Oncotarget, Advance Publications 2012
The mitochondrial citrate transporter, CIC, is essential for
Olga Catalina-Rodriguez1,*,Vamsi K. Kolukula1,*, York Tomita1, Anju Preet1,
Ferdinando Palmieri2, Anton Wellstein1,Stephen Byers1, Amato J. Giaccia3, Eric
Glasgow1, Chris Albanese1 and Maria Laura Avantaggiati1
1 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA.
2 Department of Pharmaco-Biology, University of Bari, Bari, Italy.
3 Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA.
* denotes equal contribution
Correspondence to: Maria Laura Avantaggiati, email: email@example.com
Keywords: SLC25A1, CIC, citrate, cancer, mitochondria, autophagy, Di-George Syndrome
Received: October 18, 2012, Accepted: October 18, 2012, Published: October 20, 2012
Copyright: © Catalina-Rodriguez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Dysregulation of the pathways that preserve mitochondrial integrity hallmarks
many human diseases including diabetes, neurodegeration, aging and cancer. The
mitochondrial citrate transporter gene, SLC25A1 or CIC, maps on chromosome
22q11.21, a region amplified in some tumors and deleted in developmental disorders
known as velo-cardio-facial- and DiGeorge syndromes. We report here that in
tumor cells CIC maintains mitochondrial integrity and bioenergetics, protects from
mitochondrial damage and circumvents mitochondrial depletion via autophagy, hence
promoting proliferation. CIC levels are increased in human cancers and its inhibition
has anti-tumor activity, albeit with no toxicity on adult normal tissues. The knock-
down of the CIC gene in zebrafish leads to mitochondria depletion and to proliferation
defects that recapitulate features of human velo-cardio-facial syndrome, a phenotype
rescued by blocking autophagy. Our findings reveal that CIC maintains mitochondrial
homeostasis in metabolically active, high proliferating tissues and imply that this
protein is a therapeutic target in cancer and likely, in other human diseases.
The solute family member SLC25A1, or CIC,
belongs to a family of ion transporters embedded in the
inner mitochondrial membrane, and whose defects have
been indirectly linked to various human diseases [1,2,3].
The human CIC gene maps on chromosome 22.q11.2.
Several chromosomal translocations and amplifications
involving 22q11.2 have been described in various
tumors, while microdeletions of this region give raise to
developmental disorders known as velo-cardio-facial-
(VCFS), DiGeorge syndromes (DGS) as well as to some
forms of schizophrenia [4,5]. CIC expression is induced
by insulin and by inflammation, its activity is lost in type-
1 diabetes, and early studies also suggested increased
activity in tumors of the liver [6,7,8,9]. Furthermore,
mutations of a member of the tricarboxylate transporter
family in fruit fly, INDY, promote longevity , thus
suggesting that the citrate transport pathway also controls
life-span. Although CIC has been studied in yeast, there
is currently little information on the effects of the human
protein on proliferation.
Mechanistically, CIC promotes the efflux across
the mitochondria of tricarboxylic citrate in exchange for
dicarboxylic cytosolic malate (the citrate malate antiport),
leading to important metabolic adjustments (Figure 1A)
[1,11]. Citrate, which is produced predominantly in the
mitochondria via glucose-derived pyruvate, serves as a key
substrate for the generation of energy and as an allosteric
modulator of several enzymes as well. In the mitochondria
citrate is oxidized via the Krebs cycle and Oxidative
Phosphorylation (OXOPHOS), while in the cytoplasm it
supports lipid synthesis and blunts glycolysis by inhibiting
phosphofructokinase-1 (PFK1) [12,13]. Additionally,
the entry of malate into the mitochondria in exchange
for citrate stimulates OXOPHOS and is coupled to the
Figure 1: CIC is required for tumor proliferation. A. Role of CIC in metabolism (see also text for explanation). CIC catalyzes the
electroneutral exchange across the inner mitochondrial membrane of the tricarboxylate citrate plus a proton for the dicarboxylate malate.
Once in the cytoplasm citrate is cleaved by citrate lyase (CLY), providing a source for fatty acids (FA) synthesis and producing malate
that is imported in the mitochondria. Here, malate oxidation generates NADH, which donates its electrons to the transport chain thereby
maintaining oxidative phosphorylation (OXHOPHOS) and mitochondrial membrane potential (MMP). Cytoplasmic citrate can also act
as an allosteric inhibitor of Phosphofructokinase-1 (PK1), a key glycolytic enzyme. Glycolysis in the cytoplasm generates a second key
substrate, pyruvate that is either converted into lactate to produce ATP in an anaerobic reaction, or it is transported into the mitochondria,
converted by Pyruvate Dehydrogenase (PDH) into Acetyl-Coenzyme A, recondensed to citrate, which then supports the Krebs cycle.
These activities linked to citrate, should place CIC at a nodal point in regulation of mitochondrial activity and metabolism. B. CIC mRNA
expression levels in human normal or breast tumor tissues. Data were obtained from the Oncomine database , by selecting a minimal
threshold p-value of 0.0005. C. Negative correlation between CIC expression levels and the development of metastatic disease in breast
cancer. N indicates the total number of patients. In one study conducted , 69 patients with elevated CIC levels were dead after 5
years, versus 158 patients with lower levels, who were still alive. In a second study , 18 patients with elevated CIC levels developed
metastasis after 5 years, versus 6 with lower CIC levels who remained metastasis free. p values are indicated in each panel. D. CIC
expression levels in the breast cancer cell lines indicated at the top of each panel. E-F. Clonogenic assays performed in MBA-MD-231
cells. Cells were transfected with control shRNA vector (indicated as ct), or with vectors harboring three CIC shRNA (shRNA 1, 2, 3),
each harboring a puromycin resistance gene. Vectors were transfected individually (panel E) or in different combinations (panel F) and
proliferation was assessed after one week of antibiotic selection. Representative CIC expression levels relatively to actin levels are shown in
panel E (bottom panel), and representative plates derived from the combination transfection experiments are shown at the bottom of panel F.
