Carcinogenesis vol.34 no.10 pp.2253–2261, 2013
doi:10.1093/carcin/bgt180
Advance Access publication May 29, 2013
ERRα metabolic nuclear receptor controls growth of colon cancer cells
Gérald Bernatchez, Véronique Giroux, Thomas Lassalle,
André C.Carpentier, Nathalie Rivard1 and Julie
C.Carrier*
Département de Médecine and 1Département d’Anatomie et de Biologie
Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de
Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4
*To whom correspondence should be addressed. Tel: +1 819 564 5271;
Fax: +1 819 564 5320;
Email: [email protected]
The estrogen-related receptor alpha (ERRα) is a nuclear receptor that acts primarily as a regulator of metabolic processes,
particularly in tissues subjected to high-energy demand. In
addition to its control of energy metabolism and mitochondrial
biogenesis, ERRα has recently been associated with cancer progression. Notably, increased expression of ERRα has been shown
in several cancerous tissues, including breast, ovary and colon.
However, additional studies are required to gain insight into the
action of ERRα in cancer biology, particularly in non-endocrinerelated cancers. Therefore, using a short hairpin RNA-mediated
approach, we investigated whether ERRα is required for the
rapid growth of colon cancer cells and to maintain their neoplastic metabolic state. Results show that silencing ERRα significantly
impaired colon cancer cell proliferation and colony formation in
vitro as well as their in vivo tumorigenic capacity. A pronounced
delay in G1-to-S cell cycle phase transition was observed in ERRαdepleted cells in association with reduced cyclin-dependent kinase
2 activity and hyperphosphorylated state of the retinoblastoma
protein along with disturbed expression of several cell cycle
regulators, including p15 and p27. Interestingly, ERRα-depleted
HCT116 cells also displayed significant reduction in expression of
a large set of key genes to glycolysis, tricarboxylic acid cycle and
lipid synthesis. Furthermore, using 14C isotope tracer analysis,
ERRα depletion in colon cancer cells resulted in reduced glucose
incorporation and glucose-mediated lipogenesis in these cells.
These findings suggest that ERRα coordinates colon cancer cell
proliferation and tumorigenic capacity with energy metabolism.
Thus, ERRα could represent a promising therapeutic target in
colon cancer.
Introduction
Cell metabolism is regulated by multiple mechanisms in order to fulfill the basic needs of the cell. Adequate generation of energy and synthesis of macromolecules are even more important in order to sustain
the high proliferation rate of malignant cells. Thus, reprogramming of
energy status to the biological capabilities of cancer cells is now recognized as a hallmark of cancer (1). This is often mediated by oncogenic signaling pathways involving PI3K, HIF, p53 and MYC, which
integrate nutrient and energy state signals to control the expression of
genes important for concomitant cancer cell proliferation and energy
metabolism (2). There is a growing recognition that these metabolic
changes could be exploited for targeted anticancer therapy. Therefore,
identifying new regulators of cancer cell metabolism amenable to
drugs are needed to impact future therapeutic interventions.
The estrogen-related receptor alpha (ERRα) is a well-known regulator of energy metabolism and mitochondrial biogenesis. Despite
Abbreviations: ATP, adenosine triphosphate; DMEM, Dulbecco’s modified
Eagle’s medium; ERRα, estrogen-related receptor alpha; PGC-1α/β, proliferator-activated receptor gamma coactivator-1 alpha/beta; Q-PCR, quantitative
PCR; shRNA, short hairpin RNA; TCA, tricarboxylic acid cycle.
its homology with estrogen receptors, ERRα is an orphan nuclear
receptor since it is not activated by estrogen-like molecules, while
being inhibited by synthetic inverse agonists (3). Its activity is controlled by the interaction with master regulators of energy metabolism such as peroxisome proliferator-activated receptor gamma
coactivator-1 alpha/beta (PGC-1α/β) (4,5). Interestingly, ERRα is
abundantly expressed in tissues with high-energy demand such as
the heart, kidney, intestine, skeletal muscle and brown adipose tissue
(6). Furthermore, mice lacking ERRα are unable to adapt to physiological challenges requiring high energy production such as exposure to cold or cardiac pressure overload (7,8). In-depth analyses of
genome-wide ERRα binding sites and gene expression datasets have
revealed that ERRα binds to the promoters and positively regulates
almost all genes involved in the generation of energy and intermediate metabolites (through glycolysis, fatty acid oxidation and glutaminolysis), genes required for functional tricarboxylic acid cycle (TCA)
and oxidative phosphorylation as well as genes involved in fatty acid
synthesis (4,9–11).
In addition to its participation in the regulation of energy metabolic
processes, there is rapidly accumulating evidence for a functional
action of ERRα in carcinogenesis. Indeed, high ERRα expression
has been reported in breast, ovarian and prostate cancers, correlated
with poor prognosis (12–16). Furthermore, in non-endocrine-related
tissues from colorectal cancer patient cohorts, ERRα gene and protein
expression was demonstrated to be significantly upregulated compared with adjacent normal colon, in parallel with either advanced
tumor stage (17) or tumor-promoting osteopontin expression (18). As
reviewed recently, ERRα has emerged as a transcriptional metabolic
regulator that also promotes many processes associated with cancer
development (19,20). For instance, cell-based studies have clearly
established a positive regulation of breast cancer cell proliferation
and migratory potential by ERRα (21–26) and apparent cross talk
between the EGF/HER2 and the PGC-1/ERRα signaling pathways
(23,27,28). ERRα is also involved in angiogenesis and response to
hypoxia and therefore in growth of solid tumors (29–31). Previous
studies have focused on the contribution of ERRα as a transcriptional
regulator of either energy metabolism or breast cancer cell growth
although these two functions of ERRα could very well be interrelated
and thus pertinent to colon cancers.
