LETTERS
NF-κB controls energy homeostasis and metabolic
adaptation by upregulating mitochondrial respiration
Claudio Mauro1,7 , Shi Chi Leow1,2,7 , Elena Anso3 , Sonia Rocha4 , Anil K. Thotakura1 , Laura Tornatore1 ,
Marta Moretti1,5 , Enrico De Smaele5 , Amer A. Beg6 , Vinay Tergaonkar2 , Navdeep S. Chandel3
and Guido Franzoso1,8
Cell proliferation is a metabolically demanding process1,2 . It
requires active reprogramming of cellular bioenergetic pathways
towards glucose metabolism to support anabolic growth1,2 .
NF-κB/Rel transcription factors coordinate many of the
signals that drive proliferation during immunity, inflammation
and oncogenesis3 , but whether NF-κB regulates the metabolic
reprogramming required for cell division during these processes
is unknown. Here, we report that NF-κB organizes energy
metabolism networks by controlling the balance between the
utilization of glycolysis and mitochondrial respiration. NF-κB
inhibition causes cellular reprogramming to aerobic glycolysis
under basal conditions and induces necrosis on glucose
starvation. The metabolic reorganization that results from NF-κB
inhibition overcomes the requirement for tumour suppressor
mutation in oncogenic transformation and impairs metabolic
adaptation in cancer in vivo. This NF-κB-dependent metabolic
pathway involves stimulation of oxidative phosphorylation
through upregulation of mitochondrial synthesis of cytochrome c
oxidase 2 (SCO2; ref. 4). Our findings identify NF-κB as a physiological regulator of mitochondrial respiration and establish a role
for NF-κB in metabolic adaptation in normal cells and cancer.
Transcription factors of the NF-κB/Rel family are central regulators
of immune and inflammatory responses3 . They also promote
oncogenesis3 . The best documented function of NF-κB (nuclear
factor-κ–light-chain-enhancer of activated B cells) in these processes is
the upregulation of a transcriptional program encoding inflammatory
mediators, immunoregulators and inhibitors of apoptosis, as
well as growth factors and factors stimulating cell migration
and differentiation3 .
Both immunity and tumorigenesis also involve a rapid rate of
cell division. This presents substantial bioenergetic and biosynthetic
challenges, which the cell meets by increasing glucose metabolism.
This reliance on glucose under aerobic conditions (a phenomenon
known in cancer as the Warburg effect2 ) enables dividing cells to fuel
glycolysis and the pentose phosphate pathway to generate NADPH,
macromolecules and ATP required for the doubling of biomass1,2 .
Glucose is therefore an essential nutrient for both cancer and normal
proliferating cells1 . When glucose is scarce, however, energy sensing
by AMP-activated protein kinase (AMPK) arrests anabolic pathways,
and metabolism is redirected to fatty acid oxidation and oxidative
phosphorylation (OXPHOS), thus maximizing energy efficiency with
the available resources1,5,6 . Nevertheless, mitochondrial defects, partly
due to altered expression and/or function of mitochondrial factors (for
example, cytochrome c oxidase (COX) complex proteins)7,8 , as well as
oncogenic mutations can result in glucose addiction1,2,5,8,9 . Whether the
metabolic reprogramming required for cell proliferation is controlled
by NF-κB, however, is unknown.
Strikingly, knock down of RelA, the dominant NF-κB transactivating
subunit3 , markedly enhanced glucose consumption and lactate
production in mouse embryonic fibroblasts (MEFs), under normal
culture conditions (Fig. 1a,b), thus recapitulating the Warburg
effect. Similar results were obtained using early passage knockout
MEFs (Supplementary Fig. S1a,b). RelA-deficient cells also exhibited
increased ATP levels and decreased oxygen consumption levels
(Fig. 1c,d and Supplementary Fig. S1c). Hence, basal NF-κB activity
restrains reprogramming to aerobic glycolysis.
To determine whether this glycolytic switch represented a
homeostatic response to increased proliferation, we examined the
ability of RelA-deficient cells to turn to OXPHOS for energy
provision on glucose starvation. Remarkably, RelA inhibition impaired
adaptation to glucose starvation, causing cell death (Fig. 1e and
Supplementary Fig. S1d). In contrast, the viability of wild-type cells
was virtually unaffected. Similar results were obtained with inhibitors
1
Section of Inflammation and Signal Transduction, Department of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12
0NN, UK. 2 Laboratory of NF-κB Signaling, Proteos, Singapore 138673, Singapore. 3 Department of Medicine, Division of Pulmonary and Critical Care Medicine,
Northwestern University Medical School, Chicago, Illinois 60611, USA. 4 Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences,
University of Dundee, Dundee DD1 5EH, UK. 5 Department of Experimental Medicine, Sapienza University, Rome 00161, Italy. 6 Department of Immunology, Moffitt
Cancer Center, Tampa, Florida 33612, USA. 7 These authors contributed equally to this work.
8
Correspondence should be addressed to G.F. (e-mail: [email protected])
Received 10 June 2011; accepted 22 July 2011; published online 28 August 2011; DOI: 10.1038/ncb2324
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NATURE CELL BIOLOGY VOLUME 13 | NUMBER 10 | OCTOBER 2011
© 2011 Macmillan Publishers Limited. All rights reserved.
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β-actin
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GS
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Percentage of survival
ns shRNA
RelA shRNA
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80
60
40
20
0
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2
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120
100
80
60
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20
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45
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35
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20
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GS
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60
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RelA shRNA
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Percentage of survival
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consumption rate (pmol
per min per 2.5 × 104 cells)
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(mmol per 2.5 × 104 cells)
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per min per 2.5 × 104 cells)
15
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(mmol per 2.5 × 104 cells)
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(mmol per 2.5 × 10 4 cells)
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LETTERS
Figure 1 NF-κB counters reprogramming to aerobic glycolysis and promotes
metabolic adaptation to nutrient starvation. (a–d) Glucose consumption (a,
left), lactate production (b), ATP concentration (c) and oxygen consumption
(d) in immortalized MEFs expressing non-specific (ns) or RelA-specific
shRNAs, under normal culture conditions. (a), Right, western blots with the
cells in a–d, showing levels of RelA (knockdown efficiency), c-Rel and β-actin
(knockdown specificity). (e) Left, viability of immortalized MEFs expressing
non-specific or RelA shRNAs after glucose starvation (GS). Middle, images of
representative cells. Right, western blots with non-specific and RelA shRNA
cells. Similar results were obtained using two additional non-specific shRNAs
(ns2, shc003v), two additional RelA-specific shRNAs and eGFP -specific,
luciferase-specific, laminA/C-specific and cyclophilinB-specific shRNAs.
(f–h) Lactate production (f), oxygen consumption (g) and ATP concentration
(h) in cells treated as in e. Lactate values in f after day 4 should be
interpreted with caution, owing to massive necrosis in RelA-shRNA cells.
(i,j) Survival of the cells in e after 4-day glucose starvation, either alone (GS)
or together with serum deprivation (GS + SD; i) or rapamycin (GS + Rapa; j,
left). (j) Right, representative images. In a–j the values denote mean±s.e.m.
n = 3 (a–c,e,f,h–j); n = 4 (d); n = 10 (g). In h, ∗ P < 0.05; ∗∗ P < 0.01. Scale
bars: 50 µm.
of the NF-κB-inducing kinase, IκB kinase (IKK)β (Supplementary
Fig. S1f,g). RelA-deficient cells remained dependent on glycolysis for
energy production, even during glucose starvation, shown by their
high lactate production and decreased oxygen consumption levels
(Fig. 1f,g and also Supplementary Fig. S1h). This resulted in a marked
decrease of ATP levels in glucose-starved RelA-deficient cells, but
not in starved wild-type cells (Fig. 1h and Supplementary Fig. S1e).
