European Journal of Cancer 41 (2005) 1316–1322

European
Journal ofCancerwww.ejconline.com

Co-ordinate activation of lipogenic enzymes inhepatocellular carcinoma
Naoya Yahagi a, Hitoshi Shimano d,*, Kiyoshi Hasegawa b, Kenichi Ohashi c,
Takashi Matsuzaka d, Yuho Najima a, Motohiro Sekiya a, Sachiko Tomita a,
Hiroaki Okazaki a, Yoshiaki Tamura a, Yoko Iizuka a, Ken Ohashi a, Ryozo Nagai a,
Shun Ishibashi a, Takashi Kadowaki a, Masatoshi Makuuchi b, Shin Ohnishi a,
Jun-ichi Osuga a, Nobuhiro Yamada daDepartment of Internal Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-8655, Japanb
Department of Surgery, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-8655, Japanc
Department of Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-8655, Japand
Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Received 2 December 2004; accepted 2 December 2004
Available online 24 May 2005

Abstract
Hepatocellular carcinoma is a very common neoplastic disease in countries where hepatitis viruses B and/or C are prevalent.
Small hepatocellular carcinoma lesions detected by ultrasonography at an early stage are often hyperechoic because they are composed of well-diﬀerentiated cancer cells that are rich in triglyceride droplets. The triglyceride content of hepatocytes depends in parton the rate of lipogenesis. Key lipogenic enzymes, such as fatty acid synthase, are co-ordinately regulated at the transcriptional level.
We therefore examined the mRNA expression of lipogenic enzymes in human hepatocellular carcinoma samples from 10 patients
who had undergone surgical resection. All of the samples exhibited marked elevation of expression of mRNA for lipogenic enzymes,
such as fatty acid synthase, acetyl-CoA carboxylase and ATP citrate lyase, compared with surrounding non-cancerous liver tissue.
In contrast, the changes in mRNA expression of SREBP-1, a transcription factor that regulates a battery of lipogenic enzymes, did
not show a consistent trend. In some cases where SREBP-1 was elevated, the main contributing isoform was SREBP-1c rather than
SREBP-1a. Thus, lipogenic enzymes are markedly induced in hepatocellular carcinomas, and in some cases SREBP-1c is involved inthis activation.
 2005 Elsevier Ltd. All rights reserved.
Keywords: Hepatocellular carcinoma; Lipogenesis; Triglycerides; Fatty acids; Neoplasia

1. Introduction
Hepatocellular carcinoma is a very common neoplastic disease in countries where hepatitis viruses B and/or
C are prevalent. In Japan, 90% of hepatocellular carcinomas develop in patients with chronic liver diseases*

Corresponding author. Tel.: +81 298 53 3053; fax: +81 29 853 3174.
E-mail address: shimano-tky@umin.ac.jp (H. Shimano).

0959-8049/$ - see front matter  2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ejca.2004.12.037

such as liver cirrhosis and chronic hepatitis [1]. Periodic
imaging using ultrasonography in patients with chronic
liver diseases has resulted in an increased rate of detection of small hepatocellular carcinomas of 2 cm diameter or less [2,3]. These are often hyperechoic lesions,which are composed of well-diﬀerentiated cancer cells
rich in triglyceride droplets [4,5].
The triglyceride content of hepatocytes depends inpart on the rate of fatty acid synthesis [6–8]. The fatty