The aim of this study was to characterize the function of ERRα
in colon cancer cells. We show that shRNA-mediated knockdown of
ERRα markedly reduced colon cancer cell growth and tumorigenicity in nude mice. Cell cycle studies revealed delayed G1–S transition
as well as modification of the expression and activity of certain cell
cycle regulators in ERRα-depleted cells. Furthermore, microarray
analysis and quantitative PCR (Q-PCR) confirmation indicate that
ERRα deficiency affects lipid metabolism gene expression. Finally,
ERRα-knockdown colon cancer cells exhibited lower glucose incorporation and lower lipid synthesis from glucose suggesting that ERRα
is required for adequate cell growth, possibly by controlling metabolic resources critical to sustaining cancer cell growth.
Materials and methods
Cell culture
Transformed human embryonic kidney cells (HEK293T) and human
colon carcinoma cell lines, HCT116, DLD1 and HT29, were grown in
Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA)
supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 mM N-2hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 100 U/ml penicillin and 100
μg/ml streptomycin (Wisent, St-Bruno, Canada). All cells were cultured in a
5% CO2-humidified atmosphere at 37°C. HCT116 cells are hMLH1-deficient
and therefore microsatellite unstable, express wild-type p53, wild-type APC
and carry activating K-Ras, pi3kca and β-catenin mutations. DLD1 cells are
hMLH1-deficient, express mutated p53 and APC and carry activating K-Ras
and pi3kca mutations. HT29 cells are microsatellite stable, express mutated
© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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p53 and APC and carry activating B-Raf and pi3kca mutations (information
from American Type Culture Collection and sequencing).
Lentiviral production and transduction
The lentiviral shRNA expression vector targeting hERRα (NM_004451) and
scrambled control (shCTL) were obtained from Sigma–Aldrich (Oakville,
Canada). Four shRNAs were tested: shERRα#1 (440), shERRα#3 (865),
shERRα#4 (1039) and shERRα#5 (1145). Lentiviruses were produced by
cotransfecting the shRNA expression vector and the ViraPower Lentiviral
Packaging Mix (Invitrogen) in HEK293T cells. HCT116, DLD1 and HT29
cells were infected with viral suspension containing 5 μg/ml polybrene
(Sigma–Aldrich) for 6 h. Two days after infection, cells were selected for puromycin resistance for 48 h (2 μg/ml, Sigma–Aldrich) and used for experiments
within a week.
Cell growth and soft-agarose assays
For growth kinetic assays, cells were seeded in six-well plates at a density of
30 000 cells/well, and cell number kinetics were determined using a cell particle
counter (Coulter Corporation, Miami, FL). Anchorage-independent growth was
evaluated by plating HCT116, DLD1 and HT29 populations at 15 000 cells/
well in complete DMEM containing 0.7% agarose type VII (Sigma–Aldrich)
in six-well plates with medium changes twice weekly. After 16 days, colonies
were stained with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(EMD Biosciences, Mississauga, Canada) (0.5 mg/ml) for 3 h at 37°C. Images
were acquired and the number of colonies determined by ImageJ software (NIH).
In vivo tumorigenesis assay
To assay tumor formation in nude mice, HCT116 cell populations were trypsinized and a total of 2 × 106 cells suspended in 100 μl DMEM were injected
into the dorsal subcutaneous tissue of 5-week-old female CD1 nu/nu mice (five
mice/group) (Charles River, Wilmington, MA). Tumor size was measured by
a caliper twice weekly and the volume calculated using the formula 4πR3/3.
Mice were killed 32 days postinoculation and tumors ≥2.5 mm were excised
and weighed. All experimental protocols were approved by our Institutional
Ethics Committee for Animal Experimentation.
Cell cycle analysis with flow cytometry
HCT116 and HT29 cells were seeded at 150 000 cells/well in six-well plates
and synchronized at G0/G1 phase by serum deprivation for 48 h. Cells were
released into media containing 10% fetal bovine serum with or without nocodazole (0.5 μg/ml, Sigma–Aldrich) to arrest cell cycle in mitosis, and harvested after 0, 8, 12, 16, 20, 24 and 36 h for HCT116, and after 0, 8, 16, 24, 32
and 48 h for HT29. Cells were fixed in 95% ethanol for 24 h at −20oC, washed
in phosphate-buffered saline and resuspended in a propidium iodide (50 μg/
ml)-ribonuclease A (100 μg/ml) solution for 3 h at 4°C. Cell cycle profiles
were determined using a FACS Vantage flow cytometer and the CELL Quest
Software (Becton Dickinson, San Jose, CA). Cell cycle distribution was analyzed using ModFit software (Verity Software House, Topsham, ME).
Protein expression and immunoblotting
Cells were lysed in chilled Triton buffer (50 mM Tris–HCl pH 7.5, 1% Triton
X-100, 100 mM NaCl, 5 mM ethylenediaminetetraacetic acid) supplemented
with protease inhibitors. Proteins were separated by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK) and incubated with
primary antibodies and horseradish peroxidase-coupled secondary antibodies.
