Cytosolic acetyl-CoA synthetase affected tumor cell
survival under hypoxia: the possible function intumor acetyl-CoA/acetate metabolism
Blackwell Publishing Asia

Yukie Yoshii,1 Takako Furukawa,2 Hiroshi Yoshii,3 Tetsuya Mori,1 Yasushi Kiyono,1 Atsuo Waki,1 Masato Kobayashi,1
Tetsuya Tsujikawa,1 Takashi Kudo,1 Hidehiko Okazawa,1 Yoshiharu Yonekura2 and Yasuhisa Fujibayashi1,2,4
1
Biomedical Imaging Research Center, University of Fukui, 23–3, Shimoaizuki, Eiheiji, Fukui 910-1193; 2National Institute of Radiological Sciences, 4-9-1,
Anagawa, Chiba 263-8555; 3Department of Biochemistry and Bioinformative Science, Faculty of Medical Sciences, University of Fukui, 23-3, Shimoaizuki, Eiheiji,
Fukui 910-1193, Japan(Received September 7, 2008/Revised December 21, 2008/Accepted January 5, 2009/Online publication February 17, 2009)

Understanding tumor-specific metabolism under hypoxia is importantto find novel targets for antitumor drug design. Here we found thattumor cells expressed higher levels of cytosolic acetyl-CoA synthetase(ACSS2) under hypoxia than normoxia. Knockdown of ACSS2 by
RNA interference (RNAi) in tumor cells enhanced tumor cell deathunder long-term hypoxia in vitro. Our data also demonstrated that
the ACSS2 suppression slowed tumor growth in vivo. These findings
showed that ACSS2 plays a significant role in tumor cell survival
under hypoxia and that ACSS2 would be a potential target for
tumor treatment. Furthermore, we found that tumor cells excreted
acetate and the quantity increased under hypoxia: the pattern ofacetate excretion followed the expression pattern of ACSS2.
Additionally, the ACSS2 knockdown led to a corresponding reductionin the acetate excretion in tumor cells. These results mean that
ACSS2 can conduct the reverse reaction from acetyl-CoA to acetate
in tumor cells, which indicates that ACSS2 is a bi-directional enzyme
in tumor cells and that ACSS2 might play a buffering role in tumor
acetyl-CoA/acetate metabolism. (Cancer Sci 2009; 100: 821–827)Tumor hypoxic regions have been recognized to be connectedwith poor outcome during tumor treatment, such as radiotherapy and chemotherapy.(1–4) In radiotherapy, oxygen enhances
the damage to tumor tissue resulting from exposure to ionizing
radiation, whereas the lack of oxygen reduces the damage. In thecase of chemotherapy, hypoxia-induced changes in tumor cells
make some antitumor drugs ineffective. Therefore, understanding
the characteristics of tumor cells under hypoxia is of significant
importance to find beneficial targets for tumor treatment.
Metabolic change is one of the important aspects in theelucidation of tumor hypoxia. In a tumor mass, hypoxic regions
inevitably occur because blood vessel supply cannot keep up
with the high rate of tumor cell proliferation. In order to adapt
to such mass environment, tumor cells are considered to havespecific metabolic characteristics known as the Warburg effect.(5)This refers to a general metabolic change in tumor cells thatcauses glycolysis to be used to generate energy in the place of
mitochondrial respiration, not only under hypoxia but also
normoxia.(6–10) However, the metabolic status and related gene
expression under hypoxia in tumor cells is not fully understood.
Acetyl-CoA synthetase (ACSS) (EC 6.2.1.1: adenosine triphosphate [ATP)] + acetate + coenzyme A [CoA] ↔ adenosine monophosphate [AMP] + diphosphate + acetyl-coenzyme A [CoA]) is awell-conserved universal enzyme from bacteria to humans.(11) It
has been reported that the activity of ACSS can be controlled
by acetylation on lysine residues and that this regulatory mechanism is also conserved from bacteria to humans.(12,13) Mammals
have two types of ACSS: mitochondrial ACSS1 and cytosolic
ACSS2.(14) The enzyme ACSS is generally perceived to play adoi: 10.1111/j.1349-7006.2009.01099.x© 2009 Japanese Cancer Associationrole in the incorporation of acetate into acetyl-CoA, via the forward
reaction, which is then used in the synthesis of fatty acids andamino acids, as well as in the tricarboxylic acid (TCA) cycle invivo.(14–17) On the other hand, the enzymatic reversibility of
ACSS has been reported previously from several sources, suchas mammals, yeast, plants, and bacteria.(18–22) Moreover, in yeast
and a fungus Aspergillus nidulans, it has been demonstrated thatthe ‘cytosolic’ ACSS can mediate the reverse reaction fromacetyl-CoA to acetate under the conditions of insufficient oxygen supply and would play an important role in their anaerobic
survival.(23–28) Based on these facts, we speculated that ACSS
expressed in tumor cytosol (ACSS2) might be also important intumor cell survival under hypoxia.
Here, we found that ACSS2 gene expression increased underhypoxia in tumor cells and that ACSS2 inhibition strengthened
tumor cell death under hypoxia in vitro and reduced tumor
growth in vivo. In addition, our study revealed that tumor cells
showed acetate excretion, especially under hypoxia, which was
mediated by ACSS2. Thus, this study demonstrated the significant role of ACSS2 in tumor cell survival under hypoxia andthe possible function of ACSS2 in tumor metabolism.
Materials and methods
Cell lines and growth. The following four tumor cell lines
were used in this study: LL/2 (LLC; CRL-1642, American Type
Culture Collection, ATCC, Manassas, VA, USA) mouse Lewis
lung carcinoma; B16 (RCB1283; Riken Cell Bank, Tsukuba,
Japan) mouse melanoma; Colon-26 (Colon; TKG 0518, Cell
Resource Center for Biomedical Research, Tohoku University,
Sendai, Japan) mouse colon carcinoma; and C127I (CRL-1616;
ATCC) mouse mammary carcinoma. Tumor cell lines were chosenthat had different growth rates and were derived from different
tissues (see Supporting Information, Fig. S1). Before experiments,
we confirmed metabolic characteristics of high glycolysis andlow electron transport activity in mitochondrial respiration under
normoxia, in these tumor cell lines used in this study(Figs S2,3). As reference, the mouse fibroblast cell line BALB/
3T3 clone A31 (3T3; CCL-163, ATCC) was used for gene
expression analysis and measurement of metabolites. The cells
were incubated in a humidified atmosphere of 5% CO2 in air(normoxia) at 37°C. Dulbecco’s modified Eagle’s medium(Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal
bovine serum and antibiotics was used as the cell growth
medium. Exponentially growing cells were used. Hypoxia

