3 or more independent experiments and presented as
mean SEM of triplicates ( , P < 0.05; , P < 0.01).
Alterations in glucose metabolism are important for tumor
progression (38). Mutations altering enzymatic activity of
IDH1, succinate dehydrogenase, or fumarate hydratase,
enzymes important for glucose metabolism, occur in a variety
of tumors (39, 40). Also, ectopic expression of alternative
isoforms of glycolytic enzymes that increase glycolysis, such
as PKM2, HK2, and phosphofructose kinase 4, promote tumor
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cell proliferation, survival, and invasion affecting patient outcome (29, 39, 41).
Further evidence suggesting an association between glucose
metabolism and malignancy is the observation that oncogenic
signaling molecules modulate expression of glycolytic enzymes
enhancing glycolysis and proliferation (38). For example, stimulation of the MAPK pathway, a mediator of proliferation,
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Figure 6. PI3K/AKT but not the MAPK pathway modulates tumor glycolysis in glioma-derived TSCs. A, immunoblot analysis of ERK1/2, AKT, and
PDGFR phosphorylation in lysates of TSC-S1 following 48 hours of MEK1/2 inhibitor UO126 treatments. B, growth curves of TSC-S2 following UO126
treatments. Lactate production (C) and glucose consumption (D) of TSC-S2 after 96-hour UO126 treatments. E, immunoblot analysis of PDGFR and AKT
phosphorylation in TSCs following 48 hours of AG1295 treatments. Lactate production (F) and glucose consumption (G) of TSC-Rs following 96 hours
of 10 mmol/L AG1295 (þ) or DMSO () treatment. Results are presented as a ratio normalized to DMSO-treated cells. Data in all panels are representative of 3 or
more independent experiments and presented as mean SEM of triplicates ( , P < 0.05; , P < 0.01).
alters pyruvate dehydrogenase kinase 4 expression increasing
pyruvate metabolism (34). Activated FGFR phosphorylates
PDK1 and PKM2 enhancing glycolysis (12, 34, 40). These
observations document an association between glucose
metabolism and proliferation. Some have proposed that proliferation is hard-wired to changes in glycolysis to fulfill
increased requirements for macromolecular synthesis (1,
2, 38), but modulation of cell-cycle progression alone is not
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Cancer Res; 73(6) March 15, 2013
sufficient to produce changes that resemble the Warburg effect
(42–44), raising the possibility of proliferation and glycolysis
being independently regulated in tumor cells.
The PDGF/PDGFR pathway is known to be deregulated in
multiple tumor types and it is frequently altered in glioma
(13, 16, 17). We found evidence of altered glucose metabolism by PDGF and observed that glioma-derived TSCs
exhibited enhanced glycolysis indistinguishable from the
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PDGFR-Mediated Regulation of Glioma Glycolysis
Figure 7. AKT is a key downstream mediator of PDGF-modulated glycolysis in glioma-derived TSCs. A, immunoblot analysis of PDGFR and AKT
phosphorylation in lysates of TSCs following 48 hours of LY294002 (þ) or DMSO () treatment. Lactate production (B) and glucose consumption (C)
of TSCs following 96 hours of LY294002 (þ) or DMSO () treatment. D, glycolytic rate of TSCs following 48 hours of LY294002 (þ) or DMSO () treatment.
E, left, immunoblot analysis of HA tag, pAKT, and phosphorylated p70-S6K in lysates of TSC-S3 overexpressing PCDNA3 and PCDNA3-HA-myrAKT1
transfectant. Right, bar graph of immunoblot band density measured in triplicate normalized to actin expression. Lactate production (F) and glucose
consumption (G) of TSC-S3 transfected with PCDNA3 or PCDNA3-HA-myrAKT1 following 72 hours of doxycycline treatments. Data in all panels are
representative of 3 or more independent experiments, presented as mean SEM of triplicates ( , P < 0.05; , P < 0.01; , P < 0.005).
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Warburg effect. In these TSCs, glucose uptake and lactate
production are coordinately increased (Figs. 2 and 3) and
were dependent on PDGF/PDGFR (Fig. 3). The PDGFinduced glucose uptake we observed was consistent with
previous reports showing PDGF signaling associated with
increased glucose uptake and vascular smooth muscle cell
growth (27, 45). We found that elevated levels of glucose
consumption and lactate production as well as an increased
cellular glycolytic rate (Fig. 5) in proliferating TSCs were
responsive to PDGF/PDGFR signaling. Several lines of investigation, however, indicated this regulation was independent
of proliferation (Figs. 5 and 6).