Detection was performed using the Western Lightning™ Chemiluminescence
Reagent Plus kit (PerkinElmer, Woodbridge, Canada). Specific antibodies used
were cyclin D1 sc-20044 (1/1000), cyclin E sc-481 (1/1000), E2F4 sc-866
(1/1000), E2F1 sc-22820 (1/1000), p27Kip1 sc-528 (1/500), cyclin A sc-751
(1/1000), p15INK4B sc-612 (1/500), cdc25A sc-97 (1/1000) and c-myc sc-764
(1/750) (all from Santa Cruz Biotechnologies, CA); hERRα NLS5402 (1/2000,
Novus Biologicals, Canada); pRb 554136 (1/750, Pharmingen, Mississauga,
Canada); β-actin MAB1501R (1/5000, Chemicon International, Billerica,
MA); and anti-rabbit IgG horseradish peroxidase and anti-mouse IgG horseradish peroxidase (1/5000, GE Healthcare). Protein expression was quantified
by densitometry using ImageJ software and normalized to that of actin.
Immunoprecipitation/kinase assay
Cells were synchronized by serum deprivation for 48 h before being released
into media containing 10% fetal bovine serum and harvested after 0, 2, 4 or
8 h in Triton lysis buffer. Cell lysates (500 μg) were incubated with 5 μl of
primary antibody (CDK2 sc-163, Santa Cruz Biotechnologies) for 2 h at 4°C
under agitation. Four micrograms of protein A-Sepharose (GE Healthcare) was
subsequently added for 1 h at 4°C under agitation. Immunocomplexes were
washed three times with ice-cold Triton lysis buffer, rinsed twice in kinase
buffer (40 mM Hepes pH 7.4, 20 mM MgCl2, 1 mM dithiothreitol and 10 mM
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p-nitrophenyl phosphate) and incubated for 30 min at 30°C with [γ-32P] adenosine triphosphate (ATP; PerkinElmer) and histone H1 (Sigma–Aldrich) as
substrate. Proteins were solubilized in Laemmli’s buffer, separated on sodium
dodecyl sulfate–polyacrylamide gel electrophoresis and exposed on film.
RNA extraction and real-time Q-PCR
Total RNA was isolated using the Totally RNA kit (Invitrogen) and treated with
deoxyribonuclease I according to the manufacturer’s instructions (QIAGEN,
Mississauga, Canada) before RNA reverse transcription. Q-PCRs were performed
using Brillant® II SYBR Green QPCR master mix in a MX3000P Real-Time
System (Stratagene, Cedar Creek, TX). Expression of each gene was calculated
by the ΔΔCT method and normalized to β2-microglobulin expression.
Microarray and data analysis
Six probes for microarray analysis were generated from RNA isolated
from three independent pools of HCT116 shCTL and HCT116 shERRα#5.
Affymetrix GeneChip® Human Gene 1.0 ST arrays were screened at the McGill
University and Génome Québec Innovation Center. The gene expression data
have been deposited in the National Center for Biotechnology Information’s
Gene Expression Omnibus database (accession number GSE38690, http://
www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38690). To test for statistically significant changes in signal intensity (P-value ≤0.05; significance
analysis of microarrays), compiled data (robust multi-array average analysis)
were screened using FlexArray 1.1 (Genome Quebec). Data were also analyzed through the use of ToppGene (http://toppgene.cchmc.org) to generate
networks of genes associated with biological functions and/or diseases.
Lactate production, glucose incorporation and lipid synthesis from glucose
Cells were seeded in order to obtain similar confluence; lactate levels relative
to total protein in the culture medium were measured 24 h later using a Roche
Modular P automated clinical chemistry analyzer (Roche Diagnostics, Laval,
Canada). For glucose incorporation and lipid synthesis experiments, equally
confluent cells were incubated in glucose-free DMEM for 6 h, then supplied
with 72 μM of d-[14C-]glucose (PerkinElmer) per well. After incubation at
37°C for 24 h, cells were washed three times with phosphate-buffered saline
and disrupted by adding 400 μl of 0.5% Triton X-100. Lipid synthesis from
glucose was assayed by measuring the incorporation of 14C into lipid extracted
with a hexane/isopropanol solution [500 μl, 3:2 (v:v)] (32). Incorporation of
14
C activity in cells and lipids was counted and normalized to cell number at
the end of the experiment.
Data presentation
Each experiment was conducted at least three times with consistent results. All
values in the figures are expressed as mean value ± SD. The data were analyzed
using two-tailed Student’s t-test. Significance is presented as P-value of <0.05
(*), <0.01 (**) and <0.001 (***); non-significant differences are presented as NS.
Results
Silencing of endogenous ERRα inhibits colon cancer cell growth
To better understand the role of ERRα in colon cancers, its expression
was silenced in HCT116, DLD1 and HT29 cell lines using a lentivirus-based shRNA strategy. Figure 1A shows that specific shRNAs
knocked down ERRα protein expression by 60–80%, as quantified
by densitometry. Moreover, cellular growth was strongly decreased
(by approximately 80% at the end of the experiment) in shERRαexpressing HCT116, DLD1 and HT29 cell populations, as compared
with parental cells or cells infected with a control shRNA (Figure 1B–
D). Furthermore, non-cancerous IEC-6 cells stably infected with
shERRα#1 (which silences both rat and human ERRα), displayed
reduced growth compared with shCTL-infected cells (Supplementary
Figure 1A, available at Carcinogenesis Online). These results support
an essential contribution of ERRα expression in proliferation and/
or survival of both cancerous and non-cancerous intestinal epithelial
cells. Conversely, overexpressing ERRα, with or without its surrogate
coactivator PGC-1α, did not increase IEC-6 or HCT116 cell growth
(Supplementary Figure 2, available at Carcinogenesis Online).
