ORIGINAL ARTICLE
Expression proﬁling in hepatocellular carcinoma withintrahepatic metastasis: identiﬁcation ofhigh-mobility group I(Y) protein as a molecular marker ofhepatocellular carcinoma metastasis
Makoto Chuma,1,3 Norihisa Saeki,2,5 Yoshiya Yamamoto,1,3 Tsutomu Ohta,2 Masahiro Asaka,3Setsuo Hirohashi1 and Michiie Sakamoto1,4
1Pathology Division and 2Genomics Division, National Cancer Center Research Institute, Tokyo
3Department of Gastroentelology and Hematology, Hokkaido University, Sapporo, 4Department of Pathology
School of Medicine, Keio University, Tokyo, Japan(Received for publication on January 5, 2004)

Abstract. Hepatocellular carcinoma (HCC) is one of the most common and aggressive humanmalignancies. Its high mortality rate is mainly a result of intra-hepatic metastases. To investigate thedetailed genetic mechanisms in cancer metastasis, we compared the expression proﬁles of 20 HCCs
with intrahepatic metastasis and 10 HCCs without intrahepatic metastasis using an oligonucleotide
array. Of the approximately 12600 genes that were analyzed, we identiﬁed 34 genes whose expression
levels were signiﬁcantly correlated with intrahepatic metastasis (P H 0:05). Of these genes, we further investigated the expression of high-mobility group I(Y) [HMG-I(Y)] protein. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) conﬁrmed that HMG-I(Y) was upregulated in HCC with intrahepatic metastasis, compared to its level in HCC without intrahepatic
metastasis. Further immunohistochemical examination of HMG-I(Y) revealed a signiﬁcant overexpression in HCC with intrahepatic metastasis, compared with that in HCC without intrahepatic
metastasis (P H 0:05). These results indicate that the molecular signatures of HCC with intrahepatic
metastasis and of HCC without intrahepatic metastasis are clearly different. HMG-I(Y) expression was
associated with intrahepatic metastasis and may be a predictive marker of HCC intrahepatic metastasis.
(Keio J Med 53 (2): 90–97, June 2004)
Key words: HCC, intrahepatic metastasis, HMG-I(Y), oligonucleotide array

ment.2 Pathological and genetic studies have identiﬁed
two types of HCC recurrence: the multicentric development of new tumors and intrahepatic metastasisof the original HCC.3–5 Hematogeneous intrahepatic
metastasis of HCC is frequently observed in advanced
cases and is thought to arise through tumor cell dispersal via the portal vein.6 Current studies have largely
focused on individual candidate genes, an approachthat may be insufﬁcient to precisely deﬁne the genetic
basis of metastatic HCC.7–9 A molecular understandingof intrahepatic metastasis is an important step toward
the identiﬁcation of predictive markers and more spe-

Introduction
Hepatocellular carcinoma (HCC) is one of the mostcommon malignant tumors worldwide and one of theleading causes of death from cancer in Japan.1 Recent
advances in diagnostic modalities, such as imaging
techniques and the measurement of serum tumor
markers, have improved the rate of early detection of
HCC, and therapeutic approaches to HCC have also
progressed. However, the long-term survival of HCC
patients continues to be poor because of the high incidence of recurrence within the liver after initial treat-

Present afﬁliation:5 The Organization for Pharmaceutical Safety and Research, Tokyo
Reprint requests to: Dr. Michiie Sakamoto, Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjyuku-ku,
Tokyo, 160-8582, Japan, e-mail: msakamot@sc.itc.keio.ac.jp90Keio J Med 2004; 53 (2): 90–97