4

To whom correspondence should be addressed. E-mail: yfuji@u-fukui.ac.jp

Cancer Sci | May 2009 | vol. 100 |no. 5 | 821–827

(1.2% O2, 93.8% N2, 5% CO2) was achieved with a Personal
Multi Gas Incubator (Astec, Fukuoka, Japan). The cells weretrypsinized to detach them from the plates and were counted by
the trypan blue dye-exclusion method.
Analysis of gene expression. Total RNA was isolated fromaliquots of 1–6 × 106 cells with a Micro-to-Midi total RNA
purification system (Invitrogen). Quantitative reverse transcription– polymerase chain reaction (qRT-PCR) was performed with
Taqman One-Step RT-PCR Master Mix reagents and Taqman
gene expression assays (Applied Biosystems, Foster City, CA)
using an ABI PRISM 7000 sequence detection system (Applied
Biosystems). The Taqman PCR assays were carried out in total
reaction volumes of 25 μL containing 12.5 μL 2 × Master Mix,
0.625 μL 40 × MultiScribe and RNase inhibitor mix, 1.25 μL
20 × Taqman gene-expression assay and 0.5 μg purified total
RNA as templates. The following Taqman gene-expression assays
were used: acetyl-CoA hydrolase (ACH), Mm00499641; ACSS1,
Mm00475647; ACSS2, Mm00480101; and β-actin, Mm00607939(Applied Biosystems). PCR was carried out for 30 min at 48°C
and 10 min at 95°C, followed by 40 cycles at 95°C for 15 s andat 60°C for 60 s. Target mRNA was quantified by the comparative
CT method using β-actin expression as the endogenous control.(29)
RNA interference (RNAi) experiments. To inhibit ACSS2 expression, tumor cells were transduced with lentiviral particles (Mission
lentiviral transduction particles; SHVRS-NM 019811; Sigma,
Poole, UK) carrying expression cassettes encoding single-hairpin
RNAs (shRNAs) that generate small-interfering RNAs (siRNAs).
Non-targeting shRNA (SHC002V, Sigma) was used as a negative
control.(30,31) Cells (1.6 × 104) were plated in 100 μL growth
medium per well on 96-well plates and incubated overnight. The
medium was then changed to growth medium containing hexadimethrine bromide (Sigma) at a final concentration of 8 μg/mL
to enhance the transduction efficiency. Lentiviral particles(5 μL) were added and the plates were incubated overnight.
Thereafter, we selected stable transductants expressing the shRNAs
with puromycin. Levels of mRNA expression of ACSS2 were
examined by qRT-PCR to check the efficiency of knockdown by
RNAi.
Cell survival under hypoxia. The RNAi tumor cells were plated
in 24-well plates at 5 × 105 per well in 1 mL of growth medium,and incubated for 24 h at 37°C under normoxia. In thisexperiment, we optimized the number of seeding cells to adjust
to long-term hypoxic incubation. After changing the medium,the cells were transferred to hypoxic conditions. Fresh medium
preincubated under hypoxic conditions was provided daily. The
cells were counted by the trypan blue dye-exclusion method
every 24 h from the time when they were transferred to hypoxia(day 0) for 7 days.
Allografts of RNAi tumor cells. All animal experiments andprocedures were conducted in compliance with the Animal
Treatment Regulations of the University of Fukui, Japan andthese regulations obey domestic and international guidelines andlaws. BALB/c Slc-nu/nμ male nude mice (6 weeks of age) wereobtained from Japan SLC (Shizuoka, Japan), and were maintainedin a pathogen-free environment throughout the experiments.
Before the experiments, the mice were acclimatized for at least
1 week. RNAi tumor cells suspended in phosphate-bufferedsaline (PBS) (at 8 × 106 for Colon and 1 × 107 for the other celllines) were injected subcutaneously into the flanks of the mice.
The tumor size was measured with precision callipers every 3days over a period of 12 days. The tumor volume was calculatedusing the method reported by Janik et al.(32)
Measurement of metabolites. The cells were plated in 24-wellplates at a density of 5 × 104 in 1 mL growth medium per welland were preincubated for 24 h at 37°C under normoxia. The
cells were incubated for a further 24 h in fresh growth medium
at 37°C under normoxia or hypoxia. The supernatants werecollected to measure metabolite levels, and the cell numbers
822