Seeking to identify the mediators of PDGF regulation
of glioma glycolysis, we investigated 2 pathways downstream
of PDGFR, PI3K/AKT and MAPK. We found that inhibition of
AKT resulted in decreased glycolysis, whereas modulation of
MAPK activation did not (Fig. 6). AKT, a key mediator of
insulin-induced glucose uptake, coordinates changes in glucose uptake and lactate production by altering the phosphorylation state and expression of several different glycolytic
enzymes (9, 26). We found that the mRNA or protein expression
of these enzymes correlates with PDGFB signaling (Fig. 2G and
Supplementary Fig. S2), suggesting the possibility of AKTmediating glycolysis in these cells. Overexpression of constitutively active AKT rescued decreased glycolysis associated
with inhibited PDGF/PDGFR consistent with AKT acting as a
key mediator of PDGF-regulated glycolysis (Fig. 7F and G).
PDGFR inhibition has not yet been shown clinically to impact
significantly on glioma progression as measured by tumor
growth (46). We observed among the cell cultures growth
resistant to doxycycline that some proliferated in the presence
of AKT inhibition, suggesting that these cells acquired doxycycline resistance by activation of downstream proliferative
pathways. Our finding that a PDGFR/PI3K/AKT axis regulates
glioma glucose metabolism by a pathway distinct from proliferation suggests that PDGFR inhibition altering glucose
metabolism might have therapeutic effects that cannot be
recognized in single-agent trials monitoring tumor cell proliferation. Consistent with this idea is the finding that tumor cells
may have enhanced sensitivities to apoptosis following nutriment restriction (11, 47) and the work of others to treat cancer
by inhibiting glucose metabolism (2). Other evidence shows
that alterations in glucose metabolism alone can influence key
characteristics of malignancy (48, 49) provides a strong rationale to examine the potential of PDGF/PDGFR inhibition to
contribute to tumor treatment by complementing effective,
targeted growth inhibitors.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: C. Ran, M.A. Israel
Development of methodology: C. Ran, H. Liu, Y. Hitoshi
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): C. Ran, H. Liu, Y. Hitoshi
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): C. Ran, H. Liu, M.A. Israel
Writing, review, and/or revision of the manuscript: C. Ran, M.A. Israel
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Ran, H. Liu
Study supervision: C. Ran, M.A. Israel
Acknowledgments
The authors thank Z. Reitman, Drs. D. Compton, L. Witters, and H. Yan for
suggestions and assistance and thank Dr. T. Tosteson for statistical consultation.
Grant Support
Support was generously provided by the Theodora B. Betz Foundation, Kyra
and Jordon Memorial Foundation and Hitchcock Foundation.
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 June 27, 2012; revised December 20, 2012; accepted January 4, 2013;
published OnlineFirst January 15, 2013.
References
1.
Hsu PP, Sabatini DM. Cancer cell metabolism: Warburg and beyond.
Cell 2008;134:703–7.
2. Kroemer G, Pouyssegur J. Tumor cell metabolism: cancer's Achilles'
heel. Cancer Cell 2008;13:472–82.
3. Jones RG, Thompson CB. Tumor suppressors and cell metabolism: a
recipe for cancer growth. Genes Dev 2009;23:537–48.
4. Israel M, Schwartz L. The metabolic advantage of tumor cells. Mol
Cancer 2011;10:70.
5. Warburg O. On the origin of cancer cells. Science 1956;123:309–14.
6. Hayat MA. Methods of cancer diagnosis, therapy, and prognosis. 1st
ed. Union, NJ: Springer; 2010.
7. Wolf A, Agnihotri S, Guha A. Targeting metabolic remodeling in
glioblastoma multiforme. Oncotarget 2011;1:552–62.
8. Thong FSL, Bilan PJ, Klip A. The Rab GTPase-activating protein
AS160 integrates Akt, protein kinase C, and AMP-activated protein kinase signals regulating GLUT4 traffic. Diabetes 2007;56:
414–23.
9. Whiteman EL, Cho H, Birnbaum MJ. Role of Akt/protein
kinase B in metabolism. Trends Endocrinol Metab 2002;13:
444–51.
10. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell
2000;103:211–25.
OF12
Cancer Res; 73(6) March 15, 2013
11. Weihua Z, Tsan R, Huang WC, Wu Q, Chiu CH, Fidler IJ, et al. Survival of
cancer cells is maintained by EGFR independent of its kinase activity.
Cancer Cell 2008;13:385–93.