RelA deficiency did not impair proliferation arrest following glucose
starvation (Supplementary Fig. S2a,b), thereby excluding defects in
glucose-starvation-induced cell-cycle checkpoints1 as the cause of death
in mutant cells. Hence, NF-κB inhibition hinders the ability of cells to
meet the metabolic demand during glucose starvation by impairing
OXPHOS, thus causing a metabolic crisis and cell death. Accordingly,
decreasing the metabolic demand2,5 through serum deprivation or
blockade of protein biosynthesis with the mTOR inhibitor rapamycin1,6
rescued RelA-deficient cells from glucose-starvation-induced cell death
(Fig. 1i,j and Supplementary Fig. S2c).
Survival following nutrient deprivation depends on macroautophagy,
an energy-producing auto-digestive process5,10 . Indeed, silencing
Beclin1 or ATG7, two essential macroautophagy effectors10 , or
NATURE CELL BIOLOGY VOLUME 13 | NUMBER 10 | OCTOBER 2011
© 2011 Macmillan Publishers Limited. All rights reserved.
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LETTERS
treatment with the macroautophagy inhibitors, 3-methyladenine
and chloroquine10 , induced toxicity in glucose-starved wild-type
MEFs to an extent similar to RelA inactivation (Supplementary
Fig. S3a–c). However, neither the induction of the macroautophagy
effector, LC3 (ref. 10), nor the formation of macroautophagic
vesicles10 , was impaired by RelA inhibition during glucose starvation
(Supplementary Fig. S3d,e). Instead, this inhibition basally induced
autophagic flux, shown by the enhanced rates of autophagosome
formation and degradation exhibited by untreated RelA small hairpin RNA (shRNA) cells relative to control cells (Supplementary
Fig. S3d–f, 0 h; see the increased levels of LC3 and eGFP–LC3
punctate staining, and decreased levels of the autophagic cargo
protein p62 in RelA-shRNA cells not exposed to bafilomycin A1,
inhibiting lysosomal vacuolar ATPase). Hence, defective macroautophagy is not responsible for the demise of glucose-starved RelAdeficient cells. Similarly, RelA-deficient and control cells exhibited
no obvious differences in glucose-starvation-induced caspase activation (Supplementary Fig. S4a), and blocking apoptosis using
caspase-8-specific shRNAs or the pan-caspase inhibitor z-VADfmk
(ref. 10) failed to rescue the RelA-deficient cells from programmed
cell death (PCD) after metabolic challenge (Supplementary Fig.
S4b–d). Notably, RelA inactivation retained toxicity in glucosestarved Bax−/− /Bak−/− MEFs, which are refractory to apoptosis10
(Supplementary Fig. S4e,f). Starved RelA knocked-down cells also
showed morphological signs of necrosis, lacked nucleosomal fragmentation, a hallmark of apoptosis, and their culture media contained elevated levels of the necrosis marker HMGB1 (ref. 10;
Supplementary Fig. S4g; data not shown). Thus, NF-κB-afforded
protection during glucose starvation involves suppression of a
necrotic death pathway.
Adaptation to low glucose availability is orchestrated by the energy
sensor AMPK, which suppresses anabolic pathways and induces cellcycle arrest, thus dampening ATP consumption1,5,6 . RelA deficiency,
however, had no effect on glucose-starvation-induced activation of the
Akt and mTOR pathways, promoting macromolecular biosynthesis1,5 ,
or AMPK itself (Supplementary Fig. S5a). This deficiency nevertheless
caused marked downregulation of p53 expression, in both unchallenged
and glucose-starved cells (Fig. 2a).
Basal p53 activity decreases the level of glycolysis and increases
the level of OXPHOS, hence countering the Warburg effect1,5,7,11 .
Thus, we investigated whether p53 mediated any of the metabolic
activities of NF-κB. Significantly, RelA inhibition downregulated basal
messenger RNA levels of p53, as well as of the NF-κB target A20,
consistent with a low basal NF-κB activity in MEFs (Fig. 2b and
Supplementary Figs S4f and S5c; also Supplementary Figs S1g and S5d,
phosphorylated IκBα and IKKα/β in untreated MEFs). RelA inhibition
also impaired p53 mRNA induction by TNFα (Supplementary Fig.
S5b). Conversely, p53 inactivation had no effect on RelA transcripts
(Fig. 2b and Supplementary Fig. S4f). Chromatin immunoprecipitation
showed that NF-κB/RelA complexes bound to a known κB element
in the proximal p53 promoter region12 , even under basal conditions
(Fig. 2c). This binding was specific, as it was not detected with control
genomic regions or RelA−/− cells (Fig. 2c,d). RelA binding to the p53
promoter increased after TNFα stimulation or glucose starvation,
which induce NF-κB (ref. 3; Supplementary Fig. S5d), and was more
stable at this promoter than at that of I κBα, another target of NF-κB
1274
(ref. 3; Fig. 2c). Thus, basal and induced p53 expression is under
direct NF-κB-dependent transcriptional control. In contrast, NF-κB
did not affect the relative glucose-starvation-dependent induction
of total or Ser15-phosphorylated p53 (Fig. 2a), which is controlled
by AMPK (refs 6,13), nor did it affect p53 protein stability11
(Supplementary Fig. S5e,f).
These data indicate that NF-κB is required, besides AMPK, to engage
the p53 pathway to direct the cellular response to metabolic stress.
Consistently, p53 silencing recapitulated many of the metabolic effects
of RelA inactivation, including the increase in basal lactate production,
glucose consumption and ATP levels, and the decreased oxygen
utilization level7,13,14 (Supplementary Fig. S6a–d and also Fig. 1a–d,f–h,
and Supplementary Fig. S1a–c,e, RelA-deficient cells; Supplementary
Fig. S6e, functional p53 status in immortalized MEFs). It also
markedly increased glucose-starvation-induced PCD (Supplementary
Fig. S6f,g and also Supplementary Fig. S4e), established previously6 ,
with magnitude and kinetics similar to RelA inactivation (Fig. 1e
and Supplementary Figs S1d and S4e). As with RelA deficiency,
p53 inactivation did not impair cell-cycle arrest1 under the glucose
starvation conditions used (Supplementary Fig. S2a), as reported
previously6 , and glucose-starvation-induced cell death of p53-deficient
cells was rescued by serum deprivation (Supplementary Fig. S2d),
indicating that these cells may undergo the same metabolic crisis
faced by RelA-deficient cells. Remarkably, p53 expression rescued
RelA-deficient cells from glucose-starvation-induced necrosis (Fig. 2e),
whereas RelA expression afforded no protection to glucose-starved
p53-deficient cells (Fig. 2f). p53 reconstitution also decreased the level
of lactate production and increased the level of oxygen consumption
in RelA−/− cells, thus reverting the metabolic effects of RelA loss,
both basally and during glucose starvation (Fig. 2g,h). Hence, p53 is a
crucial downstream mediator of NF-κB in the bioenergetic pathway
controlling adaptation to metabolic stress.