ERRα knockdown in colon cancer cells causes a defect in colony
formation and in vivo tumorigenesis
We next analyzed the impact of ERRα silencing on anchorageindependent growth of colon cancer cells. Reduction of ERRα
expression markedly reduced the ability of HCT116, HT29 and DLD1
ERRα and colon cancer
Fig. 1. ERRα depletion decreases growth of colon cancer cell lines. HCT116, DLD1 and HT29 cells were uninfected or infected with lentiviruses encoding
either for a non-targeting shRNA (shCTL) or four ERRα-specific shRNAs (shERRα#1, #3, #4 and #5). (A) HCT116 cells were lysed 48 h postinfection, and
ERRα expression was assayed by western blotting and normalized to actin expression. Populations of (B) HCT116, (C) DLD1 and (D) HT29 cells were seeded
at 30 000 cells/well into six-well plates, harvested at the indicated times and the number of cells counted. The experiments were performed in triplicate and
repeated twice.
cells to form colonies in soft agarose (Figure 2A). To evaluate the
impact of ERRα silencing on the in vivo tumorigenicity of HCT116
cells, HCT116 cells infected with shERRα#4, shERRα#5, shCTL
lentiviruses or uninfected cells were subcutaneously implanted into
nude mice. As shown in Figure 2B and Supplementary Figure 3,
available at Carcinogenesis Online, the growth rate, volume and
final weight of the tumor xenografts formed by shERRα cells
were significantly lower than those formed by control cells. In
fact, downregulation of ERRα expression reduced the tumorigenic
capacity of HCT116 cells since cells infected with shERRα#4 or
shERRα#5 formed tumors in only two of five injected mice while
every control cell population resulted in tumors (Figure 2C). ERRα
silencing also strongly reduced in vivo xenograft tumor development
in DLD1 cells (data not shown). Therefore, both in vitro and in vivo
assays suggest that ERRα expression in colon cancer cells is required
to sustain colony and tumor formation.
ERRα silencing delays G1-to-S phase transition in colon
cancer cells
To determine whether decreased growth of ERRα-depleted cells
is caused by a primary defect in cell cycle, cell cycle progression of
synchronous HCT116 and HT29 cell populations infected with shCTL or
shERRα#5 was first analyzed by flow cytometric assays (fluorescenceactivated cell sorting). As seen in Figure 3A (left panel, −Nocodazole),
ERRα-depleted HCT116 cells displayed a marked delay in G1-to-S
phase transition at 12 and 16 h postrelease from serum deprivation, as
compared with control HCT116 cells. Next, to more accurately analyze
the transition from G0/G1 to S phase and from S to G2/M phase without
the overlapping re-entry into the G0/G1 phase, cells were blocked into
G2/M phase by the addition of nocodazole to serum-containing medium.
As illustrated in Figure 3A (right panel, +Nocodazole), silencing of
ERRα in HCT116 cells resulted in a 4- to 8- h delay in transit from the
G0/G1 to S phase relative to control HCT116 cells such that roughly
one-fifth of ERRα knockdown cells were still in G0/G1 phase at 36 h
postserum release. These data suggest that ERRα-depleted cells have a
delayed transition from G0/G1 to S or alternatively, that a pool of this cell
population is unable to exit G0/G1 phase. Interestingly, the G0/G1 to S
progression was also delayed in ERRα-silenced HT29 cells, suggesting
that ERRα contributes to the transition between these cell cycle phases
regardless of p53 and microsatellite status (Supplementary Figure 4,
available at Carcinogenesis Online). Of note, determination of subG0
population in fluorescence-activated cell sorting profiles and caspase 3
activity (Supplementary Figure 5, available at Carcinogenesis Online)
did not provide evidence for increased apoptosis of colon cancer cells
upon shRNA-mediated ERRα repression. Overall, these results suggest
that silencing ERRα inhibits the growth of colon cancer cells through a
G1/S checkpoint arrest.
ERRα silencing affects cell cycle protein expression and activity
To gain better insight into how ERRα regulates G1 progression and G1/S
phase transition, the expression and activity of several proteins that control this portion of the cell cycle were analyzed. We first determined
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Fig. 2. ERRα knockdown reduces clonogenicity and in vivo tumorigenic properties of colon cancer cells. (A) HCT116, DLD1 and HT29 cells were infected
with lentiviruses encoding either for a non-targeting shRNA (shCTL) or shERRα#5. Forty-eight hours postinfection, populations were seeded at 15 000 cells/
well in soft agarose. After 16 days of culture, cells were stained with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and colonies were counted
using ImageJ software. The bar graph shows the mean number of colonies ± SD in three experiments, each performed in triplicate (***P < 0.001). (B) HCT116
cells were uninfected or infected with lentiviruses encoding for shCTL, shERRα#4 or shERRα#5. After 48 h, inoculation of 2 × 106 cell populations was
achieved in the subcutaneous space of five nude mice and tumors were allowed to grow until 32 days postinjection. Tumor volume was measured with a caliper
at the indicated times and expressed as mean ± SD (**P < 0.01 and ***P < 0.001 represent statistical significance between shERRα and shCTL 32 days after
inoculation). (C) Representative nude mice.