12. Hitosugi T, Kang S, Vander Heiden MG, Chung TW, Elf S, Lythgoe K,
et al. Tyrosine phosphorylation inhibits PKM2 to promote the Warburg
effect and tumor growth. Sci signal 2009;2:ra73.
13. Dai C, Celestino JC, Okada Y, Louis DN, Fuller GN, Holland EC. PDGF
autocrine stimulation dedifferentiates cultured astrocytes and induces
oligodendrogliomas and oligoastrocytomas from neural progenitors
and astrocytes in vivo. Genes Dev 2001;15:1913–25.
14. Hambardzumyan D, Squatrito M, Carbajal E, Holland EC. Glioma
formation, cancer stem cells, and akt signaling. Stem Cell Rev 2008;4:
203–10.
15. Shih AH, Dai C, Hu X, Rosenblum MK, Koutcher JA, Holland EC. Dosedependent effects of platelet-derived growth factor-B on glial tumorigenesis. Cancer Res 2004;64:4783–9.
16. TCGA. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 2008;455:
1061–8.
17. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM,
et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.
Nature 2009;462:739–44.
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 2013 American Association for Cancer
Research.
Published OnlineFirst January 15, 2013; DOI: 10.1158/0008-5472.CAN-12-2460
PDGFR-Mediated Regulation of Glioma Glycolysis
18. Hitoshi Y, Harris BT, Liu H, Popko B, Israel MA. Spinal glioma: plateletderived growth factor B-mediated oncogenesis in the spinal cord.
Cancer Res 2008;68:8507–15.
19. Gottschalk S, Anderson N, Hainz C, Eckhardt SG, Serkova NJ. Imatinib
(STI571)-mediated changes in glucose metabolism in human leukemia
BCR-ABL-positive cells. Clin Cancer Res 2004;10:6661–8.
20. Zhou BP, Hu MC, Miller SA, Yu Z, Xia W, Lin SY, et al. HER-2/neu
blocks tumor necrosis factor-induced apoptosis via the Akt/NF-kappaB pathway. J Biol Chem 2000;275:8027–31.
21. O'Neil RG, Wu L, Mullani N. Uptake of a fluorescent deoxyglucose
analog (2-NBDG) in tumor cells. Mol Imaging Biol 2005;7:388–92.
22. Elstrome RL, Bauer DE, Buzzai MKR, Karnauskas R, Harris MH, Plas
DR, et al. Akt Stimulates Glycolysis in Cancer Cells. Cancer Res
2004;64:3892–99.
23. Wlodkowic D, Skommer J, Darzynkiewicz Z. Flow cytometry-based
apoptosis detection. Methods Mol Biol 2009;559:19–32.
24. Fishbein I, Waltenberger J, Banai S, Rabinovich L, Chorny M, Levitzki
A, et al. Local delivery of platelet-derived growth factor receptorspecific tyrphostin inhibits neointimal formation in rats. Arterioscler
Thromb Vasc Biol 2000;20:667–76.
25. Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T,
et al. Sequential reduction of mitochondrial transmembrane potential
and generation of reactive oxygen species in early programmed cell
death. J Exp Med 1995;182:367–77.
26. Hoehn KL, Hohnen-Behrens C, Cederberg A, Wu LE, Turner N, Yuasa
T, et al. IRS1-independent defects define major nodes of insulin
resistance. Cell Metab 2008;7:421–33.
27. Perez J, Hill BG, Benavides GA, Dranka BP, Darley-Usmar VM.
Role of cellular bioenergetics in smooth muscle cell proliferation
induced by platelet-derived growth factor. Biochem J 2010;428:
255–67.
28. Umahara M, Okada S, Yamada E, Saito T, Ohshima K, Hashimoto K,
et al. Tyrosine phosphorylation of Munc18c regulates platelet-derived
growth factor-stimulated glucose transporter 4 translocation in 3T3L1
adipocytes. Endocrinology 2008;149:40–9.
29. Wolf A, Agnihotri S, Micallef J, Mukherjee J, Sabha N, Cairns R, et al.
Hexokinase 2 is a key mediator of aerobic glycolysis and promotes
tumor growth in human glioblastoma multiforme. J Exp Med 2011;208:
313–26.
30. Guo D, Hildebrandt IJ, Prins RM, Soto H, Mazzotta MM, Dang J, et al.
The AMPK agonist AICAR inhibits the growth of EGFRvIII-expressing
glioblastomas by inhibiting lipogenesis. Proc Natl Acad Sci U S A
2009;106:12932–7.