Reinforcing the notion that glucose addiction in NF-κB-deficient
cells results from impaired p53 signalling, RelA inactivation upregulated basal expression of p53-repressed genes, including those encoding
the glucose transporters GLUT1, GLUT3 and GLUT4 (refs 14–16),
and the glycolytic enzyme phosphoglycerate mutase 2 (PGM2; ref. 17;
Fig. 3a). It also downregulated the p53 target SCO2 (Fig. 3a), a component of COX, the main cellular site of oxygen utilization4,7 . Levels of
metabolic enzymes controlled by p53 in a species- and/or tissue-specific
manner13,18,19 were instead unaffected by NF-κB (Fig. 3a).
Knocking down GLUT1, GLUT3 or PGM2 failed to protect RelAdeficient cells against glucose-starvation-induced PCD (Supplementary
Fig. S7a), indicating that upregulation of the genes encoding these
proteins cannot account for glucose addiction in these cells. Silencing
them also promoted necrosis in glucose-starved wild-type cells. Hence,
as reported previously20 , increased glycolytic flux enhances, rather than
impairs, cell tolerance to low glucose availability. Remarkably, this
tolerance was restored in NF-κB-deficient cells by reconstitution with
SCO2 (Fig. 3b), coincident with the decreased reliance of these cells on
glucose metabolism (Supplementary Fig. S7b–d). In contrast, SCO2
downregulation to levels seen in RelA-deficient cells (Fig. 3a) induced
PCD in glucose-starved wild-type cells to an extent comparable to RelA
inactivation (Fig. 3c). SCO2 silencing also increased the levels of glucose
consumption, lactate secretion and basal ATP production in untreated
wild-type cells, and it decreased this production during glucose
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LETTERS
NM
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50
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Figure 2 p53 mediates NF-κB-dependent protection against glucose
starvation. (a) Western blots with antibodies against total or
Ser15-phosphorylated (P) p53 (equivalent to mouse p53-Ser18) in
non-specific (ns)- and RelA-shRNA-expressing immortalized MEFs after
glucose starvation (GS). exp, exposure. (b) qRT-PCR with RelA- or
p53-specific primers and RNAs from immortalized MEFs expressing the
shRNAs shown. (c,d) Chromatin immunoprecipitation with antibodies
against RelA, qPCR primers specific for κB-containing regions of the p53
and IκBα promoters or control genomic regions (control 1–3) and extracts
from untreated (UT), TNFα-treated or glucose-starved immortalized wild-type
(c,d) and RelA−/− (RelA knockout; d) MEFs. (e,f) Survival of early passage
RelA−/− (e) and p53 −/− (p53 knockout; f) MEFs expressing exogenous
eGFP, mouse (m)-p53 or human (h)-RelA before and after glucose starvation
(left). qRT-PCR with RNAs from the same cells (0 h; right). (g,h) Lactate
production and oxygen consumption in RelA−/− MEFs expressing exogenous
eGFP or mouse p53 in the presence of normal medium (NM) and during
glucose starvation (GS). In b–h the values denote mean ± s.e.m. n = 3 (b–g);
n = 15 (h). Uncropped images of blots are shown in Supplementary Fig. S8.
starvation7 (Supplementary Fig. S7e–g). It also caused a compensatory
increase in the mRNA levels of p53-repressed glycolytic genes5 in RelAdeficient cells, but not in wild-type cells (Supplementary Fig. S7h), indicating involvement of a p53-dependent mechanism. Although SCO2
downregulation also increased RelA levels (Fig. 3c), Sco2-shRNA cells
still succumbed to glucose starvation. Thus, SCO2 recapitulates many of
the effects of NF-κB on energy homeostasis, metabolic adaptation and
glycolytic gene expression. These data identify SCO2 as a downstream
effector of the NF-κB–p53 bioenergetic pathway and underscore its
importance in the protective activity of NF-κB during metabolic stress.
RelA or p53 disruption induces the Warburg effect, and deregulation
of mitochondrial proteins (for example, COX components) and
other metabolic factors is common in cancer1,2,5,7–9,19,20 . We reasoned
therefore that the metabolic reorganization resulting from RelA
deficiency may overcome the requirement for tumour suppressor
mutation in malignant transformation11 . Indeed, on expression of
oncogenic H-Ras(V12) (ref. 21), early passage RelA−/− MEFs acquired
morphological features of transformed cells (Fig. 4a), and retained a
proliferation rate comparable to their untransformed counterparts
(Fig. 4b). Similarly treated wild-type cells underwent instead growth
arrest and senescence, as expected (Fig. 4a,b). Foremost, unlike wildtype cells, H-Ras(V12)-expressing RelA−/− MEFs exhibited anchorageindependent growth (Fig. 4c), a hallmark of transformation14 . These
data demonstrate an ability of RelA-deficient cells to bypass oncogeneinduced senescence resulting in transformation even in the presence of
wild-type p53.
NATURE CELL BIOLOGY VOLUME 13 | NUMBER 10 | OCTOBER 2011
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a
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LETTERS
Figure 3 SCO2 mediates NF-κB-dependent protection against
glucose-starvation-induced PCD. (a) qRT-PCR with RNAs from non-specific
(ns)- or RelA-shRNA-expressing immortalized MEFs under basal
conditions. p53 metabolic targets referred to in the text, but not
specified: TP53-induced glycolysis and apoptosis regulator18 (TIGAR),
guanidinoacetate methyl transferase13 (GAMT ), glutaminase 2 (GLS2 ;
ref. 19). (b) Top, survival of early passage RelA−/− MEFs expressing
exogenous eGFP or mouse (m)-SCO2 after glucose starvation (GS). (c) Left,
survival of immortalized MEFs expressing the indicated shRNAs after
glucose starvation. (b) Bottom and (c) Right, qRT-PCR showing relative
Sco2 and RelA expression in the same cells (0 h). In a–c the values denote
mean ± s.e.m. (n = 3).
The effect of RelA loss on H-Ras(V12)-induced transformation was
reverted by the ectopic expression of SCO2 or p53 (Fig. 4d). Conversely,
SCO2 silencing had the same promoting effect on transformation
as the silencing of RelA or p53 (Fig. 4e). Consistent with the role of
glycolysis in proliferation and transformation, H-Ras(V12) expression
further upregulated glycolytic flux in RelA−/− MEFs, and further
decreased it in senescent wild-type cells, relative to untransformed cells
(Fig. 4f,g). These data position the p53/SCO2-dependent metabolic
pathway downstream of RelA in the suppression of oncogene-induced
transformation (see also Supplementary Fig. S5g, p53 levels in
H-Ras(V12)-expressing MEFs). They support a model whereby RelA
suppresses transformation in part by upregulating a SCO2-mediated
mechanism, which increases the rate of respiration and dampens the
rate of glycolysis, thus limiting the Warburg effect. Accordingly, SCO2
inhibition induces the same reprogramming to glycolysis exhibited by
p53- and RelA-deficient cells7 (Supplementary Fig. S7e–g), whereas
SCO2 reconstitution reverts this reprogramming in RelA−/− MEFs
(Supplementary Fig. S7b–d).
Our data indicate that the NF-κB-dependent increase in mitochondrial metabolism is a barrier for transformation. NF-κB, however,
has been shown in many studies to promote tumorigenesis. Thus,
we examined the relevance of the NF-κB metabolic function in
colon carcinoma cells, a model system in which NF-κB plays this
pro-tumorigenic role22,23 . Following RelA silencing, CT-26 colon
carcinoma cells became susceptible to metabolic challenge with glucose
starvation, either alone or in combination with the anti-type-II diabetes
drug, metformin, which inhibits mitochondrial complex I (ref. 24;
Fig. 5a,b). Moreover, data in mice showed that, although having little
or no effect on basal tumour survival and growth, NF-κB inhibition
renders CT-26-derived tumours highly susceptible to mitochondrial
stress in vivo, under conditions that do not affect NF-κB-proficient
CT-26 tumours. This effect of RelA on systemic metformin treatment
was especially pronounced at early times, when tumours expressed
higher levels of RelA-targeting shRNAs and lower levels of Sco2 mRNAs
(Fig. 5c,e). These findings indicate that suppressing mitochondrial
metabolism in certain established cancer cells through inhibition of
NF-κB and metformin diminishes tumorigenesis in vivo.