N. Yahagi et al. / European Journal of Cancer 41 (2005) 1316–1322

acid biosynthetic pathway, composed of some 25 enzymes, is now elucidated in detail [9]. Among these
enzymes, the following are of particular importance:
fatty acid synthase (FAS), the main synthetic enzymethat catalyses the condensation of malonyl-CoA toproduce the 16-carbon saturated fatty acid palmitate;acetyl-CoA carboxylase 1 (ACC1), which synthesises
malonyl-CoA from acetyl-CoA; ATP citrate lyase(ACL), which synthesises acetyl-CoA from citrate provided through glycolysis. These enzymes are known tobe co-ordinately induced when lipogenesis is needed,
and this induction of enzymes is primarily at the transcriptional level [10]. It has therefore been presumed
that these genes have a regulatory sequence in theirpromoter that interacts with common trans-acting factors such as sterol regulatory element-binding protein(SREBP)-1 [11,12].
SREBPs are members of the basic helix–loop–helix
leucine zipper family of transcription factors that regulate fatty acid and cholesterol synthesis (reviewed in[13–15]). Unlike other members of the family, SREBPs
are synthesised as precursors bound to the endoplasmic
reticulum and nuclear envelope, and are released fromthe membrane into the nucleus as mature proteins bycleavage processes. To date, three isoforms of SREBP,-1a, -1c and -2, have been identiﬁed and characterised.
The predominant SREBP-1 isoform in liver and adipose
tissue is SREBP-1c. Whereas SREBP-2 plays a crucialrole in regulation of cholesterol synthesis, SREBP-1cco-ordinately upregulates the transcription and expression of lipogenic enzymes such as FAS and ACL,
leading to elevated triglyceride synthesis (reviewed in[12,16–18]). It is remarkable that SREBP-1c controls
not only the synthetic rate of triglycerides but also theircontent in the liver [7,8,19].
As well-diﬀerentiated hepatocellular carcinomas often have abundant triglyceride droplets and SREBP-1c
is a determinant of triglyceride content in hepatocytes,
we hypothesised that the activation of SREBP-1 andthereby lipogenic enzymes might be involved in the earlystage of cancer development in hepatocytes. To test thishypothesis, we examined the expression levels of
SREBP-1 and its downstream lipogenic enzymes in surgically resected hepatocellular carcinomas.

2. Materials and methods
2.1. PatientsTen consecutive patients who had undergone surgicalresection of hepatocellular carcinoma at Tokyo University Hospital from January to June 1999 were includedin the study. Informed consent was obtained from thepatients. All cases were diagnosed as hepatocellular carcinoma by pathohistological examination.

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2.2. Northern blotting
Small pieces of hepatocellular carcinoma and noncancerous liver tissue were snap frozen in liquid nitrogen
immediately after surgical resection. Necrotic portions,
identiﬁed macroscopically, were excluded. Total RNA
from these samples was extracted using TRIzol reagent(Invitrogen), and 10 lg RNA was run on a 1% agarose
gel containing formaldehyde and transferred to a nylon
membrane. The cDNA probe for SREBP-1 was cloned
as described previously [6]. The cDNA probes for
FAS, ACC1, ACL, stearoyl-CoA desaturase (SCD)
and malic enzyme (ME) were prepared by cloning RTPCR products from human liver total RNA into
pGEM-T easy vectors (Promega). The PCR primers
used for FAS were 5 0 -TCGGAGAACTTGCAGGAGTT-3 0 and 5 0 -CCAGCCTCAAGAACTGCAC-3 0 ;for ACC1, 5 0 -CACATAAGGTCCAGCATGTCTG-3 0
and 5 0 -CCAGGCACTGGCACATAGT-3 0 ; for ACL,
5 0 -TCTCTCTGCAGCCATGTCG-3 0 and 5 0 -GGAGATAAAACTGGCCAGAATTTC-3 0 ; for SCD, 5 0 TGCTGCAGGACGATATCTCTAG-3 0 and 5 0 -CAGGGCACTGACAAAATAGTAGAAT-3 0 ; for ME,
5 0 -CATCTGAACTCTGACTTTGACAGGT-3 0and
5 0 -CCGTAGTCCAATGTAGAGTGGATC-3 0 .
Theprobes were labelled with [a-32P]dCTP using Megaprime DNA Labeling System kit (Amersham Biosciences). The membranes were hybridised with theradiolabelled probe in Rapid-hyb Buﬀer (Amersham
Biosciences) at 65 C. The membranes were washed in
0.1 · SSC, 0.1% SDS at 65 C. Blots were exposed to
Kodak XAR-5 ﬁlm and the BAS2000 Bio Imaging Analyzer (Fuji Photo Film, Tokyo, Japan).
2.3. Immunohistochemistry
The expression of FAS was immunohistochemicallydetected in formalin-ﬁxed, paraﬃn-embedded sections
as described previously [20]. As the primary antibody,
rabbit polyclonal antibody to FAS (IBL, Fujioka,
Japan) was used at 2 lg/ml IgG.
2.4. RNase protection assay
RNase protection assay was performed as described
previously [21]. Brieﬂy, after linearisation of the template plasmid DNA with HindIII, antisense RNAprobes for SREBP-1a and -1c were transcribed with[a-32P]CTP using bacteriophage T7 RNA polymerase(MAXIscript in vitro transcription kit, Ambion) andpuriﬁed by electrophoresis on 5% polyacrylamide gel.
Then aliquots of probes and total RNA (10 lg) fromeach sample were mixed and subjected to the RNase
protection assay using HybSpeed RPA kit (Ambion).
After digestion with RNase A/T1, protected fragments
were separated on 8 M urea/10% polyacrylamide gels.