G. Mitochondrial (m) and cytoplasmic (c) fractions derived from 293T cells transfected with the pcDNA4/TO control vector, or expressing
CIC or CIC-DN. The expression levels of CIC and COX IV, used as a mitochondrial marker, are shown.
Breast Cancer Cells
shRNA: ct 1 2 3
: ct 1 2 3
cell viability 10-5
cell viability 10-5
m c m c m c
Ishvinia et al.
69 vs 158
Desmedt et al.
18 vs 6
Correlation between clinical outcome and
high CIC levels
transport of one proton . This modality of exchange
maintains both electroneutrality across the mitochondrial
membrane and OXOPHOS, because malate enters into the
Krebs cycle in place of citrate. Therefore, the citrate export
pathway might impact upon mitochondrial respiration,
upon the stability of mitochondrial membrane potential
(MMP), -which is directly linked to the proton flux and to
the activity of the electron transport chain-, as well as on
glycolysis and on lipogenesis.
The majority of tumor cells display alterations in
metabolism relatively to normal cells that are intimately
connected with cancer development, progression and
invasiveness . These metabolic changes center
in large part upon the different utilization of citrate
compared to normal cells. Indeed, it has been proposed
that in cancer cells citrate is predominantly exported out
the mitochondria via CIC, being subsequently cleaved
in the cytoplasm via citrate lyase (CLY) to support lipid,
Acetyl-Coenzyme A and macromolecular biosynthesis
[15,16,17]. This switch from mitochondrial to cytoplasmic
metabolism of citrate is thought to account for the
acquisition of the lipogenic phenotype that, together with
the high rates of aeorobic glycolysis (the Warburg effect),
represents a hallmark of many cancer cells [18,19]. The
glycolytic behavior of tumors was initially attributed to
impairment of mitochondrial respiratory activity. This
hypothesis has been recently revisited in light of evidence
showing that the mitochondria of cancer cells retain some
extent of respiratory capacity . In addition, recent data
demonstrates that the tumor stroma provides a variety of
catabolized nutrients that enables the anabolic growth
of tumor cells by enhancing their mitochondrial activity
[14,20]. Thus, albeit the role of the mitochondria in
tumor proliferation has been for a long time undermined
by the Warburg hypothesis, it is now clear that these
organelles enact essential metabolic circuits needed for
tumorigenesis, including OXOPHOS, glutamine and fatty
acid oxidation .
Nevertheless, mitochondrial DNA mutations, the
hypoxic and nutrient restricted microenvironment as
well as oncogenic mutations that reduce the activity of
mitochondrial enzymes, all contribute to oxidative and
respiration stress endured by these organelles [21,22].
Consequently to these alterations, cancer cells produce
radical oxygen species (ROS), which increase oxidative
burden, but also work to the advantage of tumors because
low ROS levels produced by the stroma or within cancer
cells promote proliferation [14,22]. This oxidative
instability is at the same time also a double edge sword
and a vulnerable aspect of cancer cells, given that
enhancing ROS production leads to tumor killing and
to activation of a specialized form of autophagy, termed
mitophagy [23,24]. Although activation of mitophagy
in the adjacent stroma feeds cancer cells with nutrients
required for proliferation , avoidance of mitophagy
within the tumor appears instead to be advantageous to
cancer growth at least in some conditions [25,26].
Thus, cancer cells must maintain an adequate
equilibrium between mitochondrial activity, mitophagy,
and ROS production. Results presented here demonstrate
that CIC is important for preserving such balance.
CIC levels are elevated in human cancers and
its activity is required for tumor proliferation in
This study stemmed from the finding that the
transcription rate of the CIC promoter is enhanced by
several oncogenic pathways including mutant forms
of p53 and Myc, while it is repressed by the tumor
suppressor PTEN (Palmieri and Avantaggiati, manuscript
in preparation). Consistent with this observation, we
then found that CIC mRNA levels are elevated in various
cancer cell lines and human tumors correlating with poor
survival rates or with the development of metastatic
disease (Figure 1BC; Supplementary Figure S1AD). CIC
levels were found elevated in many, but not all of the
tumor cell lines derived from the NCI50 panel (Figure S1,
panels F-I). Significantly, CIC is almost undetectable in
a model system of ductal non-invasive carcinoma in situ
(DCIS) or in pseudo-normal MCF10A mammary cells,
compared to invasive MCF10A cells expressing the Ras/
Erb B oncogenes, or to MDA-MB-231 and BT549 cell
lines (Figure 1D). These observations suggested that CIC
expression correlates with aggressiveness. In agreement
with this idea, in MBA-MD-231 cells, expression of three
different CIC shRNAs, each transfected individually,
resulted in a small reduction in CIC protein and in a
modest growth inhibition (Figure 1E). However, co-
transfection of combination shRNAs completely blunted
proliferation (Figure 1F) and clones that survived these
experiments again displayed CIC levels nearly similar to
control (not shown). Thus, complete CIC inhibition is
incompatible with survival of MBA-MD-231 cells.