We have identified NF-κB as a physiological regulator of
mitochondrial respiration, and have established that this function of
NF-κB suppresses the Warburg effect and oncogenic transformation,
and prevents necrosis on nutrient starvation. We also identified a role
for NF-κB in metabolic adaptation in cancer in vivo. This metabolic
function of NF-κB involves the p53-dependent upregulation of SCO2,
which increases OXPHOS, thereby decreasing glycolytic flux. Our
findings position NF-κB at a nodal checkpoint tethering cell activation
and proliferation to energy sensing and metabolic homeostasis.
NF-κB–p53 crosstalk has been previously investigated during
genotoxic stress and inflammation, with different outcomes, depending
on tissue type, levels of induced nuclear activities and the nature of
post-translational modifications25 . We show here, however, that under
basal conditions, as well as in response to certain stimuli, NF-κB
and p53 cooperate to direct cellular bioenergetics. Consistently, RelA
deficiency phenocopies some of the basal effects of p53 inactivation,
including defective DNA repair and genomic instability, both in culture
and mice11,26,27 . Although NF-κB was reported to activate the p53
promoter in CAT/luciferase reporter assays12 , the fundamental roles
of NF-κB in the regulation of oxidative metabolism and metabolic
adaptation had not been investigated. Of note, our unpublished
1276
NATURE CELL BIOLOGY VOLUME 13 | NUMBER 10 | OCTOBER 2011
© 2011 Macmillan Publishers Limited. All rights reserved.
LETTERS
a RelA wild type RelA knockout p53 wild type p53 knockout
0.4
0.2
0
RelA knockout [H-Ras(V12)]
120
100
80
60
40
20
RelA wild type
RelA knockout
Lactate production
(percentage change)
20
g
10
0
–10
–20
–30
–40
RelA
wild type
150
Glucose consumption
(percentage change)
f
eGFP
m-p53
0
m-SCO2
eGFP
H-Ras(V12)
Sco2
shRNA
p53
shRNA
RelA
shRNA
50
ns
shRNA
Relative colony number
(percentage change)
100
H-Ras(V12)transformed cells:
0.6
H-Ras(V12)transformed cells:
150
0
0.8
eGFP
d
RelA wild type RelA knockout p53 wild type p53 knockout
e
1.0
p53 knockout [H-Ras(V12)]
Relative colony number
(percentage change)
H-Ras(V12)transformed cells:
1.2
wild type [H-Ras(V12)]
p53
wild type
p53
knockout
140
120
100
80
60
40
20
0
RelA
wild type
RelA
knockout
c
Relative colony number
(percentage change)
H-Ras(V12)
eGFP
Relative proliferation
(fold change)
b
100
50
0
–50
–100
Wild type [H-Ras(V12)]
Wild type [H-Ras(V12)]
RelA knockout [H-Ras(V12)]
RelA knockout [H-Ras(V12)]
Figure 4 RelA suppresses oncogenic transformation by regulating energy
metabolism. (a) Images of representative early passage RelA−/− , p53−/− and
wild-type MEFs infected with pWPT–eGFP or pWPT–H-Ras(V12), showing
transformation features (that is, higher density, spindled morphology) in
mutant cells, but not in wild-type cells. (b) Fold change in cell numbers
in H-Ras(V12)-expressing MEFs relative to the respective eGFP-expressing
controls after a 48 h culture. (c) Top, percentage change in colony numbers
with H-Ras(V12)-transformed cells relative to H-Ras(V12)-infected p53 −/−
MEFs. Bottom, images of representative colonies. (d,e) Percentage change in
colony numbers with H-Ras(V12)-transformed early passage RelA−/− MEFs
expressing eGFP, mouse (m)-p53 or m-SCO2 and H-Ras(V12)-transformed
early passage wild-type MEFs expressing eGFP relative to eGFP-expressing
RelA−/− MEFs (d), and H-Ras(V12)-transformed early passage wild-type
MEFs expressing non-specific (ns), RelA, p53 or Sco2 shRNAs relative
to RelA-shRNA-expressing MEFs (e). (f,g) Percentage change in lactate
production (f) and glucose consumption (g) in H-Ras(V12)-transformed MEFs
relative to the respective eGFP-expressing controls (see also Supplementary
Fig. S1a,b, absolute levels in the absence of H-Ras(V12)). The data in b,c,f
and g are from the cells in a. In b–g the values denote mean ± s.e.m. n = 3
(b,f,g); n = 4 (c,e). Scale bars: 50 µm.
data indicate that NF-κB may control metabolism also through
p53-independent mechanisms (C.M., S.C.L. & G.F., unpublished data),
underscoring the concept that it governs global energy metabolism
networks, beyond p53. Further studies will determine the significance
of these p53-independent functions of NF-κB. Nevertheless, here
we identify NF-κB as a central regulator of energy homeostasis and
metabolic adaptation.
The role of NF-κB in Ras-induced transformation, however, remains
controversial14,15,21,28,29 . A recent study indicated that RelA can promote
this transformation by upregulating GLUT3 (ref. 14). The status of
p53, however, is a confounding issue in this and other studies, as
the systems used were either immortalized fibroblasts harbouring a
defective p53 pathway or p53−/− models14,15,21,29 . By using early passage
p53 wild-type MEFs, we show instead that the NF-κB-imposed restraint
of glucose metabolism and transformation is mediated by endogenous
p53, which seemingly overrides other p53-independent glycolysisassociated activities of NF-κB. Consistently, NF-κB functions as an
inhibitor of proliferation and a tumour suppressor in some tissues3,28,30 .
Notwithstanding, NF-κB more often promotes progression and
survival in cancer3,29,31 . In this regard, our data indicate that NF-κB
plays this pro-tumorigenic role in part by enabling cancer growth
during metabolic stress, potentially permitting adaptation to hypoxic
and hypoglycaemic environments. These findings are consistent with
previous studies showing that mitochondrial metabolism is required
for tumorigenesis32,33 . Interestingly, the dichotomy we report for the
pro- and anti-tumorigenic metabolic activities of NF-κB has been
noted previously for many other oncogenes and tumour suppressors,
including p53 (refs 1,11,19,24). However, NF-κB is often upregulated
NATURE CELL BIOLOGY VOLUME 13 | NUMBER 10 | OCTOBER 2011
© 2011 Macmillan Publishers Limited. All rights reserved.