the kinase activity of CDK2, a cyclin-dependent kinase required for
G1/S phase transition. As illustrated in Figure 3B, both basal and seruminduced CDK2 activities were clearly lower in HCT116 cells infected
with shERRα#5 than with shCTL. Furthermore, the knockdown of
ERRα in HCT116 cells strongly reduced the hyperphosphorylated state
of retinoblastoma protein and the expression of several proteins that
control G1/S phase progression, such as cyclin D1, E2F4, E2F1 and
Cdc25A (Figure 3C). In addition, ERRα depletion in HCT116 cells
modestly reduced cyclin A (~22%) and c-myc (~30%) expression while
having no effect on cyclin E levels. Of note, most of these changes
were also observed in ERRα-depleted DLD1 and HT29 cells. Finally,
expression of cell cycle inhibitors, p27Kip1 and p15INK4B, was consistently increased in all three ERRα-depleted cell lines. Taken together,
these results indicate that ERRα deficiency affects the expression
profile of cell cycle regulatory proteins, and consequently inhibits the
growth of colon cancer cells by arresting the cell cycle at G0/G1 phase.
Transcriptome analyses of HCT116 colon cancer cells upon ERRα
depletion
To delineate the genome-wide transcriptional effect of silencing
ERRα in HCT116 colon cancer cells, the expression of 28 869 genes
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was profiled in three populations of control and ERRα-depleted
HCT116 cells, 48 h after lentiviral-mediated shRNA expression. Of
these, ERRα depletion differentially affected the expression of 1484
genes (P-value <0.01 and fold change >2), including 527 upregulated
and 957 downregulated transcripts. Gene ontology analysis of differentially expressed genes revealed many significant clusters associated with cellular bioenergetic metabolic processes, including lipid
transport, lipid metabolic processes and small molecule catabolic processes. Among the genes associated with these gene ontology terms,
Table I lists selected genes that were significantly up- and downregulated in our microarray analysis of ERRα-silenced HCT116 cells.
ERRα depletion is associated with changes in gene expression
involved in glycolysis and lipogenesis
Cancer cells exhibit an altered metabolism characterized by the
generation of ATP by glycolysis and of fatty acids by de novo synthesis
(33). The majority of genes involved in these metabolic pathways have
been reported to be targets of ERRα (4,9–11,34). Therefore, the effect
of ERRα silencing in HCT116 cells on the expression of a subset of
these genes which support the bioenergetic needs of growing cells
was further analyzed by Q-PCR. As seen in Figure 4A, knockdown of
ERRα and colon cancer
Fig. 3. ERRα depletion affects G1/S transition and cell cycle regulatory proteins in colon cancer cells. (A) HCT116 cells were infected with lentiviruses
encoding for a non-targeting shRNA (shCTL) or shERRα#5. Forty-eight hours postinfection, cells were synchronized by serum starvation for 48 h and harvested
at the indicated times after release into serum-containing media, in the presence or absence of nocodazole. Propidium iodide-stained cell populations were
analyzed for DNA content using flow cytometry. Graphs represent cell counts in y-axis and DNA content in x-axis. Percentages of cells in phases G0/G1, S and
G2/M are indicated. Experiments were repeated twice with identical results. (B) HCT116 cell populations were synchronized by serum starvation and assayed
for CDK2 kinase activity on histone H1 at 0, 2, 4 and 8 h after release in media containing serum. Phosphorylated histone H1 was visualized by autoradiography
after sodium dodecyl sulfate–polyacrylamide gel electrophoresis (upper panel). Equivalent amount of immunoprecipitated CDK2 protein was quantified by
western blot with specific antibody against CDK2 (lower panel). (C) HCT116, DLD1 and HT29 cells infected with shCTL of shERRα#5 were harvested 48 h
postinfection and cell lysates were submitted to western blot analysis with specific antibodies. In all instances, the membranes were stripped and reprobed with
β-actin antibody to confirm equal protein loading, of which a representative blot is shown.
Table I. Selected regulated genes from microarray analysis of ERRα-silenced HCT116 cells Up
AMACR
ACSL6
AGPAT9
ATP10A
BCKDHB
CFTR
Down
CYP19A1
CYP39A1
GCAT
GPAM
LDLR
OLAH
OSBP2
OSBPL7
PIGN
PLSCR1
SARDH
TNFSF11
ATP8A1
ATP8B1
ATP8B3
ATP9A
ABAT
ABCA1
ABCA2
ABCA4
ABCA12
ABCC2
ABCC3
ABCG1
ABCG4
ACAA2
ACAD11
ACLY
ACOT12
ACOX1
ACOXL
ACSL1
ADH7
ALDH1A3
ALDH1L2
ALDH5A1
ALDH7A1
CPT1A
CRAT
CYB5R1
CYP17A1
CYP46A1
CYP51A1
CYP27B1
CYP2C9
FA2H
GAD1
GLTPD2
GOT1
GPT2
IDH1
PDHA1
PDK3
PDK4
SLC27A2
SLC27A6
SLC44A1
SLC44A3
SLC44A6
SLCO3A1
SREBF1
SREBF2
Genes selected for Q-PCR analysis are given in italics.