Fig. 1. Gene-expression profiles of cytosolic acetyl-CoA synthetase(ACSS2). Levels of messenger RNA (mRNA) for ACSS2 relative to β-actin
as the endogenous control are shown. The values were normalized to
ACSS2 expressed in 3T3 non-tumor fibroblast cells under normoxia.
Quantitative reverse transcription – polymerase chain reaction was
performed using RNA isolated from cells cultured under normoxia (N,white bar) and hypoxia (H, black bar). ACSS2 expression under
normoxia was high in the tumor cells compared with the 3T3 cells(P < 0.01 in LLC, P < 0.001 in B16 and C127I, and P < 0.02 in Colon cells)
and increased in tumor cells under hypoxia (P < 0.0001 in LLC and B16,
P < 0.001 in Colon, and P < 0.02 in C127I cells) but not in 3T3 cells(P > 0.05).

were counted. Quantification of acetate and lactate was done by
enzymatic analysis using an F-kit (R-Biopharm AG, Darmstadt,
Germany) in accordance with the manufacturer’s instructions.
The growth medium itself did not contain acetate and lactate.
Statistical analysis. Data are expressed as the means andstandard deviations. P-values were calculated by the two-sided
t-test. P-values < 0.05 were considered statistically significant.
For cell studies, values from four independent experiments arepresented. For the animal studies, the number of animals in eachgroup is indicated by n.
Results
Hypoxia causes increase of cytosolic ACSS2 gene expression levels
in tumor cells. We found that the expression of transcripts encoding

cytosolic ACSS2 increased under hypoxia in the examined fourtumor cell lines (Fig. 1). The expression levels of ACSS2 was
1.8-, 2.0-, 3.3-, and 2.6-fold higher in LLC, B16, Colon, andC127I tumor cells, respectively, under hypoxia, compared withnormoxia. Under normoxia, LLC, B16, Colon, and C127I tumor
cells contained 1.5-, 1.9-, 2.4-, and 2.2-fold more ACSS2 transcripts, respectively, than 3T3 fibroblast cells as 3T3 fibroblast
cells did not show significant increase of ACSS2 expression
under hypoxia. The expression of mitochondrial ACSS1 and ACH(EC 3.1.2.1; acetyl-CoA + H2O ↔ CoA + acetate), another relating
enzyme to acetyl-CoA/acetate metabolism, was very low regardless of oxygen conditions (data not shown).
ACSS2 knockdown by shRNA affects tumor cell survival underhypoxia in vitro. To examine the significance of ACSS2 in tumor

cells under hypoxia, we performed cell-culture studies using
ACSS2-RNAi and control-RNAi tumor cells established in thisstudy. In the ACSS2-RNAi tumor cells, the expression of
ACSS2 decreased compared with control-RNAi tumor cells inall the tumor cell lines examined (Fig. 2). It was found that the
ACSS2-RNAi tumor cells exhibited considerably lower survival
than the control-RNAi tumor cells under long-term hypoxia in
doi: 10.1111/j.1349-7006.2009.01099.x© 2009 Japanese Cancer Association

Fig. 2. Suppression of messenger RNA (mRNA) for cytosolic acetyl-CoA
synthetase (ACSS2) by RNA interference (RNAi) in tumor cells. Asterisks
indicate statistical significance (*P < 0.01; **P < 0.005; ***P < 0.001).
ACSS2 mRNA expressions detected by quantitative reverse transcription– polymerase chain reaction analysis in ACSS2-RNAi tumor cells (whitebar) and control-RNAi tumor cells (black bar) are shown. These data
were expressed as relative values, compared with control-RNAi tumor
cells (%).