To specifically probe the relevance of the citrate
export activity of CIC on proliferation, we used a non-
cleavable benzene-tricarboxylate analog (BTA) that
suppresses CIC-dependent transport of citrate in vivo
[6,27]. We also designed a CIC mutant protein where
three amino acid residues necessary for citrate export,
K190, N194 and R198, were replaced with C190, I194
and C198, respectively [11,28,29]. This mutant (CIC-DN)
did not affect mitochondrial localization or embedding in
the mitochondrial membrane (Figure 1G; Figure S2AB)
but exhibited dominant negative activities, as judged by
its ability to lower the concentration of citrate in cells
expressing endogenous CIC (Figure 2A). Limited tryptic
digestion experiments further showed an impaired ability
Figure 2: CIC maintains the intracellular pool of citrate and promotes proliferation. A. Citrate levels (nM/well) were
assessed in 293T cell lines harboring the pcDNA/TO vector and treated with either DMSO or with 1 mM BTA, or in cells expressing
CIC-DN. Negative values are due to subtraction of background rates. B-C. Dose dependent growth inhibition of BTA in MDA-MB-231
or MCF10A cells. D. Control H1299 cells (pcDNA/TO), or cells expressing the CIC proteins indicated at the bottom of each panel, were
treated with DMSO (-), or with 1 mM BTA (+). Cell viability was assessed with trypan blue exclusion. E. Expression of CIC or CIC-DN
in tetracycline inducible stable clones of H1299 or 293T cells employed in this study, in presence or absence of tetracycline. F. Female
athymic balbc/ nude mice were injected with H1299 cells expressing inducible CIC in the presence or absence of tetracycline or of vehicle
control provided in the water. G. 293T cells stably transfected with the control vector (pCDNA4/TO), or expressing inducible CIC-DN
were injected in the right and left flanks of female athymic mice, respectively.
Viable cells x 10-6
Viable cells X 10-4
Viable cells X 10-4
-tet + tet
of CIC-DN to interact with the citrate analog BTA, as
it was expected (Figure S2CD). To assess the effects
of CIC and of its inhibition on cell growth, several cell
lines were treated with BTA or were transfected with the
vectors encoding CIC or CIC-DN. In MBA-MD-231, but
not in the pseudo-normal MCF10A cells, treatment with
BTA decreased proliferation rates in a dose-dependent
manner (Figure 2BC). Such differential effects suggested
a preferential sensitivity of tumor cells to CIC inhibition, a
view supported by studies shown later. Furthermore, over-
expression of wild-type CIC modestly but reproducibly
enhanced the proliferation potential of H1299 cells, and
rescued growth inhibition due to BTA treatment (Figure
2D, compare lane 2 with lane 4). By contrast expression
Figure 3: CIC is rate-limiting for tumorigenesis in vivo. A. Female athymic balbc/ nude mice were injected with H1299 cells
expressing inducible CIC in the presence or absence of tetracycline or of vehicle control as described in Figure 2F and tumor volumes were
assessed several weeks after implantation. White bars indicate control group, while black bars show tumor growth in cells expressing CIC.
B. Tumor volumes assessed after implantation in nude mice of T293 cells expressing control vector (pCDNA/TO) or dominant negative
CIC (CIC-DN). C-E. Tumor volumes in female athymic balbc/ nude mice injected with breast cancer MDA-MB-231 cells (C); with lung
cancer H1299 cell lines (D) cells; or with bladder cancer cells derived from T24 cells (E). Tumor volumes derived from mock treated (-)
and BTA treated (+) mice are shown. Bars represent standard deviations; asterisk (*) represents p-value <0.05 and double-asterisk (**)
represents p-value <0.01.
Human Bladder carcinoma
Tumor volume mm 3
Tumor volume in mm3
Tumor volume in mm3
Tumor volume in mm3
Tumor volume in mm3
Figure 4: Mitochondrial and metabolic effects arising from CIC inhibition. A. H1299 cells (panel set indicated as 1), or
cells expressing CIC (2) or CIC-DN (3) were stained with anti-CIC (red) or anti-mitochondrial Hsp70 (mHsp70, green) antibodies, and
counterstained with DAPI. The DAPI signal is shown only in the merged images. For these experiments cells were harvested 46 hours
after tetracycline induction. The arrow in panel A3 indicates mitochondria with enlarged and abnormal morphology (mega-mitochondria).
The next set of panels shows measurements of lactate levels (B), of complex I- (C); of mitochondrial membrane potential assessed with a
JC1 assay (MMP) (D); of ROS (E); of malate (F); and of isocitrate (G) levels. Measurements were performed in cells treated with DMSO
(indicated as control), or with BTA, or expressing CIC or CIC-DN at 16 hours after treatment or tetracycline induction, respectively. In the
case of ROS levels, panel D shows a dot plot or red (FL-2) versus green fluorescence (FL-1) of JC1 staining. In mitochondria with intact
membrane potential the JC1 dye concentrates into red fluorescent aggregates, while depolarized mitochondria are unable to concentrate
the JC1 and exhibit green fluorescence. The percentage of cells with depolarized mitochondrial membrane is shown in bold at the bottom
of each panel.
CIC mHsp70 Merge
% 2.82 %
Green fluorescence FL-1
Red fluorescence FL-2
% Substrate Consumption
Relative signal intensity
of CIC-DN was detrimental for cell growth (Figure 2D,
These results provide the first line of evidence that
inhibition of CIC hampers cancer cell proliferation, show
that CIC is a target of the anti-proliferative action of BTA
in vivo, and demonstrate the importance of the citrate
export pathway activity of CIC in regulation of growth.
Inhibition of CIC inhibits tumorigenesis in vivo,
but is non-toxic to adult normal tissues.
To explore whether CIC affects tumor development
in vivo, we next generated stable lung cancer (H1299)
and SV40 T-antigen transformed embryonic kidney
(293T) cell lines expressing tetracycline inducible CIC or
CIC-DN (Figure 2E), and thereby assessed their growth
capacity after implantation in immuno-deficient mice.
CIC dramatically accelerated the onset and size of tumors,
while CIC-DN led to a reduction of growth (Figure 2FG,
and Figure 3AB). CIC proteins were detectable in these
tumors (Figure S3A), arguing that their opposite effects on
proliferation are directly linked to changes in their activity.
