Published OnlineFirst January 19, 2012; DOI: 10.1158/0008-5472.CAN-11-2967
Cancer
Research
Tumor and Stem Cell Biology
PGC-1a Promotes the Growth of ErbB2/Neu–Induced
Mammary Tumors by Regulating Nutrient Supply
rie Che
nard1, Shawn McGuirk1, David Germain1, Daina Avizonis2,
Eva Klimcakova1, Vale
William J. Muller1, and Julie St-Pierre1
Abstract
Cancer cells display an increased reliance on glycolysis despite the presence of sufficient oxygen levels to
support mitochondrial functions. In this study, we asked whether ameliorating mitochondrial functions in cancer
cells might limit their proliferative capacity. Specifically, we increased mitochondrial metabolism in a murine
cellular model of ErbB2/Neu–induced breast cancer by ectopically expressing the transcriptional coactivator
peroxisome proliferator–activated receptor g coactivator 1a (PGC-1a), a master regulator of mitochondrial
metabolism. As predicted, ErbB2/Neu cells ectopically expressing PGC-1a displayed an increased level of
mitochondrial metabolism and reduced proliferative capacity in vitro, compared with controls. In contrast,
ErbB2/Neu cells ectopically expressing PGC-1a formed larger tumors in vivo. These tumors exhibited increased
concentrations of glucose and the angiogenic factor VEGF as well as higher expression of ErbB2/Neu compared
with controls. We discovered that ErbB2/Neu levels were sensitive to nutrient availability, such that reduced
glucose concentrations resulted in diminished ErbB2/Neu protein levels. Therefore, our data indicate that PGC1a prevents the nutrient-mediated downregulation of ErbB2/Neu in tumors by increasing glucose supply.
Mechanistic investigations revealed that the regulation of ErbB2/Neu levels by glucose was mediated by the
unfolded protein response (UPR). Incubation of ErbB2/Neu–induced breast cancer cells in limited glucose
concentrations or with drugs that activate the UPR led to significant reductions in ErbB2/Neu protein levels. Also,
ErbB2/Neu–induced tumors ectopically expressing PGC-1a displayed lowered UPR activation compared with
controls. Together, our findings uncover an unexpected link between PGC-1a–mediated nutrient availability,
UPR, and ErbB2/Neu levels. Cancer Res; 72(6); 1538–46. 2012 AACR.
Introduction
Cancer occurs when cells divide uncontrollably. To proliferate, cells need significant amount of energy to build all the
components necessary for making new cells. It is therefore not
surprising that cancer cells display metabolic reorganizations
tailored for their high and rapid energy demands. The best
known metabolic reorganization that cancer cells undergo is a
switch from mitochondrial to glycolytic metabolism despite
the presence of sufficient oxygen to support mitochondrial
reactions (Warburg effect).
Metabolic reprogramming plays a central role in ErbB2/
Neu–induced breast cancer. Indeed, ErbB2/Neu–induced
Authors' Affiliations: 1Department of Biochemistry and Goodman Cancer
Research Centre; and 2Metabolomics Core Facility, Goodman Cancer
Research Centre, McGill University, Montreal, Quebec, Canada
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
nard contributed equally to the work.
E. Klimcakova and V. Che
Corresponding Author: Julie St-Pierre, McGill University, 3655 Sir William
Osler, Montreal, QC, H3G 1Y6, Canada. Phone: 514-398-3474; Fax: 514398-6769; E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-11-2967
2012 American Association for Cancer Research.
1538
breast cancer cells have increased reliance on glycolysis and decreased respiration (1, 2). Limiting the activity
of the glycolytic pathway in ErbB2/Neu–induced breast
cancer cells resulted in drastically smaller tumors (1),
highlighting the glucose dependence of these cancer cells.
Importantly, ErbB2/Neu–induced breast cancer cells with
reduced glycolytic activity displayed significantly elevated
mitochondrial respiration. Overall, these experiments in
ErbB2/Neu–induced breast cancer cells show how central
to tumor maintenance is the metabolic reprogramming
of cancer cells and suggest that increasing the reliance of
cancer cells on mitochondrial metabolism could limit
tumor growth.
To modulate mitochondrial functions in cancer cells, we
need to examine key regulators of mitochondrial metabolism. The peroxisome proliferator–activated receptor g coactivators 1 (PGC-1), namely, PGC-1a (3) and PGC-1b (4), are
master regulators of mitochondrial functions. PGC-1a and
PGC-1b are expressed in highly oxidative tissues and both
stimulate mitochondrial biogenesis and increase total respiration (5). Very little work has been done on the physiologic roles of the PGC-1 transcriptional coactivators in
cancer, in particular breast cancer. Reports have shown that
the expression of PGC-1 is reduced in breast cancer patients
(6, 7) and that interference with PGC-1b activity through
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Impact of PGC-1a on Cancer Metabolism
expression of miR-378 in breast cancer cells is accompanied
by increased cell proliferation (2).
In this study, we investigated the effects of increasing PGC1a expression in ErbB2/Neu–induced breast cancer cells on
their metabolism and proliferative capacity in vitro and in vivo.