Fig. 3. Cytosolic acetyl-CoA synthetase (ACSS2) promotes tumor cell
survival under hypoxia in vitro. Cell survival under long-term hypoxic
incubation in vitro in control-RNA interference (RNAi) tumor cells (solidline) and ACSS2-RNAi tumor cells (dotted line). Asterisks indicate
statistical significance (*P < 0.01; **P < 0.001).

vitro (Fig. 3). The number of cells was not significantly different
on day 1 after hypoxic treatment. After 2 days of hypoxia, thenumber of ACSS2-RNAi tumor cells, but not control-RNAitumor cells, decreased dramatically in all the tumor cell lines
examined. The ACSS2-RNAi tumor cells examined were almost
Yoshii et al.

Fig. 4. Cytosolic acetyl-CoA synthetase (ACSS2) enhances tumor
growth in vivo. Growth of tumors derived from control-RNAinterference (RNAi) tumor cells (solid line) and ACSS2-RNAi tumor cells(dotted line) in vivo; LLC, Colon, and C127I (n = 6), and B16 cells (n = 5).
Asterisks indicate statistical significance (*P < 0.03; **P < 0.02;***P < 0.01).

dead after 3 days of hypoxia, whereas the control-RNAi tumor
cells survived. Incidentally, ACSS2-RNAi and control-RNAitumor cells showed no significant difference in growth under
normoxia (data not shown).
ACSS2 knockdown inhibits tumor growth in vivo. Next, we investigated the effect of ACSS2 knock down in tumor growth in vivo
with the RNAi tumor cells (Fig. 4). The results demonstrated
that all the ACSS2-RNAi tumors grew more slowly than thecontrol-RNAi tumors. At 12 days after injection, the volumes of
the tumors derived from the ACSS2-RNAi tumor cells were 2.9-,
5.6-, 2.9-, and 4.6-fold smaller than those from the control-RNAitumor cells for LLC, B16, Colon, and C127I cells, respectively.
Hypoxia enhances acetate excretion by tumor cells. Figure 5
showed quantification of acetate and lactate released into theculture media from cells. It was found that the pattern of acetateexcretion resembled the pattern of ACSS2 expression (Fig. 5a;compare with Fig. 1). That is, the excretion of acetate by tumor
cells was increased under hypoxia along with up-regulation of
ACSS2 expression. The values of acetate excretion underhypoxia were 1.3-fold, 1.9-, 1.7-, and 1.6-fold larger in LLC,
B16, Colon, and C127I cells, respectively, than under normoxia.
Under normoxia, tumor cells produced larger amounts of acetate
than the 3T3 fibroblast cells (Fig. 5a). 3T3 fibroblast cells did
not show any increase of acetate excretion under hypoxia. Also,
lactate excretion under hypoxia by tumor cells was largely
increased 8.1-fold, 2.7-, 3.5-, and 3.7-fold in LLC, B16, Colon,
and C127I cells, respectively, compared with normoxia, while
3T3 fibroblast cells showed only 1.9-fold increase underhypoxia (Fig. 5b).
Knockdown of ACSS2 expression by shRNA in tumor cells leads
to a corresponding reduction of acetate excretion. To verify the

involvement of ACSS2 in tumor acetate production, we quantified
acetate release from ACSS2-RNAi and control-RNAi tumor cells
established in the present study. Acetate excretion was measured
in the RNAi cells cultured under normoxia and hypoxia (Fig. 6a,b).
There was a reduction of acetate excretion in ACSS2-RNAi tumor
cells, compared with control-RNAi tumor cells, corresponding to
the decrease of ACSS2 expression, under normoxia and hypoxia,
Cancer Sci |May 2009 | vol. 100 | no. 5 | 823© 2009 Japanese Cancer Association

Fig. 5. Quantitative analysis of acetate and lactate. Measurements of metabolites released into the culture media of tumor cells (LLC, B16, Colon, andC127I) and non-tumor fibroblast cells (3T3) under normoxia (N, white bar) and hypoxia (H, black bar) are shown. (a) Amounts of acetate released per
1 × 104 cells. Acetate excretion in tumor cells was higher than in 3T3 cells under normoxia (P < 0.02 in LLC, P < 0.01 in B16, P < 0.005 in Colon, and P < 0.01
in C127I cells). Under hypoxia, acetate excretion increased in B16 (P < 0.01), Colon and C127I (P < 0.005), but not in 3T3 (P > 0.05). (b) The amounts oflactate released per 1 × 104 cells. Lactate excretion under hypoxia increased in all the examined cell lines, compared with normoxia (P < 0.001).