Similarly to CIC-DN, BTA also exhibited anti-cancer
activity in various tumor types, including in aggressive
breast cancer cell lines MDA-MB-231, lung cancer cells
H1299 as well as in bladder cancers cell lines (Figure
3CE). The anti-tumor effect of BTA observed in MDA-
MB-231 cells as a single agent (Figure 3C), is of particular
relevance given that these cells are representative of triple
receptor negative breast tumors that are often hormone-
and chemo- resistant . During the course of these
experiments, we also noticed that BTA-treated mice
showed no evidence of illness or toxicity. Therefore,
we examined potential side effects of this compound in
non immuno-compromised animals (Figure S3CE). Mice
were randomized and BTA was administered twice a
week for five consecutive months. There was a small but
statistically significant reduction of the body weight in all
of the BTA-treated animals, but animals could be treated
for a long period of time without showing any evidence of
toxicity. Thus, we conclude that inhibition of CIC activity
is non-toxic in the adult normal mouse. By contrast, CIC
is rate limiting for tumor progression in vivo, and its
inhibition has therapeutic potential.
CIC inhibition leads to mitochondrial dysfunction,
destabilizes MMP and enhances glycolysis.
We next studied the molecular mechanisms by
which CIC affects proliferation. The function of CIC has
been primarily linked to glucose-derived fatty acid (FA)
synthesis, because of its role in citrate export [11,17].
Therefore we studied the metabolism of D-[1,6-13C2]
glucose via NMR mass spectrometry. In support of the
proposed role of CIC in promoting de novo lipogenesis,
both BTA and CIC-DN severely reduced the ability of
cells to convert glucose into FA (Figure S4AB). However,
the total FA levels, which can be synthesized also from
glycerol and amino acids, were modestly reduced at 16
hours after BTA treatment (not sown), but at later time
points they were increased (Figure S4CD). Furthermore,
incubation of BTA-treated cells with the lipid precurson,
palmitic acid was not sufficient to rescue proliferation
(not shown). These observations led us to conclude that
depletion of lipids unlikely represents the only mechanism
by which CIC inhibition affects tumor growth.
Since CIC is a mitochondrial protein we examined
experiments showed that in CIC-expressing cells the
mitochondria were highly interconnected, while in cells
harboring CIC-DN the mitochondrial network was
dispersed with fewer and fragmented mitochondria (Figure
4A, indicated by arrow; compare panel 3 with panels 1
and 2). Mitochondria structure is a direct reflection of
metabolic state, and a fragmented phenotype has often
been observed in conditions of reduced OXOPHOS and
of enhanced glycolysis, as well as a consequence of ROS
production [31,32]. This suggested that CIC influences
mitochondrial activity and glucose metabolism. Indeed,
we found that BTA enhanced the levels of glucose-derived
lactate (Figure S4B), and the total levels of lactate were
elevated in cells where CIC was inhibited (Figure 4B).
In the case of CIC-DN lactate was more prominently
increased in the media than inside the cells, perhaps
suggesting that this mutant and BTA are not exactly
identical in the way they increase glucose flux (Figure
4B; Figure S4E). Consistent with a protective role of CIC
on mitochondria activity and OXOPHOS, its inhibition
led to a decline of respiratory Complex-I activity (Figure
4C). This was accompanied by a loss of mitochondrial
membrane polarity (Figure 4D) and by the production of
ROS, which was rescued by the general ROS scavenger,
N- acetyl-cysteine (NAC) (Figure 4E).
The collapse of MMP indicated that CIC dysfunction
perturbs the proton gradient, which is directly linked to
the exchange between citrate and malate promoted by
CIC, as well as the flux of anaplerotic substrates across
the mitochondrial membrane [1,2,13]. Specifically, the
depletion of citrate induced by CIC inhibition would
predictably lower the levels of malate and isocitrate, which
are produced via citrate oxidation and isomerization,
respectively. Further, the shift of metabolism towards
glycolysis due to blocking of CIC activity may consume
pyruvate in the cytoplasm for lactate production, thus
rendering pyruvate less available for mitochondrial
metabolism. Pyruvate, citrate and isocitrate can all enter
the Krebs cycle at various steps generating reducing
equivalents for the electron transport chain, which in turn
stabilize MMP . To test this hypothesis we measured
the levels of several anaplerotic substrates in cells were
CIC was inhibited. In support of our conjecture, the levels
Figure 5: Inhibition of CIC hampers survival during metabolic stress and induces mitochondrial loss via autophagy.
A. Cell death was assessed with trypan blue exclusion in cells treated with DMSO or with BTA, in the presence or absence of 5mM
methyl-pyruvate or 2 mM NAC. ATP (B) and Oxygen consumption levels (C) in cells cultured in 5 mM glucose and mock treated (DMSO,
Control) or treated with BTA, or in cells expressing CIC or CIC-DN, 16 hours after treatments. D. Cell cycle profiles of control H1299
cells, or of cells treated with 1 mM BTA, or expressing the indicated CIC proteins, cultured in either 25 mM (upper panels) or in 2 mM
Glucose (bottom panels). The percentage of cells distributed in the G1-, S-, or G2- phases of the cells cycle is shown. Apoptotic cells are
identified as the Sub-G1 (SG) population. E. Immuno-fluorescence experiments similar to those described in Figure 4A, were performed in
cells expressing epitope tagged Flag-CIC (panels 1 to 3) or Flag-CIC-DN (panels 3 to 6) proteins. Cells were stained with anti-Flag (green,
panels 1 and 4) and anti-LC3 (red, panels 2 and 5) antibodies, and counterstained with DAPI. Merged images are shown in panels 3 and 6.