1277
60
40
RelA
20
β-actin
0
2
4
6
Days after GS
120
8
Tumour volume (mm3 )
0
ns shRNA
Tumour volume (mm3 )
80
c
RelA shRNA
ns shRNA
100
ns shRNA
RelA shRNA
100
80
60
40
20
+
+
MET
PBS
e
Relative expression
–
+
ns
shRNA
+
–
350
300
250
200
150
100
50
0
100
80
60
40
20
0
1,000
2,500
800
2,000
600
1,500
400
1,000
200
500
0
0
RelA shRNA
PBS
∗∗
100
350
300
250
200
150
100
50
0
80
60
40
20
0
Time (d):
RelA
shRNA
0
GS
MET
RelA
shRNA
d
ns shRNA
RelA shRNA
120
ns
shRNA
b
Percentage of survival
a
Percentage of survival
LETTERS
7
∗∗
1,000
600
1,500
400
1,000
200
500
14
Sco2
1.2
1.2
1.2
0.8
0.8
0.8
0.8
0.4
0.4
0.4
0.4
Mouse 1
0
Sco2
ns shRNA
RelA shRNA
0
0
Mouse 2
MET
18
1.2
0
∗
0
0
RelA
2,500
2,000
11
RelA
∗
800
Mouse 1
Mouse 2
Figure 5 NF-κB promotes metabolic adaptation in cancer in vivo.
(a) Left, viability of CT-26 cells expressing non-specific (ns) or RelA
shRNAs before and after glucose starvation (GS). Right, western blots
showing RelA-knockdown efficiency. (b) Survival of the cells in a after
a 4-day treatment with glucose starvation, metformin (MET) or glucose
starvation plus metformin. (c) Growth of CT-26 tumours expressing
non-specific or RelA shRNAs in nude mice treated with metformin
or PBS. (d) Images of representative tumours from c. (e) qRT-PCR
showing the relative RelA and Sco2 levels in tumours isolated from
two representative metformin-treated mice at day 14. In a–c and e
the values denote mean ± s.e.m. n = 3 (a,b,e); n = 9 (c); ∗P < 0.05;
∗ ∗ P < 0.01.
in cancer, and p53 is often mutated. Our data, nevertheless, do not
exclude that in some tumours NF-κB may exercise its metabolic
and pro-tumorigenic functions also through mutated p53. Although
further studies are required to address this issue, our findings establish
the physiological significance of the NF-κB-dependent control of
energy metabolism to metabolic adaptation in cancer in vivo, and they
define a bioenergetic pathway by which NF-κB can promote tumour
progression and survival, independently of the regulation of apoptosis
or inflammation. These findings have therapeutic implications, as they
indicate that NF-κB inhibitors may enhance the anti-cancer efficacy of
metabolic drugs.
AUTHOR CONTRIBUTIONS
C.M. first observed the glucose addiction exhibited by RelA-null cells. C.M. and
S.C.L. carried out the further experimental characterization of this phenomenon and
most of the analyses shown. E.A. and S.R. carried out the oxygen consumption assays.
A.K.T. carried out the cell-cycle analysis and helped with in vivo studies. L.T. carried
out the immunoblot analyses of apoptosis and autophagy and the in vitro metabolic
analyses of CT-26 cells. E.D.S. and A.A.B. generated the early passage p53−/−
and RelA−/− MEFs, respectively, as well as the early passage wild-type controls
from littermates. G.F., C.M. and S.C.L. wrote the manuscript and conceived the
experiments. N.S.C. and V.T. contributed to the design of some of the experiments
and made substantial critical revision to the manuscript. C.M., E.A., A.K.T., L.T. and
M.M. carried out the experiments during revision of the manuscript. All authors
discussed and revised the manuscript.
METHODS
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/naturecellbiology
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturecellbiology
Reprints and permissions information is available online at http://www.nature.com/
reprints
Note: Supplementary Information is available on the Nature Cell Biology website
1.
ACKNOWLEDGEMENTS
We thank M. Pagano, F. Dazzi, P. Ashton-Rickardt, F. Marelli-Berg and G. Screaton
for critical comments on the manuscript. We also thank K. Ryan (Beatson Institute
for Cancer Research, Glasgow, UK) for the eGFP–LC3 plasmid; T. Lindsten
and C. B. Thompson (University of Pennsylvania, Philadelphia, USA) for the
immortalized Bax−/− /Bak−/− MEFs; D. Trono (Ecole Polytechnique Fédérale de
Lausanne, Lausanne, Switzerland) for the pWPT lentiviral vector; C. Chevtzoff and
D. G. Hardie for assistance with the use of the Seahorse machine; and K. R. Chng
for assistance with the analyses of the p53 promoter. C.M. was supported in part
by a fellowship from AIRC (Italy). S.C.L. is supported by a scholarship from
A*STAR (Singapore). M.M. is supported by a fellowship from the Pasteur Institute,
Cenci Bolognetti Foundation (Italy). This work was supported by NIH grants R01
CA084040 and R01 CA098583 and Cancer Research UK grant C26587/A8839 to G.F.,
and NIH grant R01 CA123067 to N.S.C.
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NATURE CELL BIOLOGY VOLUME 13 | NUMBER 10 | OCTOBER 2011
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METHODS
DOI: 10.1038/ncb2324
METHODS
Antibodies and reagents. The antibodies used for western blots were: anti-RelA,
(1:10,000; Assay Design); anti-caspase-8 (1:1,000; Alexis); anti-PARP1 (1:100;
Calbiochem); anti-p53 (1:2,000; Oncogene); anti-beclin1 (1:1,000), anti-p21
(1:1,000; BD Biosciences); anti-HMGB1 (1:500; Abcam); anti-LC3 (1:100; Abgent);
anti-ATG7 (1:200), anti-TSC2 (1:1,000), anti-P-TSC2 (1:1,000), anti-ACC (1:1,000),
anti-P-ACC (1:1,000), anti-Akt (1:1,000), anti-P-Akt (1:1,000), anti-P-p53(Ser
15) (1:1,000), anti-AMPKα (1:1,000), anti-P-AMPKα (1:1,000), anti-p70-S6K
(1:1,000), anti-P-p70-S6K (1:2,000), anti-IKKβ (1:1,000), anti-P-IKKα/β (1:1,000),
anti-caspase-3 (1:1,000), anti-caspase-9 (1:1,000), anti-P-IκBα (1:1,000), anti-c-Rel
(1:1,000), anti-p38 (1:1,000), anti-P-p38 (1:1,000), anti-ERK1/2 (1:1,000), antiP-ERK1/2 (1:1,000; Cell Signaling); anti-MDM2 (1:1,000), anti-β-actin (1:3,000;
Santa Cruz Biotechnology); anti-p62 (1:1,000; Sigma-Aldrich); anti-RelA (1:1,000)
and rabbit control IgG (1:1,000) for chromatin immunoprecipitation were from
Santa Cruz Biotechnology. Anti-BrdU (1:20) for cell-cycle analyses was from
eBioscience. The reagents used were: mouse TNFα (1,000 U ml−1 ; Peprotech);
the IKKβ inhibitor SC-514 (20 or 100 µM; Calbiochem); z-VADfmk (50 µM;
Alexis); MG132 (0.5 or 50 µM), 3-methyladenine (5 mM), chloroquine (5 µM),
metformin (2 mM), bafilomycin A1 (100 nM), daunorubicin (5 µM), cycloheximide
(0.1 µg ml−1 or 10 µg ml−1 ; Sigma-Aldrich); rapamycin (100 nM; Calbiochem);
BrdU (Roche); 7-AAD (BD Biosciences). Antimycin (1 µM) and rotenone (1 µM)
used for mitochondrial oxygen consumption analyses were from Sigma-Aldrich.