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N. Yahagi et al. / European Journal of Cancer 41 (2005) 1316–1322

The protected fragments for SREBP-1a and -1c contained the same number of cytidine phosphates (32 cytidines). The gels were dried and subjected to Kodak
XAR-5 ﬁlm and the BAS2000 Bio Imaging Analyzer(Fuji Photo Film, Tokyo, Japan).

3. Results
3.1. Induction of lipogenic enzymes in hepatocellular
carcinomas
Table 1 shows the outline of patients included in thecurrent study. Because we analysed 10 consecutive cases
hospitalised at random, the aetiology of backgroundchronic liver disease was diverse, but the majority patients had viral hepatitis, mostly due to the hepatitis C
virus, in accordance with epidemiological data forJapan. We performed Northern blot analysis of surgically resected samples frozen during operation and compared the mRNA expressions of lipogenic enzymes andtheir upstream transcription factor SREBP-1 between
the hepatocellular carcinoma tissue and surrounding
non-cancerous liver tissue. As shown in Figs. 1 and
5(b), all carcinoma samples exhibited marked elevationof mRNA levels for FAS, ACC1, ACL, SCD and ME,
representative enzymes in the fatty acid biosynthetic
pathway, compared with corresponding adjacent noncancerous liver tissue. The expression levels of lipogenic
enzymes were not related to the grade of carcinoma cell
diﬀerentiation or to the virus type. Meanwhile, themRNA level of SREBP-1 in carcinoma did not show
consistent diﬀerences from that in non-cancerous tissue.
Fatty changes in carcinoma were observed in cases #4,#6 and #9 by histological examination (Table 1), butthese were not inevitably accompanied by higher levelsof expression of lipogenic enzymes.

Fig. 1. Lipogenic enzymes are co-ordinately and markedly activated inhepatocellular carcinomas. Northern blot analysis of sterol regulatory
element-binding protein-1 (SREBP-1), fatty acid synthase (FAS),
acetyl-CoA carboxylase 1 (ACC1), ATP citrate lyase (ACL), stearoylCoA desaturase (SCD) and malic enzyme (ME) expressed in surrounding non-cancerous liver tissue (Control) and hepatocellular
carcinomas (HCC). Samples were resected from patients and snap
frozen during surgery. Total RNA from each sample was subjected to
Northern blotting, followed by hybridisation with the indicated cDNA
probes. Equal loading of total RNA to each lane was conﬁrmed byribosomal RNA staining with ethidium bromide. rRNA, ribosomal
ribonucleic acids.

3.2. Co-ordinate elevation of lipogenic enzymes inhepatocellular carcinoma
As shown in Fig. 1, lipogenic enzymes such as FAS,
ACC1 and ACL were co-ordinately elevated in hepatocellular carcinoma. To more clearly visualise this coordination, mRNA levels quantiﬁed by Northern blot

Table 1
Clinical characteristics of cases included in the current study
Case

Age (years)

Sex

Virus

Histological ﬁndings
Tumour diﬀerentiation

Liver cirrhosis

AFP

PIVKA2

Fatty change

1

36

M

B

ModerateEarly

20,744

8946

2

65

M

C

Moderate

Advanced



42

17

3

54

M

C

Moderate

Early



850

100,000

4

65

M

C

Moderate

Advanced

+

5

1992

5

63

M

C

Moderate

None



2543

1694

6

35

F

B

Moderate–poor

Advanced

+

1513

4794

7

81

M

nBnC

Poor

None



14

1803

8

61

F

C

Moderate

Advanced



1755

1085

9

45

M

nBnC

Well

None

+

<1

13,566

10

58

M

C

Moderate

Advanced



24

8007

nBnC, not B nor C; AFP, alpha fetoprotein; PIVKA2, protein induced by vitamin K absence 2.