Note the intense LC3 staining in the mitochondria of panel 5. The results of these immunofluorescence experiments where confirmed with
cellular sub-fractionation experiments shown in F. Cytoplasmic (c) and mitochondrial (m) fractions derived from naïve 293T cells (lanes
1,2) or 293T cells expressing CIC (lanes 3,4) or CIC-DN (lanes 5,6) or treated with BTA (lanes 7,8), were probed with anti-CIC, anti-LC3,
or anti-mitofilin antibodies, as indicated. The two arrows in the LC3 blot indicate cytosolic (-I) or activated (-II) LC3 forms. Note the
enrichment of LC3-II in the mitochondria of cells expressing CIC-DN or BTA (lanes 6 and 8, respectively). All images shown are derived
from the same autoradiogram at identical times of exposures, but lanes in between samples of interest were eliminated. G. Time course (in
hours, indicated at the top of each panel) of BTA treated- (lanes 1 to 3) or CIC-DN induced cells (lanes 4 to 6). The anti-mitofilin, anti-p62
(employed as a general autophagic marker), anti-CIC, or anti LC3 specific immuno-blots are shown.
Figure 6: CIC protects from mitochondrial damage and depletion following respiration injury. A. Assessment with NOA
staining of mitochondrial mass as a function of time (in hours) in control cells (lanes 1 and 4), or in cells treated with BTA (lanes 2-3 and
5-6) and in the absence (lanes 1-to-3) or presence (lanes 4-to-6) of 5 mM 3MA. Double asterisks refer to p<0.05. Brackets refer to the
comparison of values expressed in 3 versus 1, or 3 versus 6. B. Measurement of mitochondrial membrane depolarization with the JC1
assay in the absence (-, lanes 1,3,5) or presence (+, lanes 2,4,6) of 20 micro-M CCCP in the cell lines indicated at the bottom of each panel.
The diagram shows histograms of JC1 green fluorescence intensity, indicative of depolarization, derived from a duplicate experiment. C.
The H1299 cell lines indicated at the bottom of each panel were left untreated (lanes 1-3) or treated with CCCP (20 micro-M, lanes 4-6)
for 16 hours, at which time CCCP was removed from the media. Cells were replenished with fresh media, incubation was carried out for
additional 2 hours, after which time cell extracts were probed with the antibodies indicated at the side of each panel. Note the increase in
the endogenous CIC levels in CCCP treated versus untreated cells (lane 4 versus lane 1, respectively). The image shown is derived from the
same autoradiogram where lanes between the samples of interest were cropped. Panel D shows a quantification of the mitofilin signal. E.
Immuno-fluorescence of H1299 cells treated with CCCP for four hours (indicated as time 0, upper panels), or after 24 hours of CCCP wash-
out. All panels in this Figure show the merged signals derived from the anti-CIC (red) and anti-Hsp70 (green) staining. Approximately 70%
of cells expressing CIC-DN showed the disrupted mitochondrial network even after 24 hours of recovery from CCCP, compared to control
H1299 or CIC-expressing cells that displayed this phenotype in less 10% of cells.
293T CIC CIC-DN
1 2 3 5 4 6
0 48h 96h 0h 48h 96h
+ + +
NOA relative intensity
1 2 3 4 5 6
293T CIC CIC-DN
Mitofilin signal intensity
2 3 4 5 6
of malate and isocitrate were reduced (Figure 4FG), and
addition to the media of pyruvate, in the form of the
membrane permeable methyl-pyruvate, partially restored
mitochondrial membrane polarity (Figure S5A), as well
as complex I activity (not shown). Very importantly,
methyl-pyruvate and NAC also partially prevented loss of
viability in cells treated with BTA (Figure 5A). Thus, we
argue that CIC preserves mitochondrial activity at least in
part by maintaining adequate levels and flux of anaplerotic
substrates, which in turn prevent depolarization of the
mitochondrial membrane, while its inhibition leads to
mitochondrial dysfunction. Interestingly, impairment
of CIC activity also re-wires the metabolism towards
glycolysis, either as a consequence of mitochondrial
dysfunction (the Warburg effect) or due to the lowered
concentration of citrate that enhances PFK1 activity (see
scheme in Figure 1A).
The ability of CIC to restrain the glycolytic
addiction of tumor cells leads to a growth
The above data indicated that CIC inhibition
exacerbates the Warburg effect, a metabolic trait that is
proposed to promote malignancy. At first glance this
result appeared paradoxical, given that CIC is required
for tumorigenesis. However, glycolysis is metabolically
advantageous when glucose is abundant, because in these
conditions it can generate ATP at faster rates compared to
OXHOPHOS . By contrast, rapidly growing tumors
surpass the ability of the microenvironment to provide
nutrients, and switch towards alternative metabolic
pathways, including OXHOPHOS, for energy production
. Therefore, we hypothesized that CIC is important
for adaptation when glucose is limiting. Likely due to
the enhanced glycolytic behavior, at high concentrations
of glucose (25 mM) oxygen consumption rates and ATP
levels were not significantly modified in cells where CIC
was inhibited compared to control cells (Figure S5BC).
However, at lower, yet physiological glucose levels (5
mM), cells expressing CIC consumed more oxygen and
produced more ATP relatively to BTA-treated or CIC-
DN -expressing cells (Figure 5BC). Cell cycle analysis
further revealed that glucose restriction led to induction of
apoptosis in BTA treated cells, while expression of CIC,
but not of CIC-DN was protective in these conditions
(Figure 5D). Collectively therefore, these results provide
for a model whereby CIC restrains the excessive reliance
upon glycolysis allowing tumor cells to shift towards
OXPHOS for ATP production, ultimately favoring survival
when glucose is limiting.