Cell culture and lentiviral infections. Early passage RelA−/− , RelA+/+ , p53−/−
and p53+/+ MEFs were derived from 14-day-old mouse embryos using standard
methods and used at passage (p)1 to p5. Immortalized wild-type and RelA−/−
MEFs were described previously34 . Immortalized Bax −/− /Bak −/− MEFs were
provided by C.B. Thompson. The CT-26 wild-type mouse colon carcinoma cell
line was purchased from ATCC. Cells were cultured in high-glucose Dulbecco’s
modified Eagle’s medium (DMEM; with glutamine, without sodium pyruvate)
supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), antibiotics
(100 µg ml−1 penicillin and 100 µg ml−1 streptomycin) and 1 mM glutamine.
For glucose starvation, early passage MEFs were cultured in DMEM with no
glucose (with glutamine, without sodium pyruvate), supplemented with 10%
dialysed FBS (Sigma-Aldrich), antibiotics and 2 mM sodium pyruvate. Glucose
starvation with immortalized MEFs was carried out in the same DMEM
with no glucose, supplemented with 10% FBS, antibiotics and 2 mM sodium
pyruvate, in the presence of 6 mM d-glucose, unless otherwise specified. Glucose
starvation with CT-26 wild-type cells was carried out as for immortalized
MEFs, in the presence of 14 mM d-glucose. For serum deprivation, cells
were cultured in DMEM with no glucose, supplemented with antibiotics
and 2 mM sodium pyruvate, without FBS. Production of high-titre lentiviral
preparations in HEK293T cells and lentiviral infections were carried out as
described previously34,35 .
Lentiviral vectors. The targeting shRNAs used are listed in Supplementary
Table SI. The DNA sequences encoding shRNAs specific for eGFP, firefly
luciferase and mouse RelA (RelA shRNA), caspase-8, beclin1, Atg7, p53 (p53
shRNA, p53-2 shRNA), laminA/C and cyclophilinB, as well as the control nonspecific sequences, ns and ns2, were introduced between the BamHI and HpaI
restriction sites of the lentiviral vector pLentiLox3.7 (ref. 35). A variant of
pLentiLox3.7 (pLL–red), expressing enhanced red fluorescent protein (eRFP), was
used to deliver non-specific and RelA-specific shRNAs to immortalized MEFs
in Supplementary Fig. S3e. The DNA-coding sequences of the shRNAs specific
for mouse Glut1 (also known as Slc2a1), Glut3 (also known as Slc2a3), Pgm2
or Sco2, and the additional RelA-specific sequences, RelA-2 shRNA and RelA-3
shRNA, as well as the control sequence, shc003v, in the pLKO.1 lentiviral vector
were purchased from Sigma-Aldrich. The pWPT lentiviral vector, expressing
eGFP, has been described previously36 . The full-length complementary DNAs
of human H-Ras(V12), human RelA and mouse p53 were introduced between
the BamHI and SalI restriction sites of pWPT to replace eGFP. The cDNAs
of mouse Sco2 and LC3 were purchased from Eurofins MWG Operon and
obtained from K. Ryan, respectively, then cloned between the same restriction sites
of pWPT.
Measurements of glucose, lactate, ATP and oxygen. Glucose consumption,
lactate secretion and intracellular ATP levels were measured as described
previously13,14 . Briefly, cells were seeded onto 60-mm tissue-culture dishes, media
were changed 6 h later and assays were carried out after a 48-h culture in normal
medium (basal determinations) or in medium containing no or low glucose (glucose
starvation), as specified. Glucose and lactate concentrations were measured in the
culture media using the Glucose Assay Kit and the Lactate Assay Kit II (Biovision),
respectively, according to the manufacturer’s instructions. Glucose consumption
was extrapolated by subtracting the measured glucose concentrations in the media
from the original 25 mM glucose concentration. Intracellular ATP levels were
determined in cell lysates using the ATP Bioluminescent Somatic Cell Assay Kit
(Sigma-Aldrich), according to the manufacturer’s instructions. Oxygen uptake was
measured in 24-well plates using a Seahorse XF24 extracellular flux analyser9 . Cells
were seeded at 2–5 × 104 cells per well in 8.3 g l−1 DMEM base medium (pH 7.4)
supplemented with 200 mM GlutaMax-1, 100 mM Na pyruvate, 32 mM NaCl and
40 µM phenol red, in the presence of either 25 mM (basal determinations) or 6 mM
(glucose starvation) glucose, and oxygen consumption was measured continuously
as described previously9 . Mitochondrial oxygen consumption rates were calculated
by subtracting the residual oxygen consumption in the cells after treatment with
antimycin (1 µM) plus rotenone (1 µM). All values were finally normalized on a
per-cell basis.
Soft-agar colony assays. Soft-agar colony formation was assessed using standard
methods14 . Briefly, early passage MEFs were infected with the viruses indicated and
poured 6 days later at a density of 2 × 104 cells per dish together with 0.5%-agarose
F12 medium onto a pre-solidified bottom layer of 0.5% agarose in the same medium,
in 60-mm tissue-culture dishes. Colony formation was assessed 3 weeks later.
Cell death and macroautophagy analyses. Cell viability and HMGB1 extracellular release were assessed by trypan blue exclusion assays and western blotting,
respectively, as reported previously34,37 . HMGB1 assessment was carried out both in
media and cellular fractions. For the analyses of macroautophagy, cells were infected
with pWPT–eGFP–LC3, expressing an eGFP fusion protein of LC3, and eGFP–LC3
translocation was monitored as described previously38 . Macroautophagic vesicles
were quantified by counting 100 cells and scoring as positive those showing any signs
of punctuate, rather than diffuse, eGFP–LC3 fluorescence, using an inverted Leica
SP5 confocal microscope.
Cell-cycle analyses. Cell-cycle analyses were carried out using BrdU and 7-AAD
labelling, as described previously39 . Briefly, early passage or immortalized MEFs
were seeded at a density of 1 × 106 cells per dish in 100-mm tissue-culture dishes
and 24 h later either left untreated or subjected to glucose starvation (0 mM and
6 mM glucose, respectively) for a further 48 h. Cells were then incubated with
BrdU (30 µM) for 3 h, trypsinized, fixed in 70% ethanol and permeabilized with
2N HCl and 0.5% Triton X-100, followed by neutralization in 0.1 M sodium borate.
Samples were finally stained with FITC-labelled anti-BrdU antibody and 7-AAD,
and fluorescence signals were acquired using a FACS Caliber (Beckman).
Quantitative real-time polymerase-chain reaction. Total RNA was extracted
with Trizol, and purified using the PureLink RNA mini-kit (Invitrogen). RNA
(1 µg) was added as a template to reverse-transcriptase reactions carried out using
the GeneAmp RNA PCR Kit (Applied Biosystems). Quantitative real-time PCRs
(qRT-PCRs) were carried out with the resulting cDNAs in triplicate using SYBR
Green PCR Master Mix (Applied Biosystems), the primers listed in Supplementary
Table SII and an ABI 7900 real-time PCR machine. Experimental Ct values
were normalized to hypoxanthine–guanine phosphoribosyltransferase (HPRT), and
relative mRNA expression was calculated versus a reference sample.
Chromatin immunoprecipitation. Chromatin immunoprecipitation was carried
out as described previously40 . Briefly, unstimulated, TNFα-treated and glucosestarved immortalized RelA−/− and wild-type MEFs were treated with 1%
formaldehyde for 10 min at room temperature to crosslink protein–DNA complexes;
then reactions were quenched using 125 mM glycine. Chromatin extracts were
sonicated with a Bioruptor sonicator (Diagenode), and protein–DNA complexes
were immunoprecipitated using anti-RelA or rabbit control IgG antibodies.