We uncovered an unexpected link between PGC-1a–mediated
glucose supply, unfolded protein response (UPR) and ErbB2/
Neu levels. We show that ErbB2/Neu–induced tumors ectopically expressing PGC-1a are larger than controls and display
elevated levels of glucose and ErbB2/Neu. We discovered that
ErbB2/Neu levels are reduced during glucose shortages. We
conclude that PGC-1a–mediated glucose supply in tumors
limits ErbB2/Neu downregulation, which in turn favors
tumorigenesis.
Materials and Methods
Tissue culture and generation of stable cell lines
NMuMG and SK-BR-3 cells were obtained from the American Type Culture Collection. The NT2196 and TM15 cells are
described in ref. 8. To generate NT2196-PGC-1a stable cell
lines, NT2196 cells were transfected with a PGC-1a vector
(Addgene #1026) and clones resistant to G418 were selected.
NT2196-PGC-1a clones were independently derived twice to
confirm reproducibility of data. NT2196-PGC-1a cells were
cultured in Dulbecco's Modified Eagle's Medium, 10% FBS, 10
mg/mL insulin, 20 mmol/L HEPES, penicillin/streptomycin,
1 mg/mL puromycin, 400 mg/mL G418, pH 7.5, at 37 C and
5% CO2.
Gene expression
Total RNA was extracted with RNeasy Mini Kit and RNaseFree DNase Set (QIAGEN) or Aurum Total RNA Mini Kit
(Biorad) and reverse transcribed with SuperScript II Reverse
Transcriptase kit or iScript cDNA Synthesis kit (Invitrogen or
Biorad). mRNA expression was assessed by real-time quantitative PCR using Brilliant SYBR Green QPCR Master Mix or iQ
SYBR green Supermix (Stratagene or Biorad), gene-specific
primers (Supplementary Table S1; ref. 8–11) and a Stratagene
Mx3005P (Agilent Technologies) or MyiQ2 Real-Time Detection System (Biorad). Tbp was used as endogenous control
gene.
Immunoblotting
Proteins were extracted with lysis buffer (50 mmol/L TrisHCl pH7.4, 1% Triton X-100, 0.25% sodium deoxycholate, 150
mmol/L NaCl, 1 mmol/L EDTA) with inhibitors (2 mg/mL
pepstatin, 1 mg/mL aprotinin, 1 mg/mL leupeptin, 0.2 mmol/
L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate) and quantified. The blots were incubated according to
the manufacturer's instructions with the following primary
antibodies: PGC-1a (Calbiochem; ST1202), Neu (Santa Cruz;
sc-284), eIF2a (Cell Signaling; 9721,9722) and Actin (Santa
Cruz; sc-1616) and with horseradish peroxidase–conjugated
secondary antibodies (GE Healthcare or Santa Cruz). The
results were visualized by Western Lightning Plus-ECL (Perkin
Elmer). Densitometry analyses were conducted with ImageJ
software (NIH).
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Respiration
Cellular respiration was measured as previously published
(1). The inhibitors were from Sigma.
In vitro cell proliferation assay
To determine proliferation, cells were grown in their respective medium, washed, trypsinized, and counted by a TC10
automated cell counter (Biorad). Viability was determined by
exclusion of trypan blue dye.
In vivo tumorigenesis assays
Cells were resuspended at 5 104 cells per 30 mL in PBS and
injected into the fourth mammary gland fat pad of 5 to 7-weekold athymic nude female mice (Taconic). For NT2196-derived
clones, 3 groups of 5 mice were injected bilaterally with NT2196
empty vector Control-1 or Control-2 clones on the left side and
NT2196-PGC-1a-1.1 (group 1), PGC-1a-1.2 (group 2), or PGC1a-2 (group 3) cells on the right side. Tumor growth was
monitored weekly with a caliper. Tumor volumes were calculated with the following formula: volume (mm3) ¼ width2 length/2. All animal studies were approved by the Animal
Resource Centre at McGill University and comply with guidelines set by the Canadian Council of Animal Care. The transgenic MMTV/neu deletion mouse model (NDL2-5 strain) is
described in ref. 12.
VEGF quantification
VEGF was quantified by the VEGF ELISA Kit (Calbiochem)
according to the manufacturer's instructions.
Glucose concentration
The samples were extracted from tumor tissues according to
previously established protocols (13), dried and stored at
80 C until nuclear magnetic resonance (NMR) data collection. NMR data collection was carried out on a 500 MHz Inova
NMR system (Agilent Technologies) equipped with a cryogenically cooled probe. One-dimensional NMR spectra of samples
were collected using the first increment of the standard NOESY
experiment supplied with the instrument. Glucose chemical
shift assignments were confirmed by 2-dimensional 75 ms
mixing time total correlation spectroscopy. Targeted profiling
of glucose was achieved with a 500 MHz metabolite library
from Chenomx NMR Suite 7.0 (Chenomx). The area fit for the
glucose peaks was compared with that of the internal concentration standard (DSS) resulting in a concentration based
on the Chenomx library glucose compound as described
previously (14). The amount of glucose was normalized to the
weight of the tumor.