31. Ko YH, Pan W, Inoue C, Pedersen PL. Signal transduction to
mitochondrial ATP synthase: Evidence that PDGF-dependent
phosphorylation of the delta-subunit occurs in several cell lines,
involves tyrosine, and is modulated by LPA. Mitochondrion 2002;
1:339–48.
32. Kang SG, Chung H, Yoo YD, Lee JG, Choi YI, Yu YS. Mechanism of
growth inhibitory effect of Mitomycin-C on cultured human retinal
pigment epithelial cells: apoptosis and cell cycle arrest. Curr Eye Res
2001;22:174–81.
33. Liu K-W, Feng H, Bachoo R, Kazlauskas A, Smith EM, Symes K, et al.
SHP-2/PTPN11 mediates gliomagenesis driven by PDGFRA and
INK4A/ARF aberrations in mice and humans. J Clin Invest 2011;121:
905–17.
www.aacrjournals.org
34. Grassian AR, Metallo CM, Coloff JL, Stephanopoulos G, Brugge JS.
Erk regulation of pyruvate dehydrogenase flux through PDK4 modulates cell proliferation. Genes Dev 2011;25:1716–33.
35. Ramaswamy S, Nakamura N, Vazquez F, Batt DB, Perera S, Roberts
TM, et al. Regulation of G1 progression by the PTEN tumor suppressor
protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt
pathway. Proc Natl Acad Sci U S A 1999;96:2110–5.
36. Yecies Jessica, Zhang HuiH, Menon S, Liu S, Yecies D, Lipovsky AI,
et al. Akt stimulates hepatic SREBP1c and lipogenesis through parallel
mTORC1-dependent and independent pathways. Cell Metabolism
2011;14:21–32.
37. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.
Cell 2011;144:646–74.
38. Vander Heiden MG, Cantley LC, Thompson CB. Understanding
the Warburg effect: the metabolic requirements of cell proliferation.
Science 2009;324:1029–33.
39. Goidts V, Bageritz J, Puccio L, Nakata S, Zapatka M, Barbus S, et al.
RNAi screening in glioma stem-like cells identifies PFKFB4 as a key
molecule important for cancer cell survival. Oncogene 2012;31:
3235–43.
40. Hitosugi T, Fan J, Chung TW, Lythgoe K, Wang X, Xie J, et al.
Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism. Mol Cell
2011;44:864–77.
41. Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A,
Gerszten RE, Wei R, et al. The M2 splice isoform of pyruvate kinase
is important for cancer metabolism and tumour growth. Nature
2008;452:230–3.
42. Lemons JMS, Feng X-J, Bennett BD, Legesse-Miller A, Johnson EL,
Raitman I, et al. Quiescent fibroblasts exhibit high metabolic activity.
PLoS Biol 2010;8:e1000514.
43. Funes JM, Quintero M, Henderson S, Martinez D, Qureshi U,
Westwood C, et al. Transformation of human mesenchymal
stem cells increases their dependency on oxidative phosphorylation for energy production. Proc Natl Acad Sci U S A 2007;
104:6223–8.
44. Weinberg F, Hamanaka R, Wheaton WW, Weinberg S, Joseph J, Lopez
M, et al. Mitochondrial metabolism and ROS generation are essential
for Kras-mediated tumorigenicity. Proc Natl Acad Sci U S A 2010;107:
8788–93.
45. Livnat T, Chen-Zion M, Beitner R. Platelet-derived growth factor
(PDGF) rapidly stimulates binding of glycolytic enzymes to muscle
cytoskeleton, prevented by calmodulin antagonists. Biochem Med
Metab Biol 1994;53:28–33.
46. Razis E, Selviaridis P, Labropoulos S, Norris JL, Zhu MJ, Song DD,
et al. Phase II study of neoadjuvant imatinib in glioblastoma: evaluation
of clinical and molecular effects of the treatment. Clin Cancer Res
2009;15:6258–66.
47. Sengupta S, Peterson TR, Sabatini DM. Regulation of the mTOR
complex 1 pathway by nutrients, growth factors, and stress. Molecular
Cell 2010;40:310–22.
48. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis?Nat Rev Cancer 2004;4:891–9.
49. Ward PS, Thompson CB. Metabolic reprogramming: a cancer
hallmark even Warburg did not anticipate. Cancer Cell 2012;21:
297–308.
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Proliferation-Independent Control of Tumor Glycolysis by
PDGFR-Mediated AKT Activation
Cong Ran, Huan Liu, Yasuyuki Hitoshi, et al.
Cancer Res Published OnlineFirst January 15, 2013.
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