Quantitative PCR (qPCR) assays were then carried out using anti-RelA and IgG
precipitates and the primers listed in Supplementary Table SII, encompassing the
κB-containing regions of the p53 and I κBα promoters or control genomic regions
(control 1–3), to assess RelA DNA-binding specificity. RelA-specific DNA binding
was calculated relative to the background signal yielded by control IgG and expressed
as fold change.
Mouse allografts. Early passage CT-26 wild-type cells expressing non-specific or
RelA shRNAs were collected, washed twice with PBS, and resuspended in PBS
at a concentration of 1 × 106 cells ml−1 before being inoculated subcutaneously
into both flanks (non-specific shRNA cells on the right flank, and RelA-shRNA
cells on the left flank; 1 × 105 cells per flank) of nude mice (6-week-old females;
Charles River). Metformin was dissolved in PBS (50 mg ml−1 ) and administered
intraperitoneally daily (250 mg × kg body weight) from day 3 after tumour cell
injection. The control group received PBS, using the same administration schedule
and route. Tumour volume (mm3 ) was measured twice a week at the indicated
times and estimated from caliper measurements using the following formula:
volume = A × B2 /2 (with A being the larger diameter, and B the smaller diameter
of the tumour). Experiments were carried out under the Home Office Authority
(Cambridge, UK; PPL 70/6874).
NATURE CELL BIOLOGY
© 2011 Macmillan Publishers Limited. All rights reserved.
METHODS
DOI: 10.1038/ncb2324
Statistical analysis. Results are expressed as mean ± s.e.m. from an appropriate
number of samples as indicated in the figure legends. Student’s t -test was used to
determine statistical significance.
34. Pham, C. G. et al. Ferritin heavy chain upregulation by NF-κB inhibits TNFαinduced apoptosis by suppressing reactive oxygen species. Cell 119,
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35. Yang, H. et al. TNF-α inhibits asbestos-induced cytotoxicity via a NF-κB-dependent
pathway, a possible mechanism for asbestos-induced oncogenesis. Proc. Natl Acad.
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36. Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system.
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37. Zong, W. X. et al. Alkylating DNA damage stimulates a regulated form of necrotic
cell death. Genes Dev. 18, 1272–1282 (2004).
38. Crighton, D. et al. DRAM, a p53-induced modulator of autophagy, is critical for
apoptosis. Cell 126, 121–134 (2006).
39. Zinszner, H. et al. CHOP is implicated in programmed cell death in response to
impaired function of the endoplasmic reticulum. Genes Dev. 12, 982–995 (1998).
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DOI: 10.1038/ncb2324
Figure S1 NF-kB counters aerobic glycolysis and promotes adaptation
to GS in MEFs. (a-c) Glucose consumption, lactate production and
intracellular ATP concentration in early-passage RelA-/- (KO) and RelA+/+
(WT) MEFs, under normal culture conditions. Similar results were obtained
using two additional clones of mutant and wild-type MEFs. (d) Viability
of early-passage RelA-/- and RelA+/+ MEFs after GS. (e) Intracellular ATP
concentration in MEFs treated as in (d). Lactate concentration for either
RelA-/- or RelA+/+ MEFs could not be determined following complete glucose
withdrawal because they were below the detection limit of the assay. (f)
Viability of immortalized MEFs after a 3-day culture in normal medium (NM)
or medium containing 6 mM glucose (GS), either in the presence (SC-514)
or absence (UT) of the IKKb inhibitor, SC-514 (20 mM). (g) Western blots
showing phosphorylated (P) IkBa, and total and phosphorylated p38 and
ERK in immortalized MEFs, either left untreated or treated with TNFa, in the
presence (+) or absence (-) of SC-514 (100 mM). P-IkBa shows the efficacy
of NF-kB inhibition, whereas P-p38 and P-ERK1/2 show the specificity of
this inhibition. b-actin is shown as loading control. ns, non-specific. (h)
Oxygen consumption in immortalized MEFs expressing sh-RelA-3 (a different
RelA-specific hairpin from that used in Figs. 1d and 1g) or sh-ns sh-RNAs
under normal culture conditions and during GS (left). Western blots with the
same cells showing RelA knockdown efficiency and specificity (right). (a-f, h)
Values denote mean ± s.e.m. (a-f) n=3; (h) n=12.
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Figure S2 NF-kB or p53 deficiency does not impair cell-cycle arrest
following GS, and the cytotoxic effects of this deficiency during GS are
blocked by serum deprivation. (a) FACS analyses of early-passage RelA-/-,
p53-/- and wild-type (WT) MEFs cultured in normal medium (NM) or in
medium containing no glucose (GS) for 48 hours, then labelled with 7-AAD
and BrdU. (b) FACS analyses of immortalized MEFs expressing sh-ns or
RelA-specific sh-RNAs cultured either in NM or in medium containing 6
mM glucose (GS) for 48 hours, then labelled as in (a). (a-b) Depicted in
the dot plots are the percentages of cells in the different phases of the cell
cycle (G1, bottom, left; S, top; G2/M, bottom, right). FL1-H, BrdU staining;
FL3-H, 7-AAD staining. (c-d) Survival of early-passage RelA-/- (c), p53/- (d), and wild-type MEFs after GS, either alone (GS) or in combination
with serum deprivation (GS+SD) for 96 hours. (c-d) Values denote mean ±
s.e.m. (n=3).
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Figure S3 NF-kB does not impair the induction of macroautophagy by GS.
(a) Survival of immortalized MEFs expressing sh-ns, sh-RelA, sh-Beclin1,
sh-ATG7, and sh-ATG7-2 sh-RNAs during GS. (b) Western blots with
antibodies against RelA, Beclin1 or ATG7 and extracts from the untreated
cells shown in (a) (0-hour time point). (c) Survival of sh-ns- and sh-RelAexpressing immortalized MEFs after GS for 120 hours, either alone (GS) or
in combination with 3-methyladenine (GS+3-MA) or chloroquine (GS+CQ)
treatment. (d) Western blots showing the upregulation of LC3 in immortalized
MEFs expressing sh-ns or RelA-specific sh-RNAs before and after GS. (e)
Percentages of immortalized sh-RelA and sh-ns MEFs expressing a eGFPfusion protein of LC3 (labelling macroautophagic vesicles10,38) and displaying
punctuate intracellular fluorescence, before and after GS (left). The analysis
of sh-RelA cells was stopped at day 3, due to massive cell death beyond
this time. Representative images of the same cells (right). Of note, whilst
normally supporting macroautophagy induction by GS, RelA-deficient cells
displayed increased autophagosome formation under basal conditions (see
the 0-hour time points; see also the increased basal protein expression of LC3
in sh-RelA cells (d)). (f) Western blots showing the levels of the autophagic
cargo protein, p62, in immortalized sh-ns and sh-RelA MEFs before (-) and
after (+) treatment with the lysosomal vacuolar ATPase inhibitor, bafilomycin
A1. The data show that p62 levels are reduced in RelA-deficient cells under
basal conditions compared to control cells, consistent with an enhanced rate
of autophagosome degradation in sh-RelA cells. Accordingly, blocking this
degradation with bafilomycin A1 increased p62 levels in RelA-deficient cells.
Together with the data shown in (d) and (e), these results demonstrate that
RelA inhibition causes an increase in basal autophagic flux, possibly owing to
an enhancement of protein/organelle turnover10. (b, d, f) b-actin is shown as
loading control. (a, c, e) Values denote mean ± s.e.m. (n=3).