Results
Neu-transformed NT2196 cells display the Warburg
effect
Several ErbB2/Neu–initiated mammary tumor cell lines
have been shown to display increased reliance on glycolysis
even in the presence of normal levels of oxygen, a phenomenon
called the Warburg effect (1, 2). We used NT2196 cells as
ErbB2/Neu experimental cell line. These cells are ex vivo
explants originating from tumors expressing an activated form
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Klimcakova et al.
of ErbB2/Neu (8). To determine the metabolic properties of
NT2196 cells, we first examined the gene expression profile of
various glycolytic and mitochondrial enzymes (Fig. 1A). The
expression of the glycolytic genes hexokinase II (Hk2) and
lactate dehydrogenase A (Ldha) was upregulated in NT2196
cells compared with parental controls. Conversely, the expression of genes involved in mitochondrial metabolism, such as
cytochrome c oxidase subunit VIIa 1 (Cox7a1), uncoupling
protein 2 (Ucp2), and NADH dehydrogenase (ubiquinone) 1
beta subcomplex 5 (Ndufb5), was downregulated in NT2196
cells compared with controls. In addition, the expression of
PGC-1a was significantly decreased in NT2196 cells. To determine the physiologic relevance of these changes in gene
expression, we quantified the activity of the glycolytic and
mitochondrial pathways by measuring respectively lactate
production and cellular respiration. NT2196 cells showed
increased lactate production and decreased respiration compared with parental control cells (Fig. 1B) illustrating that
NT2196 cells display the Warburg effect and are thus a good
experimental model to study the impact of increasing mitochondrial metabolism on tumor growth.
Figure 1. Neu-induced mammary tumors cells NT2196 display the
Warburg effect. A, NT2196 cells display increased expression of
glycolytic genes hexokinase 2 (Hk2) and lactate dehydrogenase A (Ldha)
and decreased expression of mitochondrial metabolism genes
cytochrome c oxidase subunit 7A1 (Cox7a1), uncoupling protein 2
(Ucp2), and NADH dehydrogenase (ubiquinone) 1 beta subcomplex 5
(Ndufb5) compared with parental controls. PGC-1a mRNA levels are
lower in NT2196 cells compared with parental controls, paralleling the
reduced expression of mitochondrial metabolism genes. B, uncoupled
respiration and lactate production of NT2196 cells and their parental
controls. Data are presented as means SEM. , P < 0.05, Student's
t test, n ¼ 3 to 6 for mRNA expression, n ¼ 4 for respiration, n ¼ 9 for
lactate production.
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Ectopic expression of PGC-1a in NT2196 cells increases
mitochondrial metabolism and lowers proliferative
capacities in vitro
To ameliorate mitochondrial functions in NT2196 cells, we
created stable NT2196 cell lines ectopically expressing PGC-1a.
We independently derived 2 sets of clones to ensure reproducibility in the results. In the first set, we isolated 2 PGC-1a
clones called PGC-1a-1.1 and PGC-1a-1.2 and one control
clone called Control-1. In the second set, we isolated one
PGC-1a clone called PGC-1a-2 and one control clone called
Control-2.
First, we confirmed that PGC-1a expression was increased
in the PGC-1a clones. All 3 PGC-1a clones displayed increased
expression of PGC-1a at the mRNA and protein levels compared with their respective controls (Fig. 2A and B). Importantly, the expression of PGC-1b, another member of the PGC-1
family, remained unchanged (Fig. 2A). The expression of the
mitochondrial electron transport chain gene Cox7a1, which is a
known target of PGC-1a, was increased in all 3 clones (Fig. 2A).
The expression of the glycolytic gene Ldha was unchanged (Fig.
2A). It is important to mention that the clones from the first set
(Control-1, PGC-1a-1.1, and PGC-1a-1.2) had lower levels of
Neu compared with those from the second set (Control-2 and
PGC-1a-2; Fig. 2B). To evaluate the physiologic impact of
PGC-1a in NT2196 cells, we carried out bioenergetics analyses
of mitochondrial functions. All PGC-1a clones displayed significantly higher rates of total and uncoupled respiration
compared with controls (Fig. 2C). Finally, we assessed the
impact of PGC-1a ectopic expression on the growth properties
of NT2196 cells in vitro. All 3 PGC-1a clones displayed reduced
proliferation rates compared with controls (Fig. 2D). Together,
these results show that elevated PGC-1a levels in NT2196 cells
enhance mitochondrial functions and limit their proliferative
capacity in vitro.