N. Yahagi et al. / European Journal of Cancer 41 (2005) 1316–1322

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analysis were plotted in a scatter diagram (Fig. 2). Therewas signiﬁcant correlation between the mRNA expression of FAS, ACC1, ACL and SCD, whose P-value
by FisherÕs formula was less than 0.001. In contrast,
there was no signiﬁcant correlation between the mRNAlevel for SREBP-1 and that for lipogenic enzymes suchas FAS, ACC1 and ACL.
3.3. Immunohistochemical detection of FAS
To conﬁrm the elevated expression of FAS protein in
hepatocellular carcinoma, immunostaining was performed on formalin-ﬁxed, paraﬃn-embedded sections
using anti-FAS antibody. In all of the 10 cases, FAS
protein was strongly stained in hepatocellular carcinoma
specimens. Moreover, although FAS was also detectedin non-cancerous liver tissues, there was a tendency forstronger staining in carcinoma samples (Fig. 3).
3.4. RNase protection assay reveals SREBP-1cdominance

Fig. 2. Correlation among mRNA levels of sterol regulatory elementbinding protein-1 (SREBP-1) and lipogenic enzymes. Correlation
between (a) mRNA level of SREBP-1 and fatty acid synthase (FAS)
and (b) FAS and acetyl-CoA carboxylase 1 (ACC1) or ATP citratelyase (ACL). Northern blot analysis data presented in Fig. 1 were
quantiﬁed and plotted. A regression line was plotted in (b), where
signiﬁcant correlations were detected. Con, control non-cancerous
liver tissue; HCC, hepatocellular carcinoma.

For further analysis of SREBP-1 isoform, we selectedfour cases out of 10 covering each virus type (B, C, or
non-B non-C), and conducted an RNase protection assay with probes that can discriminate SREBP-1a and 1c. Because the protected fragments for SREBP-1aand -1c contained the same number of cytidine phosphates (32 cytidines), the intensity of each band was proportional to the amount of each isoform in the samples.

Fig. 3. Immunostaining of fatty acid synthase (FAS). Immunohistochemical expression of FAS is shown in two representative cases. Case #4 (upper
panels) has the highest FAS mRNA expression and case #10 (lower panels) has the second lowest FAS mRNA. The carcinoma in case #4 shows clear
cell change that indicates lipid accumulation. Control, surrounding non-cancerous liver tissue; HCC, hepatocellular carcinoma.

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N. Yahagi et al. / European Journal of Cancer 41 (2005) 1316–1322

Fig. 4. RNase protection analysis of SREBP-1 isoforms in noncancerous liver tissue (Control) and hepatocellular carcinomas (HCC).
SREBP-1c is the dominant isoform. The assay was performed on four
samples selected to cover each type of virus. The protected fragments
for SREBP-1a and -1c contain the same number of cytidine
phosphates (32 cytidines), so the intensity of each band is proportional
to the amount of each isoform in the samples.

As shown in Fig. 4, the RNase protection assay did notexhibit a deﬁnite pattern of isoform change, and both in
non-cancerous tissue and in hepatocellular carcinoma
SREBP-1c was more abundant than SREBP-1a. In
two cases (cases 7 and 9), where SREBP-1 mRNA was
increased by malignant transformation as shown by
Northern blot analysis (Fig. 1), the change in the expression of SREBP-1c was more prominent than that of
SREBP-1a. These results were quantiﬁed and presented
in a graph in Fig. 5(a).