ERRα in HCT116 cells was associated with lower expression of genes
critical for the glycolytic process, such as HK1, G6PC, PFKFB1,
PFKFB2, ALDOC, GPT2 and PGM2, likely reducing pyruvate
production. However, the fate of pyruvate produced from glycolysis
in ERRα-depleted HCT116 cells remained unclear based on the gene
expression profile. Indeed, pyruvate is converted to acetyl-CoA by
pyruvate dehydrogenase, for which gene expression is unaffected by
ERRα depletion. However, pyruvate dehydrogenase is inhibited by a
family of pyruvate dehydrogenase kinases 1–4 (33), encoded by genes
significantly downregulated by ERRα depletion. This could result in
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Fig. 4. ERRα depletion decreases energy metabolism gene expression. (A) HCT116 cells were infected with lentiviruses encoding for a non-targeting shRNA
(shCTL) or shERRα#5. After 48 h, indicated gene expression was measured by Q-PCR and normalized to β2-microglobulin expression. The relative expression
of each gene was presented as log 2 transformed mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). (B) Schematic illustration
of metabolic pathways affected by ERRα in which boxes highlight specific genes found to be significantly downregulated by ERRα knockdown in HCT116 cells
following Q-PCR.
preferential conversion of pyruvate into acetyl-CoA instead of lactate
in cells knocked down for ERRα. In cancer cells, glycolysis is linked
to de novo lipogenesis by the availability of cytosolic acetyl-CoA,
which is produced from citrate by ATP citrate lyase (33). Herein,
gene expression of this target enzyme for cancer therapy was strongly
reduced in ERRα-depleted HCT116 cells. Furthermore, the expression
of other key genes of fatty acid and sterol metabolism, including
ACCA and ACCB (acetyl-CoA carboxylase α and β), FASN (fatty acid
synthase), SCD1 (stearoyl-coA desaturase-1) and ACSL1 (acetyl-CoA
synthase long-chain), was significantly reduced in ERRα-depleted
cells. Of note, ERRα depletion also reduced the expression of genes
involved in the regulation of glutaminolysis and in the generation of
intermediate metabolites from amino acids and glutamate, namely,
glutamic pyruvate transaminase (GPT2), aldehyde dehydrogenase
4, member A1 (Aldh4a1) and glutamate oxaloacetate transaminase
(Got1). Silencing ERRα in non-cancerous IEC-6 cells also significantly
reduced the expression of key metabolic genes (Supplementary
Figure 1B, available at Carcinogenesis Online). Altogether and
as schematized in Figure 4B, cellular depletion of ERRα alters the
expression of genes involved in bioenergetic pathways, such as energy
production from glucose, generation of intermediate metabolites and
promotion of de novo lipogenesis.
ERRα depletion is associated with reduced glucose uptake and
lipid synthesis from glucose
To better characterize the consequence of ERRα knockdown on
energy metabolism in colon cancer cells, we first investigated glucose
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incorporation in HCT116, DLD1 and HT29 cells. In keeping with
reduced expression of key genes associated with glycolysis, glucose
incorporation was significantly reduced in ERRα-depleted versus
control cells (Figure 5A). Of note, lactate production was modestly
reduced only in ERRα-silenced HT29 cells (Figure 5B). Given that
aerobic lactate production is preserved in some ERRα-depleted cells
in which glucose incorporation, and possibly pyruvate production, is
reduced, this lack of substrate could impact anabolic processes such as
lipid synthesis. Accordingly, ERRα-depleted cells displayed reduced
lipid synthesis from glucose (Figure 5C). Collectively, these results
suggest that ERRα is required for adequate glucose incorporation and
lipid synthesis from glucose in colon cancer cells.
Discussion
ERRα is a regulator of metabolic function that controls multiple biosynthetic pathways involved in energy metabolism. Herein, we provide new insights into the protumorigenic action of ERRα. Using
shRNA to silence ERRα expression, we demonstrate that this constitutively active nuclear receptor is required for colon cancer cell
growth in vitro and in vivo and for the expression of cell cycle regulatory proteins controlling G1/S phase transition. Our data also show
that silencing ERRα in colon cancer cells affects the expression of
genes involved in bioenergetic functions and reduces glucose incorporation and lipogenesis from glucose. These results are consistent
with a growth-promoting action of ERRα in colon cancer and suggest that ERRα contributes to the coordination of energy-generating
ERRα and colon cancer
Fig. 5. Glucose incorporation and de novo lipid synthesis from glucose are
impaired upon ERRα knockdown. HCT116, DLD1 and HT29 cells were
infected with lentivirus encoding for a non-targeting shRNA (shCTL) or
shERRα#5. Forty-eight hours postinfection, cells were used for metabolic
assays. (A) d-[14C]-glucose incorporation in 24 h was measured as described
in the experimental procedure and normalized to cell number. (B) Lactate
production was quantified in supernatants from 48 h-incubated cells and
normalized to protein levels. (C) d-[14C]-glucose-derived lipid synthesis
was assessed as described in the experimental procedure and normalized
to cell number. (A–C) Graphs represent mean ± SD of three independent
experiments (*P < 0.05, **P < 0.01, ***P < 0.001).
and biosynthetic pathways to effectively promote colon cancer cell
proliferation.