PGC-1a increases Neu-mediated tumorigenesis in vivo
To determine whether the PGC-1a–mediated improved
mitochondrial functions in NT2196 cells impacted tumor
growth in vivo, PGC-1a clones and controls were injected in
the mammary fat pad of female nude mice. Each mouse was
injected on the left side with a control clone and on the right
side with a PGC-1a clone. Contrary to the decreased proliferation of PGC-1a clones in vitro, Neu-initiated mammary
tumors ectopically expressing PGC-1a grew faster and were
larger than controls (Fig. 3A). The growth rates of the PGC-1a-2
and Control-2 tumors were higher than those of PGC-1a-1 and
Control-1 tumors (Fig. 3A), consistent with their higher Neu
protein levels (Fig. 2B). Given the difference between the in
vitro and in vivo results, we first confirmed that PGC-1a
endpoint tumors displayed higher PGC-1a mRNA levels than
control tumors (Fig. 3B). Furthermore, the expression of
Cox7a1, a PGC-1a target gene, was higher in PGC-1a endpoint
tumors compared with controls (Fig. 3B). The expression of
PGC-1b and LdhA was not different between PGC-1a and
control endpoint tumors (Fig. 3B). Together, these gene expression data illustrate that PGC-1a and control endpoint tumors
display the same expression profile as the PGC-1a and control
clones (Figs. 2A and 3B).
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Impact of PGC-1a on Cancer Metabolism
Figure 2. Ectopic expression of
PGC-1a in NT2196 cells ameliorates
mitochondrial functions. A, PGC-1a
and Cox7a1 mRNA levels are
increased in clones of NT2196
ectopically expressing PGC-1a,
while the expression of PGC-1b and
Ldha remains unchanged. B, protein
levels for PGC-1a and Neu in
NT2196-PGC-1a clones. Note that
we independently derived 2 sets of
clones to ensure reproducibility in the
results. C, total and uncoupled
respiration of NT2196-PGC-1a
clones normalized to their controls.
D, proliferation curves for NT2196PGC-1a clones and their controls.
Data are presented as means SEM.
, P < 0.05, Student's t test, n ¼ 3 for
mRNA expression, n ¼ 6 to 7 for total
and uncoupled respiration, n ¼ 3 to 5
for proliferation.
Next, we quantified Neu protein levels in endpoint tumors
(Fig. 3C and D). Strikingly, we noticed that a large fraction
(11 of 15) of PGC-1a endpoint tumors displayed higher levels
of Neu compared with their paired control (P < 0.05). Indeed,
all mice of the group injected with the PGC-1a-1.1 clones,
2 mice of the group injected with the PGC-1a-1.2 clones, and
4 out of 5 mice of the group injected with PGC-1a-2 clones
displayed higher levels of Neu compared with their paired
controls. Importantly, there was no difference in the protein
levels of Neu between controls and PGC-1a clones in vitro
(Fig. 2B). To further highlight the differences between the
in vitro and in vivo conditions, we loaded side by side on 1 gel
the controls and PGC-1a clones next to the controls and
PGC-1a tumors (Fig. 3F). As shown in Fig. 2B, the protein
levels of Neu for the clones were similar between PGC-1a
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and controls, whereas for the tumors the levels of Neu were
higher in the PGC-1a compared with controls (Fig. 3F).
Furthermore, the Neu protein levels for both PGC-1a and
controls were lower in the tumors compared with the clones
(Fig. 3F).
To determine whether there was a correlation between Neu
levels and tumor size, we graphed the relative Neu protein
levels of all control and PGC-1a tumors in relation with their
tumor size and carried out a correlation analysis (Fig. 3E).
There was a significant positive correlation between Neu
protein levels and tumor volumes across all tumors (r ¼
0.685, P < 0.0001). Furthermore, for each group of mice, the
mice with the highest fold changes in tumor volume were the
ones with the highest fold changes in Neu levels (data not
shown). Together, these data show that a large fraction of PGC-
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Figure 3. NT2196-PGC-1a clones
form larger tumors during in vivo
tumorigenesis assays compared
with controls. A, tumor growth
curves for NT2196-PGC-1a and
control clones injected in
mammary fat pad of nude mice. B,
PGC-1a and Cox7a1 mRNA levels
are increased in NT2196-PGC-1a
tumors, while the expression of
PGC-1b and Ldha remains
unchanged. C, Neu protein levels in
NT2196-PGC-1a and control
tumors. Each mouse was injected
on the left with control clone and on
the right with NT2196-PGC-1a
clone. D, densitometry analyses for
(C). Fold changes were calculated
by dividing the relative Neu signal
values for the NT2196-PGC-1a
clones by those for the control
clones. Note that 11 of 15 of
NT2196-PGC-1a tumors display
higher Neu levels compared with
their paired controls. E, correlation
between tumor size and Neu
protein levels across NT2196PGC-1a and control tumors (n ¼
30). Relative Neu levels were
calculated by dividing the signal of
each band by that of the loading
control. The correlation analysis
was conducted by Spearman test.
F, comparison of Neu protein levels
between NT2196-PGC-1a clones
and their derived tumors,
contrasting the identical Neu levels
between NT2196-PGC-1a and
control clones with the different
Neu levels between NT2196-PGC1a and control tumors. A and B,
data are presented as means SEM. , P < 0.05, Student's paired t
test. D, the horizontal bars
represent the averages. A–D, n ¼ 5
mice per group. E, n ¼ 15 mice.
1a tumors displayed higher protein levels of Neu compared
with controls, and that there was a link between Neu levels and
tumor volumes.
PGC-1a regulates Neu levels indirectly by increasing
nutrient availability
One key difference between in vitro and in vivo conditions
is the control of nutrient availability through blood supply.