CIC is induced by mitochondrial respiration
injury and prevents mitochondrial depolarization
The mitochondria of tumor cells undergo oxidative
stress due to mtDNA and to oncogenic mutations that
affect the activity of mitochondrial enzymes involved
in respiration [21,22,23]. Mitochondrial damage in turn
triggers quality control systems consisting of mitochondria
degradation via autophagy/mitophagy [34,35]. In keeping
with our previous results we suspected that CIC might
exert a protective role on mitochondrial damage and
disposal. First, immuno-fluorescence and cellular sub-
fractionation experiments demonstrated recruitment of the
autophagic machinery in the form of the active- lipidated
form of LC3 and of lysosomes, to the mitochondria of
cells expressing CIC-DN or treated with BTA, but not in
CIC containing cells (Figure 5E, compare panels 2 and
5; Figure 5F, compare lanes 6 and 8 with lanes 2 and
4; Figure S6AB). Furthermore, kinetic studies where
mitochondrial content was assessed with the marker of
mitochondrial mass, mitofilin [36,37], showed a reduction
of mitochondrial quantity as a function of time, following
BTA treatment or CIC-DN induction (Figure 5G, compare
lanes 2 and 3, and lanes 5 and 6 with lanes 1 and 4,
respectively). This mitochondrial depletion coincided with
activation of autophagy, as judged based on LC3 activation
and conversion (Figure 5G, lower panel). Similar results
were obtained when mitochondrial amount was studied
with a second marker of mitochondrial content, namely
Nonyl-Acridine Orange (NOA)  (Figure 6A, compare
lanes 2 and 3 with lane 1). Blocking of autophagy with
3-Methyladenine (3MA), restored mitochondrial amount
(Figure 6A, compare lanes 5 and 6 with lanes 2 and 3),
thus suggesting clearance of these mitochondria by
mitophagy/autophagy. Importantly, 3MA also prevented
cell death induced by BTA in glucose-restricted conditions
Autophagy can be organelle selective or proceed
as a bulk degradation process of multiple cellular
compartments. CIC did not interfere with global
autophagic flux in response to the mTOR inhibitor,
rapamycin, or to glucose restriction, suggesting that
it might specifically affect mitophagy (Figure S6D).
To test this hypothesis further, we employed the
mitochondrial respiration uncoupler carbonyl-cyanide-
depolarization of the mitochondrial membrane and
mitochondria fragmentation followed by mitophagy
[i.e., 37]. Cells where CIC was abundant were protected
from MMP loss induced by CCCP, while expression of
CIC-DN destabilized MMP independently of CCCP and
failed to restore mitochondrial membrane polarity (Figure
6B, compare lanes 3 and 4 with lanes 1 and 2; and lane
6 with lane 4, respectively). Furthermore, in conditions
CCCP, which induces
in which CCCP was washed out from the media after
treatment, H1299 and CIC-expressing cells maintained
stable mitofilin content (Figure 6C, compare lanes 4 and
5 with lanes 1 and 2; quantified in Figure 6D) as well as
normal mitochondrial structure and morphology (Figure
6E). The protection seen in naïve H1299 cells correlated
with an induction of the expression levels of endogenous
CIC by CCCP (Figure 6C, compare lane 1 versus lane 4
in the CIC immuno-blot). Other types of mitochondrial
stressors, such as rotenone, also induced CIC expression
levels (Figure S6E).
Viewed as a whole, these results indicate that CIC
is a sensor and an effector of mitochondrial stress. Via its
citrate export ability, CIC stabilizes membrane potential
minimizing mitochondrial depletion. By contrast, cells
where CIC is inhibited trigger autophagic clearance of
The knock-down of the CIC orthologous gene
in zebrafish induces mitochondrial loss and
activation of autophagy.
Genes that promote tumorigenesis are often
necessary for embryonic development, and some of these
genes are metabolic modulators, given that embryogenesis
and tumorigenesis are
[39,40]. Additionally, in keeping with the hypothesized
involvement of CIC in chromosome 22q11.2 microdeletion
syndromes in humans , we deemed important to
Figure 7: The knock-down of CIC in zebrafish results in mitochondrial DNA depletion and activation of autophagy.
A. Zebrafish embryos injected with control morpholino (Control MO) or with three different doses of the CIC specific morpholino (CIC
MO), resulting in a progressively abnormal phenotypes classified from mild to severe. B. Phenotypes of zebrafish injected with control
or CIC MO at 4 days post-development. Images were captured at identical magnification. C. Immuno-blot of pools of 15-30 embryos of
each phenotype shown in A, with antibodies listed at the side of each panel. Two different exposures of the CIC-specific immuno-blot are
shown. Like in human cell extract, in zebrafish extracts the two main LC3 forms, I and II, could be detected and are indicated by arrows. D.
DNA extracted from pooled embryos representative of each phenotype was probed in semi-quantitative PCR by using primers that amplify
either 1Kb of mtDNA encompassing the mitochondrial gene NADH-dehydrogenase (NADH-D; top panel), or the nuclear p53 gene (bottom
panel). E. Survival of embryos injected with control MO or with CIC-MO grown in media containing (+) or lacking (-) 5 mM 3MA. The
percentages of each phenotype classified as normal, slight, severe and dead are indicated by bars of different colors.
Control MO CIC MO - mild
CIC MO - intermediate CIC MO - severe
Phenotype variations (%)
investigate whether changes of CIC dosage affects
embryogenesis in the model organism zebrafish. This
approach was possible because human and zebrafish CIC
proteins display a high degree of homology (Figure S7).
By using a morpholino-based (MO) knock-down strategy,
we found that the CIC MO led to a stark dose-dependent
phenotype, and produced a relatively small percentage of
dead embryos (34%, Figure 7A, Figure 7E). At 24 hours
post-fertilization the head was flattened and reduced along
the ventral-dorsal axis and at later steps of development
CIC morphants displayed a prominent reduction of the
entire cranial region, involving the size of the brain and
the jaw. The heart was small and surrounded by pericardial
edema, indicative of cardiac dysfunction (Figure 7B).