Fig. 6. Knockdown of cytosolic acetyl-CoA synthetase (ACSS2) expression leads to a corresponding reduction of acetate excretion in tumor cells. Reductionof acetate excretion under normoxia (a) and hypoxia (b) in ACSS2-RNA interference (RNAi) tumor cells (white bar) compared with control-RNAi tumor
cells (black bar). These data were expressed as relative values compared with control RNAi (%). Asterisks indicate statistical significance (*P < 0.005;**P < 0.001).

respectively (Fig. 6; compare with Fig. 2). This tendency was similarin all the tumor cell lines examined (LLC, B16, Colon, and C127I).
Discussion

Tumor hypoxia has been known to be an obstacle for traditional
tumor treatment, such as radiotherapy and chemotherapy.(1–4)
824Hence, understanding of the properties of tumor cells underhypoxia, including specific gene expression and metabolicstatus, is of significant importance to develop novel antitumor
strategies targeting tumor hypoxia. Here we found that gene
expression of cytosolic ACSS (ACSS2) in tumor cells wasincreased under hypoxia compared with normoxia and that
ACSS2 knockdown results in shortage of tumor cell survival
doi: 10.1111/j.1349-7006.2009.01099.x© 2009 Japanese Cancer Association

under long-term hypoxia in vitro. This means that ACSS2 plays
an important role in tumor cell survival under hypoxia and that
ACSS2 would be a potential target to discover novel strategies
against tumor hypoxia.
In addition, we revealed that ACSS2 knockdown inhibitedtumor growth in vivo, which indicates that ACSS2 is also significant in tumor growth. So far, it is shown that tumor hypoxia
frequently occurs in tumor mass and the tumor hypoxia isconsidered to be caused by deficiency in blood vessel supply
and shifting during active proliferation of tumor mass.(33–36) To
understand the role of ACSS2 in tumor growth in vivo, further
studies on the exact relationships between ACSS2 expression
and hypoxic regions shifting within tumors would be necessary.
The present study revealed that tumor cells excreted acetate
and the quantity was increased under hypoxia, compared withnormoxia, that is, the pattern of acetate excretion followed thepattern of ACSS2 expression. Also, the ACSS2 knockdown intumor cells led to a corresponding reduction of the acetateexcretion by tumor cells. These evidences indicate that ACSS2
is responsible for the acetate production via the reverse reaction
in tumor cells; namely, ACSS2 could be a bi-directional enzyme
in tumor cells (Fig. S4).
So far, it is generally accepted that ACSS mediates the forward
reaction to incorporate acetate into acetyl-CoA.(14–17) On theother hand, under anaerobic conditions or respiratory deficiency,
it is known that cytosolic ACSS expressed in yeast and fungus
Aspergillus nidulans displays considerably higher affinity toacetyl-CoA than acetate and mediates reverse reaction leading toacetate production.(23–28) This suggested that the reverse reactionof cytosolic ACSS plays a role in reducing intracellular acetyl-CoA
concentration and generating ATP to survive under anaerobic
conditions in these organisms.
There was a similarity between these anaerobic organisms
and tumor cells: they produced acetate under insufficient oxygen
supply by the mediation of cytosolic ACSS. However, ATPgeneration by ACSS2 reaction might not be so substantial intumor cells, since the amount of acetate production was small
compared with that of lactate production: the ratios of lactate
production to acetate production were 23.6 in LLC, 13.4 in B16,
15.9 in Colon, and 12.2 in C127I, respectively, under hypoxia(Fig. 5a,b).
Meanwhile, acetyl-CoA metabolism by ACSS2 might beimportant in tumor cells, because acetyl-CoA is known to be a
References
1 Brizel DM, Scully SP, Harrelson JM et al. Tumor oxygenation predicts forthe likelihood of distant metastases in human soft tissue sarcoma. Cancer
Res 1996; 56: 941–3.
2 Brown JM. The hypoxic cell: a target for selective cancer therapy –eighteenth Bruce F. Cain Memorial Award lecture. Cancer Res 1999; 59:5863–70.
3 Hockel M, Schlenger K, Aral B et al. Association between tumor hypoxia
and malignant progression in advanced cancer of the uterine cervix. Cancer
Res 1996; 56: 4509–15.
4 Pitson G, Fyles A, Milosevic M et al. Tumor size and oxygenation are
independent predictors of nodal diseases in patients with cervix cancer. Int J
Rad Onco Biol Phys 2001; 51: 699–703.
5 Dang CV, Semenza GL. Oncogenic alterations of metabolism. Trends
Biochem Sci 1999; 24: 68–72.
6 Gambhir SS. Molecular imaging of cancer with positron emission
tomography. Nat Rev Cancer 2002; 2: 683–93.
7 Warburg O. On respiratory impairment in cancer cells. Science 1956; 124:
269–70.
8 Warburg O, Posener K, Negelein E. Uber den Stoffwechsel der
Carcinomzelle. Biochem Z 1924; 152: 309–44.
9 Clavo AC, Brown RS, Wahl RL. Fluorodeoxyglucose uptake in humancancer cell lines is increased by hypoxia. J Nucl Med 1995; 36: 1625–
32.
10 Shaw RJ. Glucose metabolism and cancer. Curr Opin Cell Biol 2006; 18:
598–608.