Given that PGC-1a impacted Neu levels in vivo but not in vitro
and that PGC-1a is a powerful regulator of VEGF expression
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and angiogenesis (15), we hypothesized that PGC-1a regulates
Neu levels in vivo by controlling nutrient availability through
angiogenesis.
To assess the ability of PGC-1a to regulate VEGF expression, we measured the amount of VEGF secreted in culture
medium for all experimental clones (Fig. 4A). All PGC-1a
clones secreted larger amounts of VEGF in culture medium
compared with controls. Furthermore, PGC-1a tumors displayed higher levels of VEGF compared with their paired
controls (Fig. 4B). Specifically, 12 of the 15 PGC-1a tumors
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Impact of PGC-1a on Cancer Metabolism
had higher levels of VEGF compared with their paired control.
In support of the increased VEGF levels in the PGC-1a tumors,
they also displayed elevated concentration of glucose compared with their paired controls (Fig. 4C). Indeed, 11 out of the
15 PGC-1a tumors displayed higher glucose levels compared
with their paired controls.
To assess whether there was a link between the elevated
levels of Neu, VEGF, and glucose in PGC-1a tumors, we
determined whether Neu levels are regulated by nutrient
availability. PGC-1a clones and controls were incubated in
various glucose concentrations (25, 2.5, and 0.5 mmol/L) for 5
days to mimic the low nutrient levels that can be found in solid
tumors. Neu protein levels were drastically reduced in PGC-1a
and control clones incubated in 2.5 and 0.5 mmol/L glucose
compared with those incubated in 25 mmol/L glucose (Fig.
4D). Together, these data illustrate that PGC-1a positively
regulates the angiogenic factor VEGF and glucose levels in
tumors, and that Neu levels are regulated by glucose
availability.
Regulation of ErbB2/Neu levels by the UPR
To gain further insight into the regulation of Neu protein
levels upon glucose availability, we studied the impact of UPR.
The UPR is a response to endoplasmic reticulum (ER) stress
that can be initiated by various stress stimuli, notably glucose
deprivation (16). Acute glucose removal drastically reduced
Neu levels in PGC-1a and control clones (Fig. 5A). The reduction in Neu levels occurred at the posttranslational level as
neuNT mRNA levels remained unchanged after glucose withdrawal (Supplementary Fig. S1). Decreased ErbB2/Neu protein
levels upon glucose withdrawal were also observed in another
mouse ErbB2/Neu–induced breast cancer cell line (TM15;
ref. 8) as well as in the human ErbB2-positive SKBR3 breast
cancer cell line (Fig. 5A), indicating the generality of ErbB2/Neu
regulation by glucose levels.
To assess whether the UPR was activated upon glucose
withdrawal in our experimental conditions, we measured
the splicing of X-box binding protein1 (XBP-1) mRNA, which
is an indicator of ER stress. The ratio of spliced XBP-1/total
XBP-1 mRNA was significantly increased after glucose withdrawal in all PGC-1a and control clones (Fig. 5B). In addition
to glucose withdrawal, various drugs can be used to stimulate
the UPR, in particular DTT and thapsigargin. Incubation of
PGC-1a clones and controls with either DTT or thapsigargin
led to significant reduction in Neu protein levels (Fig. 5C and
Supplementary Fig. S2). There was similar reduction in Neu
levels upon DTT or thapsigargin treatment between the PGC1a clones and controls. These data illustrate that Neu levels
are regulated by the UPR. Given that Neu is regulated at the
protein level during glucose withdrawal-induced ER stress
and that a central way by which the UPR halts protein synthesis is through phosphorylation of the eukaryotic initiation
factor 2a (eIF-2a), we quantified the phosphorylation of
eIF-2a in PGC-1a tumors and their paired controls to assess
whether UPR activation was lowered in PGC-1a tumors in
accordance with their higher Neu and glucose levels. Indeed,
the PGC-1a tumors displayed significantly less phosphorylation of eIF-2a compared with their paired controls (P ¼
0.0012; Fig. 5D and E). Specifically, 13 out of 15 PGC-1a
tumors showed this effect, illustrating lowered UPR activation in these tumors. Furthermore, 11 out of 15 PGC-1a
tumors displayed reduced XBP-1 mRNA splicing (P ¼ 0.076;
Figure 4. Neu levels are regulated by glucose availability. A, amount of VEGF secreted in the medium for the NT2196-PGC-1a clones normalized to controls. B,
fold change in tumor VEGF content. For each mouse, the content of VEGF in the NT2196-PGC-1a tumor was divided by the content of VEGF in the
paired control tumor. C, glucose levels in NT2196-PGC-1a tumors compared with their paired controls. Glucose levels were expressed as fold changes
representing glucose concentration values in NT2196-PGC-1a tumors divided by those of their paired control tumors. D, Neu protein levels are regulated
by glucose availability. NT2196-PGC-1a and control clones were grown for 5 days in 0.5, 2.5, or 25 mmol/L glucose. A, data are presented as means SEM.
n ¼ 9. , P < 0.05, Student's t test. B and C, the horizontal bars represent the averages. B and C, n ¼ 5 mice per group.