There is increasing evidence supporting a role for ERRα as a protumorigenic factor. In hormone-responsive cancers such as breast
cancer, high ERRα expression has been found to correlate with poor
clinical outcome and/or unfavorable biomarkers such as ERα negativity and elevated ErbB2 expression (11,12,15). As direct evidence
of its protumorigenic action, ERRα deficiency significantly delays
tumor formation in a mouse model of ErbB2-induced mammary
tumorigenesis (34). Furthermore, in non-endocrine-related colorectal
cancers, ERRα gene expression also parallels advanced tumor stage,
suggesting that ERRα participates in cancer progression either by
tissue-specific mechanisms or by promoting key processes required
for tumor growth. Colorectal cancer evolves through the addition of
mutation in tumor suppressors, including APC and p53, and oncogenic activation of intracellular signaling pathways downstream of
growth factor receptors (35). In this study, in spite of their heterogeneous nature, knocking down ERRα expression in HCT116, HT29 or
DLD1 colon cancer cells resulted in profound anchorage-dependent
and -independent growth retardation, suggesting that ERRα is necessary to sustain their high level of proliferation. Furthermore, very few
small tumors developed when ERRα-silenced HCT116 cells were
implanted into nude mice. Since ERRα is known to induce vascular endothelial growth factor messenger RNA expression in breast
cancer cell lines (31), ERRα depletion may have further effects on
tumor development by reducing angiogenesis. Overall, our observations are in keeping with the growth inhibitory impact of reducing
ERRα expression or activity in breast, liver and prostate cancer cells
(21,23,26,36). Of note, silencing ERRα reduced the growth of noncancerous intestinal IEC-6 cells, suggesting that ERRα is also required
for normal cell growth. This observation is of particular interest since
ERRα knockout mice are viable, in spite of intestinal lipid malabsorption, possibly through compensatory mechanisms (37). In turn, we
found that increasing ERRα expression, together with its surrogate
coactivator PGC-1α, was not sufficient to impact either HCT116 or
IEC-6 cell growth. This finding is in agreement with the observations
of Fisher et al. (38), whereby the expression of constitutively active
VP16-ERRα stimulated metabolism but not growth in KSR1−/− MEFs
expressing H-rasV12.
As commonly observed for factors for which the activity is coupled
to the availability of bioenergetic substrates, ERRα is required for
colon cancer cell cycle progression and its depletion markedly delays
G1/S phase transition following serum stimulation. In contrast,
silencing ERRα did not result in significant cell death in colon cancer
cells, in concordance with breast cancer cells transfected with miR137 in which ERRα expression is downregulated (26), but in contrast
to HepG2 liver carcinoma treated with an ERRα inverse agonist
(36). G1/S phase transition involves a variety of regulatory factors
including cyclins (D and E), cyclin-dependent kinases (CDK4/6 and
CDK2) and CDK inhibitors (p15INK4B, p21Cip1 and p27Kip1 inhibitors)
(39). In colorectal cancer cells, this transition is frequently activated
by aberrant cell-proliferative signaling pathways or inactivation
of cell cycle-regulating proteins (40). Herein, CDK2 activity was
found to be weaker in serum-starved and synchronized HCT116
ERRα knockdown cells than in control cells, resulting in reduced
phosphorylated retinoblastoma protein and a likely release of E2F
factors from phosphorylated retinoblastoma protein during the
progression from the G1 to S phase. In addition, silencing ERRα also
decreased E2F4 and E2F1 expression, which could contribute to the
observed reduction in proliferation, as these transcription factors are
required for S-phase entry into the colon cancer cell cycle (41). The
reduction in CDK2 activity in ERRα knockdown cells could also be
due to lower levels of Cdc25A phosphatase, which removes inhibitory
phosphorylations on CDK2 (42). Another mechanism whereby
silencing ERRα expression could lead to CDK2 inhibition and G0/G1
arrest would be via the strong induction of p27Kip1 and p15INK4B
expression. These CDK inhibitors are frequently downregulated in
various tumors, and anticancer agents have been reported to restore
p27Kip1 and/or p15INK4B expression in colorectal cancer cells (43,44).
Furthermore, the induction, stabilization or nuclear localization of
p27 has been shown to be key in inhibiting cyclin E-CDK2 activity
(43,45,46). Of note, knocking down the expression of ERRα
markedly reduced the levels of phosphorylated retinoblastoma
protein in all three cell lines studied, regardless of their microsatellite,
p53, APC, K-Ras, pi3kca and β-catenin mutation status. This effect
may be cell type specific since some reports have shown that, in
breast cancer cells, antagonizing ERRα activity upregulates p21
expression in a p53-dependent manner (21), and reducing ERRα
expression decreases cyclin E levels in a ERα-dependent manner
(26). Furthermore, ERRα could also be a downstream target of
various oncogenic factors in certain cell types, since it has been
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G.Bernatchez et al.
shown that (i) ERRα is required to mediate the increased metabolism
and anchorage-independent growth of oncogenic Ras in fibroblasts
(38); (ii) extracellular signal-regulated kinase 8 interacts with and
represses ERRα, possibly by controlling its cellular localization
(47); (iii) ERRα is responsive to EGF and ErbB2 signaling pathways
(27,28); and finally (iv) PGC-1β expression is controlled by ErbB2,
IGF-1 and PI3K signaling pathways in breast cancer cells (23). This
suggests a finely tuned regulation of ERRα activity in response to
the availability of energetic substrates. In turn, ERRα interacts with
MYC, HIFα and β-catenin in breast cancer cells, contributing to their
oncogenic signals and metabolic reprogramming (48).