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Figure S4 NF-kB-afforded protection against GS-induced killing depends
on the suppression of a necrosis-like pathway of cell death. (a) Western
blots showing the processing of Caspase-8, Caspase-9, and Caspase-3, and
the cleavage of the Caspase-3 substrate, PARP1, after GS in immortalized
MEFs expressing either sh-ns or RelA-specific sh-RNAs. Filled arrowheads,
unprocessed proteins; open arrowheads, specific cleavage products; asterisks,
non-specific bands. (b) Survival of immortalized sh-RelA MEFs after GS, in
the presence (z-VADfmk +) or absence (z-VADfmk -) of z-VADfmk. (c) Images
of representative cultures from (b) (i.e. z-VADfmk +, day 5) and immortalized
sh-RelA MEFs expressing either sh-ns (sh-ns/sh-RelA) or Casapse-8-specific
sh-RNAs (sh-Casp8/sh-RelA) after GS (left). Western blots showing the
expression of RelA and Caspase-8 in the same cells (0-hour time points;
right). (d) Survival of immortalized sh-ns and sh-RelA MEFs after treatment
with TNFa plus cycloheximide (CHX; 0.1 mg/ml), in the presence or absence
of z-VADfmk, at the same concentration used in (b) and (c). These data
demonstrate that RelA-deficient cells can be rescued by z-VADfmk under
conditions in which they are known to succumb to apoptosis34. (e) Survival of
immortalized Bax-/-/Bak-/- (DKO) MEFs expressing sh-ns, sh-RelA, or sh-p53
sh-RNAs before and after GS. (f) qRT-PCR showing the relative expression
of RelA and p53 in the cells in (e). (g) NF-kB inhibits the GS-induced
extracellular release of the specific necrosis marker, HMGB1. Shown are
Western blots with anti-HMGB1 antibody and culture media or total lysates
from immortalized sh-ns and sh-RelA MEFs after GS. (a, c, g) b-actin is
shown as loading control. (b, d-f) Values denote mean ± s.e.m. (n=3).
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Figure S5 NF-kB upregulates p53 following TNFa stimulation or oncogenic
H-Ras(V12) expression, without affecting p53 protein stability or AMPK, Akt
and mTOR signalling. (a) Western blots showing total and phosphorylated
(P) levels of upstream regulators and downstream effectors of the AMPK,
Akt and mTOR pathways, controlling metabolism and nutrient uptake1,5, in
immortalized sh-ns and sh-RelA MEFs before and after GS (1 mM glucose).
The proteins analyzed included: AMPK and its substrate, Acetyl CoA
Carboxylase (ACC), Akt, the mTOR inhibitor, Tuberin (TSC2), and the mTOR
substrate, p70-S6 Kinase (p70-S6K). (b) qRT-PCR with primers specific for
p53 (left) or IkBa (right) and RNAs from unstimulated and TNFa-treated,
immortalized MEFs expressing sh-ns or RelA-specific sh-RNAs. (c) qRTPCR with primers specific for A20 and RNAs from immortalized sh-ns and
sh-RelA MEFs under basal culture conditions. (b, c) Values denote mean
± s.e.m. (n=3). (d) Western blots with antibodies against total IkB kinase
b (IKKb) and phosphorylated (P) IKKa and IKKb (P-IKKa/b), showing
activation of the NF-kB-inducing IKK complex in immortalized MEFs
expressing sh-ns or RelA-specific sh-RNAS after GS. (e, f) NF-kB does
not affect p53 protein stability in cells. Western blots showing p53 and
RelA expression in immortalized sh-ns and sh-RelA MEFs before and after
treatment with the proteasome inhibitor, MG132 (0.5 mM) (e), or the protein
synthesis inhibitor, cycloheximide (CHX; 10 mg/ml) (f). The data show that
the levels of p53 proteins decrease with similar kinetics in sh-ns and shRelA MEFs, thus demonstrating the similar half-life of p53 in these cells.
(g) RelA deficiency impairs the induction of p53 by oncogenic H-Ras(V12).
Western blots with antibodies against p53 or b-actin and early-passage
RelA-/- (KO) and wild-type (WT) MEFs expressing exogenous H-Ras(V12) or
eGFP, in the presence (+) or absence (-) of MG132 (50 mM). The data show
that the levels of p53 are decreased in H-Ras(V12)-expressing RelA-/- MEFs
relative to H-Ras(V12)-expressing WT MEFs. (a, d-g) b-actin is shown as
loading control.
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Figure S6 p53 recapitulates the effects of NF-kB on energy homeostasis
and metabolic adaptation. (a-d) Lactate production, glucose consumption,
intracellular ATP concentration, and oxygen consumption in immortalized
MEFs expressing sh-ns or p53-specific sh-RNAs, under basal conditions.
Similar results were obtained using an additional p53-specific sh-RNA
(sh-p53-2). (e) Western blots with antibodies against p53 and the p53
transcriptional targets, p21 and MDM2, in immortalized MEFs expressing
sh-ns or p53-specific (sh-p53) sh-RNAs before and after treatment with
the DNA damaging agent, daunorubicin (Dauno). The data show that
daunorubicin treatment induces a vigorous upregulation of p21 and MDM2,
as well as of p53 (a target of itself), in immortalized sh-ns MEFs, but not
in sh-p53 MEFs. Hence, immortalized MEFs are capable of mounting a
normal p53-dependent DNA-damage response, and so contain a functional
p53 pathway. (f, g) p53 promotes cell survival during GS. (f) Viability of
early-passage p53-/- (KO) and p53+/+ (WT) MEFs after GS. Similar results
were obtained using two additional clones of mutant and wild-type MEFs.
(g) Survival of sh-ns- and sh-p53-expressing immortalized MEFs after GS
(left). Western blots showing p53 and RelA expression in the same MEFs (0hour time points; right). (e, g) b-actin is shown as loading control. (a-d, f, g)
Values denote mean ± s.e.m. (a-c, f, g) n=3; (d) n=5.
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Figure S7 SCO2 mediates the metabolic activities of NF-kB. (a) Survival of
immortalized, sh-ns and sh-RelA MEFs infected with pLKO.1-shc003v, -shGLUT1, -sh-GLUT3 or -sh-PGM, then subjected to GS. Values express the
percentage of live cells in each culture relative to the viability observed in the
respective sh-ns/shc003v control for each time point. (b-d) SCO2 expression
reverts the metabolic effects of RelA loss. Basal glucose consumption, lactate
production, and intracellular ATP concentration in early-passage RelA-/- MEFs
infected with pWPT-eGFP or pWPT-SCO2. (e-g) SCO2 silencing mimics the
metabolic effects of RelA inhibition. Glucose consumption, lactate production,
and intracellular ATP concentration in immortalized MEFs expressing shc003v
or SCO2-specific sh-RNAS before and/or after GS. (h) SCO2 silencing
upregulates expression of p53 glycolytic targets in RelA-deficient, but not in
wild-type cells. qRT-PCR with primers specific for GLUT1, GLUT3, GLUT4
or PGM and RNAs from immortalized, sh-ns and sh-RelA MEFs infected with
either pLKO.1-shc003v (not depicted) or pLKO.1-sh-SCO2 (sh-ns/sh-SCO2
and sh-RelA/sh-SCO2, respectively). Shown is the percent change in glycolytic
gene expression in sh-ns and sh-RelA cells after infection with pLKO.1-shSCO2 relative to expression in the respective pLKO.1-shc003v-infected
controls (see also Fig. 3a, the basal gene expression in sh-ns and sh-RelA
immortalized MEFs). (a-h) Values denote mean ± s.e.m. (n=3).
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Figure S8 Full scans.
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