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Figure 5. Regulation of ErbB2/Neu levels by the UPR. A, acute glucose deprivation reduces Neu protein levels. NT2196-PGC-1a and control clones as well as
TM15 cells were deprived of glucose overnight. SK-BR-3 cells were deprived of glucose for 45 hours. B, the ratio of spliced XBP-1 mRNA is increased upon
glucose withdrawal in all control and NT2196-PGC-1a clones. The fold change in XBP-1 splicing was calculated by dividing the ratio of spliced XBP-1/total
XBP-1 mRNA in no glucose condition by that of the control condition. C, Neu protein levels are reduced upon induction of the UPR by glucose withdrawal, DTT,
or thapsigargin. D, phosphorylated eIF2a (eIF2a-P) and total eIF2a (eIF2a-T) protein levels in NT2196-PGC-1a and control tumors. Each mouse was injected
on the left with control clone and on the right with NT2196-PGC-1a clone. E, densitometry analyses for (D). The data represent the average of 4 independent
loadings and quantifications. Fold changes were calculated by dividing the ratio eIF2a-P/eIF2a-T for the NT2196-PGC-1a tumors by that of their paired
control tumors. Note that 13 of 15 of NT2196-PGC-1a tumors display lower ratios eIF2a-P/eIF2a-T compared with their paired controls. B, data are presented
as means SEM, n ¼ 7. , P < 0.05; , P < 0.001, Student's t test. D and E, n ¼ 5 mice per group. E, the horizontal bars represent the averages.
Supplementary Fig. S3). Together, the results presented here
suggest that PGC-1a, by regulating glucose availability, would
reduce ER stress, thereby alleviating the UPR, preventing Neu
downregulation, and favoring tumorigenesis.
Discussion
In this article, we examined the role of PGC-1a in ErbB2/
Neu-induced breast cancer. There is currently much interest
to investigate whether ameliorating mitochondrial functions
in cancer could limit tumor growth (17). The PGC-1a transcriptional coactivator is a well known master regulator of
mitochondrial metabolism (5). However, it is important to
appreciate that in addition to increasing mitochondrial functions, PGC-1a can stimulate VEGF expression and angiogenesis (15). These 2 functions of PGC-1a can have opposing
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consequences on tumorigenesis. Elevated mitochondrial metabolism could in theory reduce tumor growth, while increased
angiogenesis would support tumorigenesis. Therefore, PGC-1a
may play a positive or negative role in cancer depending on
the balance between these 2 processes in a given tumor.
Reports on the expression of PGC-1 in cancer show that it is
dependent on tumor types. The expression of PGC-1 is reduced
in breast (6, 7), colon (18), ovarian (19), and liver (20) tumors,
and low levels of PGC-1 are associated with poor clinical
outcome in breast cancer patients (6). Overexpression of
PGC-1a in ovarian and intestinal cell lines stimulated apoptosis (19), while interference with PGC-1b activity through
expression of miR-378 in breast cancer cells was accompanied
by increased cell proliferation (2). On the contrary, PGC-1a
expression is increased in endometrial cancer (21) and renal
tumors (22), and PGC-1 activity has been shown to promote the
Cancer Research
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Impact of PGC-1a on Cancer Metabolism
growth of prostate cancer cells (23). A recent paper by Tiraby
and collaborators (24) showed no impact of PGC-1a on the
growth of the MDA-MB-231 breast cancer cell line in vitro and
in xenograft experiments. Given that MDA-MB-231 cells do not
overexpress ErbB2, these results support our conclusion that
PGC-1a promotes the growth of ErbB2/Neu tumors by preventing ErbB2/Neu levels downregulation through increasing
nutrient supply. Importantly, there are in vivo situations in
which increased ErbB2/Neu signaling is associated with elevated levels of PGC-1a. Indeed, mammary tumors from
transgenic mice expressing an activated form of ErbB2/Neu
(NDL2-5) displayed elevated mRNA levels of PGC-1a compared with adjacent mammary gland (Supplementary Fig. S4).
However, it is also possible that PGC-1a promotes the growth
of ErbB2/Neu tumors independently of its impact on ErbB2/
Neu levels. In our study, 4 out of the 15 PGC-1a tumors were
larger than their paired controls, but did not display increased
levels of ErbB2/Neu. It is important to appreciate that PGC1a levels are highly sensitive to many environmental factors
and this could explain the reported variability in PGC-1a
expression in different tumor types. In agreement with this,
PGC-1a levels were found to be regulated by oxygen and
nutrient levels (15), which can fluctuate greatly during tumor
growth.
A central discovery of this study is that ErbB2/Neu levels are
regulated by glucose availability and the UPR. The UPR is
activated when misfolded proteins accumulate in the ER upon
stressful cellular environmental conditions like glucose limitations. The UPR is particularly relevant for solid tumors, which
experience nutrient stress due to poor vascularization. Upon
an environmental insult, the UPR initially halts translation and
activates signaling cascades aiming at restoring proper protein
folding. If the insult persists or proper folding is not achieved,
the UPR leads to activation of apoptosis. Therefore, the UPR
can have prosurvival and prodeath functions depending on the
severity of the environmental insult (16). It is of paramount
importance to understand how modulation of the UPR in
cancer affects the balance between its prosurvival and prodeath functions. Indeed, activation of the prosurvival functions
of the UPR would promote tumorigenesis, while activation of
the prodeath functions of the UPR would limit tumorigenesis.