Yoshii et al.

key metabolite in lipid synthesis of tumor cytosol. Generally, in
non-tumor cells under normoxia, acetyl-CoA produced frompyruvate enters the TCA cycle: this reaction is mediated by
pyruvate dehydrogenase (PDH). Under respiratory deficiency or
hypoxia, the glycolytic flow increases and the TCA cycle slows,which leads to increased acetyl-CoA levels in mitochondria. In
response, the excess of acetyl-CoA leads to suppression of PDH.
In the case of tumor cells, it is also reported that, under hypoxia,
hypoxia-inducible factor 1 (HIF-1) induces the expression of
pyruvate kinase 1 (PDK1), which is known as an suppressor of
PDH.(37,38) However, other observations revealed that, in tumor
cells, respiration deficiency or hypoxia causes activation ofprotein kinase B (Akt) resulting in up-regulation of glycolysisrelated enzymes and ATP citrate lyase (ACL), which produces
cytosolic acetyl-CoA pool as a component of lipid synthesis andplays an important role in tumor cell survival (Fig. S4).(39–46)
According to these studies related to ACL, the enzyme ACL canconvert citrate transported from mitochondria into acetyl-CoA
in cytosol and the citrate is provided through a truncated TCA
cycle via continued acetyl-CoA production by PDH. Together withthese studies and our present study, it may be suggested that tumor
cells possess a certain size of acetyl-CoA pool in cytosol andthat ACSS2 might play a buffering role in acetyl-CoA/acetate
metabolism as a basis of lipid synthesis in cytosol of tumor cells(Fig. S4). To understand the exact role of tumor ACSS2 metabolism, further detailed analysis of the related metabolites such asacetyl-CoA and lipids would be necessary.
Finally, this study showed that cytosolic ACSS2 plays animportant role in tumor cell survival under hypoxia. This insightshould help to clarify the behavior of tumors and provide anovel basis for metabolic targeting against tumor hypoxia.
Acknowledgments
This work was supported by Grants-in-Aid for Young Scientists (B) fromthe Japan Society for the Promotion of Science (JSPS), Japan, in part by
the 21st Century Centers of Excellence (COE) program from the JSPS,
the Leading Project from the Ministry of Education, Culture, Sports,
Science and Technology, Japan, CREATE from the Japan Science andTechnology Agency, Japan, and a Grant-in-Aid for Tumor Research from
the Ministry of Health, Labour and Welfare, Japan. We thank membersof our laboratory for helpful discussions, and H. Takagi, Y. Kishimoto,and H. Maeda for technical help.

11 Karan D, David JR, Capy P. Molecular evolution of the AMP-forming
acetyl-CoA synthetase. Gene 2001; 265: 95–101.
12 Starai VJ, Celic I, Cole RN et al. Sir2-dependent activation of acetyl-CoA
synthetase by deacetylation of active lysine. Science 2002; 298: 2390–2.
13 Schwer B, Bunkenborg J, Verdin RO et al. Reversible lysine acetylation
controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2.
Proc Natl Acad Sci USA 2006; 103: 10224–9.
14 Fujino T, Kondo J, Ishikawa M et al. Acetyl-CoA synthetase 2, a
mitochondrial matrix enzyme involved in the oxidation of acetate. J Biol
Chem 2001; 276: 11420–6.
15 Howard BV, Howard WJ, Bailey JM. Acetyl coenzyme A synthetase and theregulation of lipid synthesis from acetate in cultured cells. J Biol Chem
1974; 249: 7912–21.
16 Sone H, Shimano H, Sakakura Y et al. Acetyl-coenzyme A synthetase is alipogenic enzyme controlled by SREBP-1 and energy status. Am J Physiol
Endocrinol Metab 2002; 282: E222–30.
17 Luong A, Hannah VC, Brown MS et al. Molecular characterization ofhuman acetyl-CoA synthetase, an enzyme regulated by sterol regulatory
element-binding proteins. J Biol Chem 2000; 275: 26458–66.
18 Beinert H, Green DE, Hele P et al. The acetate activating enzyme system ofheart muscle. J Biol Chem 1953; 203: 35–45.
19 Jones ME, Black S, Flynn RM et al. Acetyl coenzyme a synthesis throughpyrophosphoryl split of adenosine triphosphate. Biochim Biophys Acta 1953;
12: 141–9.
20 Barak R, Prasad K, Shainskaya A et al. Acetylation of the chemotaxis
response regulator CheY by acetyl-CoA synthetase purified from
Escherichia coli. J Mol Biol 2004; 342: 383–401.