Confirming the efficiency of the knockdown, a progressive
decline of the expression levels of CIC was seen with
increasing doses of MO, paralleled by a reduction in the
levels of mitofilin and of mitochondrial, but not nuclear
DNA (Figure 7C and Figure 7D, respectively). Similarly
to what observed in human tumor cells, LC3 total levels
and the conversion to the lipidated form were starkly
increased (Figure 7C, compare lanes 2 to 4 with lane 1 in
the LC3 immunoblot), and the phenotypes of the CIC-MO
were largely rescued by 3MA (Figure 7D). Noticeably,
CIC morphants recapitulated features of VCFS, which is
characterized by multiple abnormalities including cardiac
defects, facial and jaw anomalies and cleft palate .
The effects of CIC on the development of the brain as
well as on the mitochondria could also explain the delay in
cognitive functions and the learning disabilities that occur
in some forms of this disease. Together with other gene
products within chromosome 22q11.2, CIC deficiency
might therefore contribute to- or act as a modifier of- the
complex spectrum of pathological features occurring in
human velo-cardio-facial syndromes.
In this study, we have conducted, for the first
time, a functional characterization of the mitochondrial
Figure 8: CIC maintains mitochondrial homeostasis in tumor cells. The diagram summarizes the findings (see also the
Discussion). The mitochondria of tumor cells endure oxidative and respiration stress caused by the nutrient restricted microenvironment,
by oncogenic activation, and by altered activity of mitochondrial enzymes. We have shown here that high CIC levels in tumors allows for
adaptation to nutritional stress and resistance to mitochondria respiration injury. This protective effect of CIC on the tumor mitochondria
relies upon an inhibition of glycolysis, promotion of mitochondrial OXOPHOS and ATP production, and stabilization of the mitochondrial
membrane potential (MMP). We have mechanistically linked these activities of CIC to its ability to promote the export of citrate and to
maintain adequate levels and flux of other anaplerotic substrates. By contrast, genetic or pharmacologic inhibition of CIC increases ROS
levels, compromises respiratory capacity and leads to mitochondrial dysfunction and depletion via autophagy, preventing adaptation to
oxidative or respiration stress, and ultimately leading to tumor killing.
ADAPTATION TO STRESS
HYPERSENSITIVITY TO STRESS
transporter CIC, a gene product highly conserved
throughout evolution from yeast to humans. Our findings
have identified CIC as an important determinant of the
homeostatic control of tumor mitochondria, through
which activity CIC becomes essential to the cancer
promoting metabolic program (depicted in Figure 8).
We have shown that high CIC levels in tumors preserve
a critical threshold of mitochondrial activity and amount
that allows adaptation during metabolic and respiration
stress. Importantly, embryonic tissues and tumor cells
appear selectively sensitive to CIC inhibition compared
to adult normal tissues. This sensitivity may be due to the
already high extent of oxidative damage, of ROS, and of
mitochondrial stress due to differentiation programs during
development  and to oncogenic signal pathways
in tumors respectively, which could then reach levels
incompatible with proliferation when CIC is inhibited.
Importantly, new evidence now demonstrates the synthetic
lethal activity of compounds that increase ROS production
in various tumors . These agents achieve tumor killing
with a remarkable degree of selectivity.
Based on these considerations, we believe that the
CIC inhibitor compound, BTA, provides a reasonable
platform for the design of a new class of agents that
target mitochondrial metabolism and turnover. There
are currently few examples of drugs that exploit the
intrinsic vulnerability of tumor mitochondria to achieve
selective cancer killing  and the development of
such agents might improve therapeutic responses .
Given the inability of cells where CIC is dysfunctional
to tolerate nutrient restriction, it can also be predicted
that CIC inhibitors might act synergistically with drugs
that interfere with glucose metabolism, for example with
2-deoxyglucose or metformin, that are currently being
evaluated in cancer therapy . Therefore, our results
should provide a rationale for testing of CIC inhibitors,
alone or in combination with other metabolic modulators,
as anti-cancer agents.
Albeit the fact that CIC inhibition exacerbates
the Warburg effect and yet has anti-tumor activity may
appear paradoxical, there are several explanations for this
observation. Based on our findings, we propose that CIC
functions to balance the potential detrimental effects that
arise when the excessive dependence upon glycolysis of
tumor cells overpowers the ability of the environment
to adequately supply nutrients, and/or if the extent of
oxidative stress endured by tumor cells reaches levels
such to trigger excessive degradation via mitophagy .
However, given the incredible plasticity of tumor cells
and their ability to adapt to metabolic pressure, it will be
very important to interrogate the long term effects of the
anti-tumor activity of BTA in many other tumor types of
different tissue origin as well as in tumor prone animal
Based on data presented here and available in
literature, it further appears reasonable to predict the
involvement of CIC in other human pathogenic conditions
hallmarked by mitochondrial oxidative damage and
by alterations of mitochondrial turnover. Aside from
chromosome 22q11.21 microdeletion syndromes that alter
mitochondrial plasticity , mitochondrial homeostasis
is perturbed in human disease states ranging from
diabetes, neurodegeneration and aging. Interestingly, an
analysis of existing gene expression databases derived
from Parkinson’s disease, shows statistically significant
alterations of CIC expression levels in various regions
of the brain of affected patients relatively to normal
healthy control individuals (http://www2.cancer.ucl.
and region=1 and region=1 and region=1 and choice=slist
and list=SLC25A1 and Pvalue=0.05 and LogFC= and
NegLogFC= and AdjPvalue=). A common denominator
of neurodegeneration, aging and cancer consists in
alterations of mitochondrial activity and turnover [47, 48].