So far, reports have shown both increased and decreased UPR
in different tumor types (16).
PGC-1a has been shown to mediate the UPR in skeletal
muscle, a response particularly important during exercise training (11). The UPR is activated during exercise, and muscle
tissues from MCK-PGC-1a transgenic mice that have increased
expression of PGC-1a display less ER stress after exercise than
wild-type mice (11). In this study, we did not detect any
difference in the downregulation of ErbB2/Neu protein levels
upon activation of the UPR during glucose withdrawal between
ErbB2/Neu–induced breast cancer cells ectopically expressing
PGC-1a and controls (Fig. 5A and C; Supplementary Fig. S2).
However, ErbB2/Neu–induced mammary tumors ectopically
expressing PGC-1a displayed less ER stress than their paired
controls (Fig. 5D and E; Supplementary Fig. S3).
ErbB2-positive breast tumors display a large increase in
ErbB2 expression (25). It is important to appreciate that cells
spend approximately 30% of their energy budget for protein
translation, with mRNA processing also making a significant
contribution (26). Strictly from an energy budget standpoint, it
would make sense under harsh cellular conditions to halt the
translation of a protein that is significantly overexpressed and
hence costly to maintain. Therefore, reducing the supply of
nutrients in solid growing tumors would be an efficient way to
limit ErbB2 levels. In support of this point, VEGF inhibitors are
currently showing promise in breast cancer patients in combination therapy with trastuzumab, a humanized her2 antibody (27). Clearly, the results presented in this article provide a
strong rationale for reduction of angiogenesis in ErbB2/Neu
breast tumors.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors thank Drs Vincent Giguere, John Bergeron, Peter Siegel, Nicole
Beauchemin, and Damien D'Amours for helpful discussion. The authors also
thank Erzsebet Nagy Kovacs and Vasilios Papavasiliou for technical assistance.
Grant Support
This work was supported by grants from the Canadian Institutes of Health
Research to J. St-Pierre (MOP-106603) and W.J. Muller (MOP-93525). E. Klimcakova was supported by a Tomlinson fellowship (McGill University) and a
fellowship from the McGill Integrated Cancer Research Training Program. S.
McGuirk was supported by a studentship from the McGill Integrated Cancer
Research Training Program (McGill University). NMR experiments were
recorded at the Quebec/Eastern Canada High Field NMR Facility, supported
by the Natural Sciences and Engineering Research Council of Canada and
Canada Foundation for Innovation. J. St-Pierre is an FRSQ research scholar and
W.J. Muller holds a Canada Research Chair in Molecular Oncology.
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 September 1, 2011; revised January 6, 2012; accepted January 11, 2012;
published OnlineFirst January 19, 2012.
References
1.
2.
3.
Fantin VR, St-Pierre J, Leder P. Attenuation of LDH-A expression
uncovers a link between glycolysis, mitochondrial physiology, and
tumor maintenance. Cancer Cell 2006;9:425–34.
Eichner LJ, Perry MC, Dufour CR, Bertos N, Park M, St-Pierre J, et al.
miR-378( ) mediates metabolic shift in breast cancer cells via the PGC1beta/ERRgamma transcriptional pathway. Cell Metab 2010;12:352–61.
Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM. A
cold-inducible coactivator of nuclear receptors linked to adaptive
thermogenesis. Cell 1998;92:829–39.
www.aacrjournals.org
4.
5.
6.
Lin J, Puigserver P, Donovan J, Tarr P, Spiegelman BM. Peroxisome
proliferator-activated receptor gamma coactivator 1beta (PGC-1beta),
a novel PGC-1-related transcription coactivator associated with host
cell factor. J Biol Chem 2002;277:1645–8.
Handschin C, Spiegelman BM. Peroxisome proliferator-activated
receptor gamma coactivator 1 coactivators, energy homeostasis, and
metabolism. Endocr Rev 2006;27:728–35.
Jiang WG, Douglas-Jones A, Mansel RE. Expression of peroxisomeproliferator activated receptor-gamma (PPARgamma) and the
Cancer Res; 72(6) March 15, 2012
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research.
1545
Published OnlineFirst January 19, 2012; DOI: 10.1158/0008-5472.CAN-11-2967
Klimcakova et al.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
1546
PPARgamma co-activator, PGC-1, in human breast cancer correlates
with clinical outcomes. Int J Cancer 2003;106:752–7.
Watkins G, Douglas-Jones A, Mansel RE, Jiang WG. The localisation
and reduction of nuclear staining of PPARgamma and PGC-1 in human
breast cancer. Oncol Rep 2004;12:483–8.
Ursini-Siegel J, Rajput AB, Lu H, Sanguin-Gendreau V, Zuo D, Papavasiliou V, et al. Elevated expression of DecR1 impairs ErbB2/Neuinduced mammary tumor development. Mol Cell Biol 2007;27:
6361–71.