Cancer Sci |May 2009 | vol. 100 | no. 5 | 825© 2009 Japanese Cancer Association

21 Hiatt AJ, Evans HJ. Influence of certain cations on activity of acetic
thiokinase from spinach leaves. Plant Physiol 1960; 35: 673–7.
22 Frenkel EP, Kitchens RL. Purification and properties of acetyl coenzyme A
synthetase from bakers’ yeast. J Biol Chem 1977; 252: 504–7.
23 Duntze W, Neumann D, Gancedo JM et al. Studies on the regulation andlocalization of the glyoxylate cycle enzymes in Saccharomyces cerevisiae.
Eur J Biochem 1969; 10: 83–9.
24 Klein HP, Jahnke L. Variations in the localization of acetyl-coenzyme A
synthetase in aerobic yeast cells. J Bacteriol 1971; 106: 596–602.
25 Klein HP, Jahnke L. Effects of aeration on formation and localization of theacetyl coenzyme A synthetases of Saccharomyces cerevisiae. J Bacteriol
1979; 137: 179–84.
26 Polakis ES, Bartley W. Changes in the enzyme activities of Saccharomyces
cerevisiae during aerobic growth on different carbon sources. Biochem J
1965; 97: 284–97.
27 Rogers PJ, Stewart PR. Respiratory development in Saccharomyces
cerevisiae grown at controlled oxygen tension. J Bacteriol 1973; 115: 88–97.
28 Takasaki K, Shoun H, Yamaguchi M et al. Fungal ammonia fermentation, anovel metabolic mechanism that couples the dissimilatory and assimilatory
pathways of both nitrate and ethanol. Role of acetyl CoA synthetase inanaerobic ATP synthesis. J Biol Chem 2004; 279: 12414–20.
29 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using
real-time quantitative PCR and the 2–ΔΔCT Method. Methods 2001; 25: 402–
8.
30 Scholl C, Bansal D, Dohner K et al. The homeobox gene CDX2 is aberrantly
expressed in most cases of acute myeloid leukemia and promotes
leukemogenesis. J Clin Invest 2007; 117: 1037–48.
31 Patel JH, McMahon SB. BCL2 is a downstream effector of MIZ-1 essential
for blocking c-MYC-induced apoptosis. J Biol Chem 2007; 282: 5–13.
32 Janik P, Briand P, Hartmann NR. The effect of estrone-progesterone
treatment on cell proliferation kinetics of hormone-dependent GR mouse
mammary tumors. Cancer Res 1975; 35: 3698–704.
33 Brizel DM, Rosner GL, Harrelson J et al. Pretreatment oxygenation profilesof human soft tissue sarcomas. Int J Rad Onco Biol Phys 1994; 30: 635–42.

34 Brizel DM, Sibley GS, Prosnitz LR et al. Tumor hypoxia adversely affects
the prognosis of carcinoma of the head and neck. Int J Rad Onco Biol Phys
1997; 38: 285–9.
35 Hockel M, Schlenger K, Knoop C et al. Oxygenation of carcinomas of theuterine cervix: evaluation by computerized O2 tension measurements. Cancer
Res 1991; 51: 6098–102.
36 Vaupel P, Schlenger K, Knoop C et al. Oxygenation of human tumors:evaluation of tissue oxygen distribution in breast cancers by computerized O2
tension measurements. Cancer Res 1991; 51: 3316–22.
37 Kim JW, Tchernyshyov I, Semenza GL et al. HIF-1-mediated expression of
pyruvate dehydrogenase kinase: a metabolic switch required for cellular
adaptation to hypoxia. Cell Metab 2006; 3: 177–85.
38 Papandreou I, Cairns RA, Fontana L et al. HIF-, 1 mediates adaptation tohypoxia by actively downregulating mitochondrial oxygen consumption.
Cell Metab 2006; 3: 187–97.
39 Yoshimoto M, Waki A, Yonekura Y et al. Characterization of acetate
metabolism in tumor cells in relation to cell proliferation: acetate metabolism
in tumor cells. Nucl Med Biol 2001; 28: 117–22.
40 Hatzivassiliou G, Zhao F, Bauer DE et al. ATP citrate lyase inhibition can
suppress tumor cell growth. Cancer Cell 2005; 8: 311–21.
41 Bui T, Thompson CB. Cancer’s sweet tooth. Cancer Cell 2006; 9: 419–20.
42 Bauer DE, Hatzivassiliou G, Zhao F et al. ATP citrate lyase is an importantcomponent of cell growth and transformation. Oncogene 2005; 24: 6314–22.
43 Young CD, Anderson SM. Sugar and fat – that’s where it’s at: metabolicchanges in tumors. Breast Cancer Res 2008; 10: 202.
44 Pelicano H, Du Xu RH, M et al. Mitochondrial respiration defects in cancer
cells cause activation of Akt survival pathway through a redox-mediatedmechanism. J Cell Biol 2006; 175: 913–23.
45 Berwick DC, Hers I, Heesom KJ et al. The identification of ATP-citratelyase as a protein kinase B (Akt) substrate in primary adipocytes. J Biol
Chem 2002; 277: 33895–900.
46 Furuta E, Pai SK, Zhan R et al. Fatty acid synthase gene is up-regulated byhypoxia via activation of Akt and sterol regulatory element binding protein-1.
Cancer Res 2008; 68: 1003–11.