4. Discussion
Our present studies clearly demonstrate that lipogenic enzymes are induced in hepatocellular carcinomas
compared with background liver tissue. It is tempting to
speculate that this induction is associated with fattychanges often seen in well-diﬀerentiated hepatocellular
carcinomas. Notably, however, there was no correlation
between the increase in lipogenic enzyme expression andthe degree of carcinoma diﬀerentiation.
In general, cancers show high levels of glycolysis andlipogenesis (reviewed in [22]). Accelerated fatty acid synthesis in tumour tissues was ﬁrst reported in the 1950s[23]. Since then, elevated expression of FAS in malignant cells has been documented in various cancers
including breast [24–28], prostate [29–31], ovarian [32],endometrial [33], colon [34], stomach [20], tongue [35]and lung carcinoma [36]. Our study demonstrates, forthe ﬁrst time, that hepatocellular carcinoma can be
added to this list.
As an underlying mechanism for this upregulation, it
has been assumed that there is a link between cellular
proliferation and fatty acid synthesis, because fatty acidsynthesis has been observed to be activated when cells

Fig. 5. Quantiﬁed results of Northern blotting and RNase protection
assay. (a) RNase protection assay results presented in Fig. 4 arequantiﬁed by BAS2000 imaging analyser system. (b) Northern blot
analysis presented in Fig. 1 quantiﬁed and shown as graphs. Bars, S.E.
SREBP1, sterol regulatory element-binding protein-1; FAS, fatty acidsynthase; ACC1, acetyl-CoA carboxylase 1; ACL, ATP citrate lyase;
SCD, stearoyl-CoA desaturase; ME, malic enzyme; Con, control noncancerous liver tissue; HCC, hepatocellular carcinoma.

enter into proliferative phase in several systems [37–40]. Our results are in accordance with these data andprovide further evidence for the link between cellular
proliferation and lipogenesis. Furthermore, it would beof therapeutic value if inhibitors of fatty acid synthesis
such as cerulenin were found to be useful for the treatment of hepatocellular carcinoma.
It has been established that the lipogenic enzymes
are in general regulated co-ordinately at mRNA level[10], and our current ﬁnding that lipogenic enzymes
such as FAS, ACC1 and ACL are at the same time
activated in carcinoma is in agreement with this.
Although the precise molecular mechanism for thisregulation is currently unclear, this co-ordinated control of gene expression is believed to be achieved bya regulatory sequence that interacts with common
trans-acting factors [10]. As documented previously[11,12], SREBP-1 is one of the candidate transcription
factors responsible for this regulation. In fact, theN. Yahagi et al. / European Journal of Cancer 41 (2005) 1316–1322

potential involvement of SREBP-1 has been demonstrated in some cancers [41–43]. Hence, we examinedthe expression of SREBP-1 at the mRNA level, but,
unexpectedly, there was no consistent trend in
SREBP-1 mRNA elevation in hepatocellular carcinoma. It is possible, however, that SREBP-1 might
be increased post-translationally, that is, at the processing level, and be involved in lipogenic gene induction. To address this issue, it is necessary to quantify
the amount of nuclear SREBP-1 protein in the active
form by nuclear protein extraction. This analysis
could not be performed, however, due to the limited
amounts of surgically resected human samples
available.
The 1a isoform of SREBP-1 is known to be expressed
relatively abundantly in organs where cells are rapidly
turned over, such as in the spleen, thymus and small
intestine as well as in whole embryos [21]. It has also
been shown that transformed cell lines usually express
SREBP-1a dominantly. We therefore expected that
hepatocellular carcinomas would exhibit a 1a-dominantpattern of SREBP-1 isoform. This did not seem to be thecase, however, in our study. In the samples we examined, 1c was more prominently induced, a phenomenon
that may be related to the highly diﬀerentiated feature ofthis cancer.
In conclusion, we have shown that lipogenic enzymes
are markedly induced in hepatocellular carcinomas, andthat SREBP-1c rather than SREBP-1a might be partly
involved in this activation.

Conﬂict of interest statement
None declared.

Acknowledgements
We thank Alyssa H. Hasty for critical reading of themanuscript. This work was supported by grants-in-aid
from the Ministry of Science, Education, Culture, andTechnology of Japan, and by a grant from the Research
Fellowships of the Japan Society for the Promotion of
Science (to N. Yahagi).

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