Dufour CR, Wilson BJ, Huss JM, Kelly DP, Alaynick WA, Downes M,
et al. Genome-wide orchestration of cardiac functions by the orphan
nuclear receptors ERRalpha and gamma. Cell Metab 2007;5:345–56.
St-Pierre J, Drori S, Uldry M, Silvaggi JM, Rhee J, Jager S, et al.
Suppression of reactive oxygen species and Neurodegeneration by
the PGC-1 transcriptional coactivators. Cell 2006;127:397–408.
Wu J, Ruas JL, Estall JL, Rasbach KA, Choi JH, Ye L, et al. The unfolded
protein response mediates adaptation to exercise in skeletal muscle
through a PGC-1alpha/ATF6alpha complex. Cell Metab 2011;13:
160–9.
Siegel PM, Ryan ED, Cardiff RD, Muller WJ. Elevated expression of
activated forms of Neu/ErbB-2 and ErbB-3 are involved in the induction
of mammary tumors in transgenic mice: implications for human breast
cancer. EMBO J 1999;18:2149–64.
Wu H, Southam AD, Hines A, Viant MR. High-throughput tissue
extraction protocol for NMR- and MS-based metabolomics. Anal
Biochem 2008;372:204–12.
Weljie AM, Newton J, Mercier P, Carlson E, Slupsky CM. Targeted
profiling: quantitative analysis of 1H NMR metabolomics data. Anal
Chem 2006;78:4430–42.
Arany Z, Foo SY, Ma Y, Ruas JL, Bommi-Reddy A, Girnun G, et al. HIFindependent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha. Nature 2008;451:1008–12.
Healy SJ, Gorman AM, Mousavi-Shafaei P, Gupta S, Samali A. Targeting the endoplasmic reticulum-stress response as an anticancer
strategy. Eur J Pharmacol 2009;625:234–46.
Cancer Res; 72(6) March 15, 2012
17. Jeninga EH, Schoonjans K, Auwerx J. Reversible acetylation of PGC-1:
connecting energy sensors and effectors to guarantee metabolic
flexibility. Oncogene 2010;29:4617–24.
18. Feilchenfeldt J, Brundler MA, Soravia C, Totsch M, Meier CA. Peroxisome proliferator-activated receptors (PPARs) and associated transcription factors in colon cancer: reduced expression of PPARgammacoactivator 1 (PGC-1). Cancer Lett 2004;203:25–33.
19. Zhang Y, Ba Y, Liu C, Sun G, Ding L, Gao S, et al. PGC-1alpha induces
apoptosis in human epithelial ovarian cancer cells through a PPARgamma-dependent pathway. Cell Res 2007;17:363–73.
20. Ba Y, Zhang CN, Zhang Y, Zhang CY. [Down-regulation of PGC-1alpha
expression in human hepatocellular carcinoma]. Zhonghua Zhong Liu
Za Zhi 2008;30:593–7.
21. Cormio A, Guerra F, Cormio G, Pesce V, Fracasso F, Loizzi V, et al. The
PGC-1alpha-dependent pathway of mitochondrial biogenesis is upregulated in type I endometrial cancer. Biochem Biophys Res Commun
2009;390:1182–5.
22. Klomp JA, Petillo D, Niemi NM, Dykema KJ, Chen J, Yang XJ, et al. BirtHogg-Dube renal tumors are genetically distinct from other renal
neoplasias and are associated with up-regulation of mitochondrial
gene expression. BMC Med Genomics 2010;3:59.
23. Shiota M, Yokomizo A, Tada Y, Inokuchi J, Tatsugami K, Kuroiwa K, et al.
Peroxisome proliferator-activated receptor gamma coactivator-1alpha
interacts with the androgen receptor (AR) and promotes prostate cancer
cell growth by activating the AR. Mol Endocrinol 2010;24:114–27.
24. Tiraby C, Hazen BC, Gantner ML, Kralli A. Estrogen-related receptor
gamma promotes mesenchymal-to-epithelial transition and suppresses breast tumor growth. Cancer Res 2011;71:2518–28.
25. Marcotte R, Muller WJ. Signal transduction in transgenic mouse
models of human breast cancer–implications for human breast cancer.
J Mammary Gland Biol Neoplasia 2008;13:323–35.
26. Rolfe DF, Brown GC. Cellular energy utilization and molecular origin of
standard metabolic rate in mammals. Physiol Rev 1997;77:731–58.
27. Jones KL, Buzdar AU. Evolving novel anti-HER2 strategies. Lancet
Oncol 2009;10:1179–87.
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 2012 American Association for Cancer Research.
Published OnlineFirst January 19, 2012; DOI: 10.1158/0008-5472.CAN-11-2967
PGC-1α Promotes the Growth of ErbB2/Neu−Induced Mammary
Tumors by Regulating Nutrient Supply
Eva Klimcakova, Valérie Chénard, Shawn McGuirk, et al.
Cancer Res 2012;72:1538-1546. Published OnlineFirst January 19, 2012.
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