Supporting Information
Additional Supporting Information may be found in the online version of this article:
Fig. S1. Cell growth. Cell numbers are shown every 24 h during the growth of tumor cells (LLC, B16, Colon, and C127I) and non-tumor fibroblast
cells (3T3).
Fig. S2. 3H-deoxyglucose (DG) uptake. Data are shown as 3H-DG uptake per h per 104 cells in tumor cells (LLC, B16, Colon, and C127I) and nontumor fibroblast cells (3T3) under normoxia. The tumor cells took up 3H-DG 1.9-fold to 2.3-fold faster than the 3T3 cells (P < 0.001).
Fig. S3. Mitochondrial electron transport activity, assayed by reduction of Alamar Blue dye. Data represent percentages of reduced Alamar Blue dye
in tumor cells (LLC, B16, Colon, and C127I) and non-tumor fibroblast cells (3T3) under normoxia. The tumor cells reduced Alamar Blue dye lessefficiently than the 3T3 cells (P < 0.001).
Fig. S4. Diagram illustrating proposed tumor metabolic pathway in this study. Glycolysis is a universal metabolic pathway by which glucose is broken
down to pyruvate (blue line). Pyruvate is changed to acetyl-CoA, which is transfered into mitochondria, by the mediation of pyruvate dehydrogenase(PDH) (orange line) or converted to lactate in the cytosol by lactate fermentation (pink line). Mitochondrial citrate provided by truncated tricarboxylic
acid cycle in tumor cells is exported to the cytosol and changed to acetyl-CoA for lipid synthesis via adenosine triphosphate (ATP) citrate lyase (ACL)(green line).(1–7) Cytosolic acetyl-CoA synthetase (ACSS2) mediates interconversion between acetyl-CoA and acetate to keep metabolic balances intumor cells (red line). Respiration deficiency or hypoxia causes activation of glycolysis-related enzymes, ACL, and ACSS2 (For details, see main text).
Supporting text
Cell growth
Cells (3 × 104) were seeded in 1 mL growth medium in 24-well plates. The numbers of cells were counted every 24 h for 3 days.
3
H-deoxyglucose (DG) uptake
Cells (1 × 105) were seeded in 24-well plates in growth medium and incubated for 24 h. The medium was then changed to 500 μL of fresh growth
medium containing 74 kBq of 3H-DG (GE Healthcare, Pollards, UK). 3H-DG uptake is an indicator of glucose utilization.(8) After 1 h of incubation,
the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lyzed in 500 μL of 0.2 N NaOH at room temperature for 2 h, then thelysates were mixed with aqueous counting scintillant (ACS)II (GE Healthcare). Radioactivity was measured with a liquid scintillation counter(LSC5100, Aloka, Tokyo, Japan). The number of cells treated in the same way before cell lysis was also counted.
Alamar Blue assay
The Alamar Blue dye (Serotec, Sapporo, Japan) was used to estimate the mitochondrial oxidation–reduction potential of electron transport. Cells(1 × 105) were incubated for 4 h at 37°C in 1 mL serum-free RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% Alamar Blue dye.
Medium alone was prepared in the same manner and used as a blank. The absorbances at 570 and 600 nm were used to calculate the percentages of
reduced Alamar Blue dye.
References
1

826

Yoshimoto M, Waki A, Yonekura Y et al. Characterization of acetate metabolism in tumor cells in relation to cell proliferation: acetate metabolism
in tumor cells. Nucl Medical Biol 2001; 28: 117–22.

doi: 10.1111/j.1349-7006.2009.01099.x© 2009 Japanese Cancer Association

2 Hatzivassiliou G, Zhao F, Bauer DE et al. ATP citrate lyase inhibition can suppress tumor cell growth. Cancer Cell 2005; 8: 311–21.
3 Bui T, Thompson CB. Cancer’s sweet tooth. Cancer Cell 2006; 9: 419–20.
4 Bauer DE, Hatzivassiliou G, Zhao F et al. ATP citrate lyase is an important component of cell growth and transformation. Oncogene 2005; 24: 6314–
22.
5 Young CD, Anderson SM. Sugar and fat – that’s where it’s at: metabolic changes in tumors. Breast Cancer Res 2008; 10: 202.
6 Pelicano H, Xu RH, Du M et al. Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redox-mediated mechanism. J Cell Biol 2006; 175: 913–23.
7 Berwick DC, Hers I, Heesom KJ et al. The identification of ATP-citrate lyase as a protein kinase B (Akt) substrate in primary adipocytes. J Biol
Chem 2002; 277: 33895–900.
8 Sokoloff L, Reivich M, Kennedy C et al. The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat. J Neurochem 1977; 28: 897–916.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries(other than missing material) should be directed to the corresponding author for the article.

Yoshii et al.

Cancer Sci |May 2009 | vol. 100 | no. 5 | 827© 2009 Japanese Cancer Association