The coordination of cell cycle and metabolic circuits is essential
to sustain proliferation in cancerous cells. In particular, continuous
cell growth requires rapidly available supplies of ATP, simultaneously
with the conversion of biosynthetic precursors into building blocks for
biomass production (1,2,33). Furthermore, several transcription factors and oncogenes have been involved in coordinating the induction
of a gene expression program that allocates nutrients to provide free
energy while fueling the anabolic processes (2,49). These observations have raised growing interest in exploiting the metabolic dependencies of cancer cells to target key metabolic enzymes as a selective
anticancer strategy (50). Using gene expression profiling, we found
that ERRα deficiency in colon cancer cells significantly affected the
expression of gene clusters implicated in a very large set of biological functions, ranging from cellular development and adhesion to
lipid metabolism and small molecule catabolic processes. More specifically, our ensuing Q-PRC analysis revealed that ERRα depletion
significantly reduced the expression of key genes coding for proteins
controlling glycolysis, TCA cycle, oxidative phosphorylation and
fatty acid synthesis. The expression of the majority of these selected
genes are concordant with a larger list of published ERRα target
genes in human breast cancer cells, and in metabolically active mouse
tissues, showing ERRα occupancy and/or activation of the promoter
of genes encoding enzymes involved in virtually every step of energy
and biomass generating pathways (3,4,9–11).
Reminiscent of that observed in this study in cancer cells, functional studies have elegantly shown that the Drosophila ERR ortholog
is required to coordinate developmental growth and cellular proliferation with metabolism (51). Indeed, dERR mutants display decreased
expression of genes involved in carbohydrate metabolism and die
during larval growth with high sugar but low ATP and triglyceride
levels, possibly due to defective conversion of dietary nutrient into
biomass. In our three colon cancer cell lines tested, ERRα silencing markedly reduced glucose incorporation and lipid synthesis from
glucose, suggesting parallel defects in glycolysis and lipogenesis
along with loss of neoplastic proliferative advantage. Interestingly,
lactate production in the presence of sufficient oxygen, another hallmark of cancer, was not consistently affected by ERRα depletion in
colon cancer cells. Furthermore, the expression of lactate dehydrogenase, an ERRα target (52), was unaffected by ERRα silencing.
This may be related to the culture conditions used, in which oxygen,
glucose and glutamine levels are not limiting. In these proliferating
cells, glycolytic ATP production is high and the main role of the TCA
cycle is unlikely to maximize ATP production from pyruvate. Rather,
the TCA cycle serves as an important source of biosynthetic precursors, including citrate used for lipid biosynthesis and oxaloacetate
and α-ketoglutarate used to generate amino acids (33). Based on published ERRα regulon and the present Q-PCR analysis of gene expression in colon cancer cells, impaired glucose-mediated lipogenesis in
ERRα-depleted cells is likely due to a defect in the gene network
regulating energy metabolism and the supply of biosynthetic precursor for lipid synthesis.
The observed requirement of ERRα for glucose-mediated lipogenesis in a context of cancer is also in agreement with metabolic studies showing that ERRα null mice display reduced anabolic pathways
such as lipogenesis in adipose tissues, associated with resistance to
high-fat diet-induced obesity and altered expression of genes driving lipid, eicosanoid and steroid synthesis (53). As an orphan nuclear
2260
receptor, the ability of ERRα to regulate energy metabolism has been
tied to the expression and activity of members of the PGC-1 family
of coactivators (3). In primary adipocyte cultures, ERRα-induced adipogenesis has been shown to be triggered by modulating the expression of PGC-1β (54). At the present time, the effect of PGC-1α in
colorectal cancer remains controversial since a recent study showed
that PGC-1α promotes apoptosis in colorectal cancer cells (55) while
another demonstrated that loss of PGC-1α protects mice from azoxymethane-induced carcinogenesis by inducing gene expression programs supporting lipogenesis (56).
Finally, the vast majority of studies showing that ERRα promotes
cancer cell growth have been performed in breast cancer-derived
cells. Findings from the present study using a powerful loss-offunction strategy suggest that ERRα also promotes the proliferation of colon cancer cells through a gene expression network that
facilitates the incorporation of nutrients into cell building blocks,
such as glucose-mediated lipogenesis. Such findings provide valuable insight into the biological actions of ERRα and could establish
a molecular basis for the development of new cancer therapeutic
agents.
Supplementary material
Supplementary Figures 1–5 can be found at http://carcin.
oxfordjournals.org/
Funding
Canadian Institutes of Health Research (MOP-89970); Cancer
Research Society.
Acknowledgements
We thank Pierre Pothier for reviewing the manuscript. A.C.C., N.R. and J.C.C.
are members of the Fonds de la Recherche en Santé du Québec (FRSQ)funded Centre de Recherche Clinique Étienne-Le Bel. V.G. was the recipient of a CIHR Frederick Banting and Charles Best fellowship. A.C.C. is the
recipient of the CIHR-GlaxoSmithKline Chair in Diabetes. N.R. is a recipient
of a Canadian Research Chair in Colorectal and Inflammatory Cell Signalling.
J.C.C. was an FRSQ research scholar.
Conflict of Interest Statement: None declared.
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Received October 1, 2012; revised May 5, 2013; accepted May 23, 2013
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