Importantly, in the fruit fly a member of the tricarboxylate
transporter family, INDY, promotes longevity .
Thus, in addition to being detrimental to tumor growth,
CIC inhibition might -perhaps advantageously- interfere
with other life-shortening conditions. Based on these
considerations, it is possible that CIC is central to many
pathways linked to human health and thus, targeting of
its activity, positively or negatively, could be beneficial to
Cells, reagents, antibodies, primers.
The cell lines employed in this study were obtained
from the tissue culture core facility at LCCC. H1299
cells were obtained from ATCC. Cells were grown in
Dulbecco’ s modified Eagle’s medium (DMEM, 5 mM
glucose, with glutamine and pyruvate from Invitrogen)
and supplemented with 10% fetal calf serum (FCS).
Early passage cell lines or frozen pellets were used for
screening of CIC expression levels (Fig.S1 and Fig.1A).
The normal and tumor breast cancer specimens employed
for CIC analysis were obtained from the Histopathology
and Tissue Shared Resource, Georgetown University
Hospital. All the reagents for the study of complex I
activity were purchased from Sigma-Aldrich and were
as follows. Succinate: #S3674-100G; KCN: #60178-
25G; Coenzyme Q1: #C7956-10MG; Decylubiquinone:
D1878-5G; Thenoyltrifluoroacetone: 88300-5G; rotenone:
#R8875; NADH: #N4505. 1,2,3, Benzenetricarboxylica
acid (BTA), was from Sigma (# B4201). The CIC
specific shRNA vectors were purchased from Origene
(#TG316728 and #TR316728, untagged and GFP-tagged).
The vectors expressing human CIC untagged or Flag-Myc
epitope tagged were also from Origene (#SC120727 and
RC200657, respectively). The antibodies used in this
study were as follows. The anti-CIC antibody from Santa
Cruz Biotech, # sc-86392 employed at 1:1000 dilution in
immuno-blot and at 1:100 in immuno-fluorescence; the
anti-mHsp70 and anti-mitofilin antibody were from Novus
Biological (#NB300-527 and #NB100-1919, respectively)
employed at 1:500 in immuno-fluorescence and 1:1000 in
immuno-blot; for the LC3 immuno-blot a mixture of two
antibodies was used each at 1:1000 dilution (LC3 MBL
#PM036; LC3 Novus Biol. # NB100-2220). The LAMP-
1 antibody was from Abcam (#H4A3). The sequences
of primers used were: the human cytochome c oxidase:
The zebrafish NADH-dehydrogenase:
The zebrafish p53:
Strategy for the generation of CIC wild-type and
CIC mutant expressing vectors, and of stable cell
The two cDNA clones expressing human untagged
CIC and epitope tagged CIC were cloned into the
pcDNA4/TO tetracycline regulated vector (Santa Cruz,
T-Rex system). Stable tetracycline inducible cell lines
were obtained as described previously.
Quantification of metabolites, ATP, oxygen, ROS,
MMP and mitochondrial mass.
The concentration of citrate, lactate, isocitrate,
and malate were assessed using specific kits from
BioVision. Cellular ATP levels were determined via the
ATP kit (Promega), while oxygen consumption rates
were measured using the BD oxygen biosensor systems
(OBS) from BD Bioscience. Triplicate samples of 50,000
cells were seeded onto 96-well OBS plates. The number
of cells was determined at each time point by sampling
cells seeded into side-by-side plates. Fluorescence was
measured from the bottom of the well every 24 h and
measurements were normalized by subtracting the reading
from the same well prior to the addition of the cells
(blank). For measurement of ROS we used H2DCFDA
(Invitrogen). Mitochondrial mass was assessed by using
mitotracker green (Invitrogen), as per manufacturer
instructions, or Nonyl Acridine Orange at a final
concentration of 300 nM, followed by analysis by flow
cytometry. Mitochondrial Membrane Potential (MMP)
was studied with the JC1 assay kit from Cayman.
Mice and tumors.
To produce tumor xenografts 5 × 106 cells were
resuspended in PBS and injected subcutaneously in the
flanks of female nude mice. For drug treatments mice were
randomized to receive either PBS or a PBS solution of
BTA at a concentration 26 mg/kg which was administered
via intra-peritoneal route three times a week. Mice were
pre-treated twice prior to the inoculation of tumor cell
lines. Once detectable tumors started to form, their size
was measured with a caliper in three dimensions. Serial
measurements were made at two-three day intervals after
the identification of the initial cellular mass to determine
growth curves in vivo. Tumor volume was calculated
using the formula for a prolate spheroid: volume = (4/3) x
a2b, where a is the width and b is the length. All animals
were sacrificed when the tumors exceeded 1.5 cm. At the
completion of experiments, tumors were excised, weighed
and statistical significance of differences in tumor volume
were made using two factor repeated measures analysis of
variance followed by Fisher’s last significant difference
test for multiple comparisons. The trial administration of
BTA to explore its toxic effects, was conducted on 8 non
immuno-compromized mice, randomized in two groups
and injected as described before for five consecutive
months. Animals were monitored once a week for the
presence of signs of disease, particularly neurological
disturbances or weight loss, and they were weighted
periodically. All animal studies were approved by the
Georgetown University Institutional Animal Care and Use
Data are expressed as means ± standard deviations
(SD). The two-tailed Student t test was used for all
statistical analysis of experiments presented and Excel was
used for statistical calculations. Significant differences are
indicated using the standard Michelin Guide scale (P <
0.05, significant; P < 0.01, highly significant; P < 0.001,
Additional materials and methods are provided as
This paper was accepted based in part on extensive
peer-review in another journal as well as extensive
editorial peer-review in Oncotarget.
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