ciﬁc targets for HCC recurrence. The establishment of
microarray methods enabling the large-scale analysis ofgene expression has made it possible to seek molecularmarkers for cancer classiﬁcation and outcome prediction in addition to identifying molecules involved in
carcinogenesis in a variety of tumor types.10–12 Several
studies have compared gene expression between nontumorous liver tissue and HCC and shown that the gene
expression patterns present in HCC are speciﬁc to thatcell type.13–16 Here, we compared the expression proﬁles of 20 HCCs with intrahepatic metastasis and 10
HCCs without intrahepatic metastasis using an oligonucleotide array to identify genes generally involved in
intrahepatic metastasis. Among the genes identiﬁed, we
further investigated high-mobility group I(Y) (HMGI(Y)). HMG-I(Y) is a family of low molecular mass,
nonhistone nuclear DNA binding proteins. Overexpression of HMG-I(Y) has been described in several
cancers, and a signiﬁcant correlation between elevated
HMG-I(Y) expression and a poor prognosis has beenreported in patients with prostate and breast cancers.
However, HMG-I(Y) expression has rarely been mentioned and its role is not clearly understood in HCC.
Therefore we further investigated whether HMG-I(Y)expression is associated with intrahepatic metastasis
using reverse transcription polymerase chain reaction(RT-PCR) and immunohistochemistry.
Materials and Methods
Patients and tissue samples
Thirty HCCs and corresponding noncancerous livertissues were obtained from patients who had undergone
surgical resections at the National Cancer Center Hospital, Japan. Specimens were immediately cut into small
pieces, snap-frozen in liquid nitrogen, and stored until
further use. The clinicopathological characteristics ofthe 20 HCCs with intrahepatic metastasis and the 10
HCCs without intrahepatic metastasis are shown in Table 1. There were no signiﬁcant differences in factors
between the two groups of HCCs. For the immunohistochemical analysis, 102 HCCs were analyzed. Sections
were prepared from formalin-ﬁxed, parafﬁn-embedded
tissue samples that were resected surgically between
1990 and 2002. The main clinicopathological featuresof the specimens are presented in Table 2. The histopathological grade of tumor differentiation was assessedusing a modiﬁcation of the Edmondson Grading System.17,18 Macroscopically, HCC was subclassiﬁed into
three types: single nodule (type 1), single nodule withextranodular growth (type 2), and conﬂuent multinodule (type 3). Histological diagnosis of intrahepatic
metastases was made according to the WHO criteria.19
Nodules apparently growing from portal vein tumor

91
Table 1

Patient Characteristics per Study Group

Number of cases
Mean ages (years)aSex
Male
FemaleVirus maker
HBs-Ag(þ)
HCV Ab(þ)
NBNCb
Maximal size (mm)Differentiation
Well
Moderate
Poor
Macroscopic type1
2
3

vp(þ)/im(þ)

vp()/im()20
59:5 G 13:9

10
60:3 G 7:5

15
5

7
3

7
9
4
63:9 G 30:7

3
6
1
44:1 G 20:4

0
10
10

2
5
3

2
8
10

2
6
2

p value

0.87
0.77

0.69

0.08
0.1

0.28

a Mean G SE. b NBNC; patients without HBs-Ag(þ) nor HCVAb(þ).

Table 2

Immunohistochemical Examination of HMG-I(Y) in HCC
HMGI expression

Number of cases
Mean ages (years)aSex
Male
FemaleVirus maker
HBs-Ag(þ)
HCV Ab(þ)
NBNCb
Maximal size (mm)Differentiation
Well
Moderate
Poor
Macroscopic type1
2
3
Intrahepatic metastasisc
Present
Absent

()

(þ)

60
58:6 G 9:54

42
60:3 G 10:2

38
22

25
17

16
36
8
47:2 G 22:4

12
26
4
52:5 G 24:2

11
36
13

3
21
18

13
30
17

5
18
19

24
36

26
16

p value

0.72
0.84

0.91

0.34
p < 0:05

p ¼ 0:182

p < 0:05

a Mean G SE. b NBNC; patients without HBs-Ag(þ) nor HCV-Ab(þ)
c included tumor thrombus in the portal vein.

thrombi or satellite nodules surrounding a large main
tumor represent intrahepatic metastases, whereas othernodules can be considered multicentric HCCs if theysatisfy any of the following three criteria (1) multiple,
small early-stage HCCs or concurrent small early-stage

92

Chuma M, et al: Expression proﬁling in hepatocellular carcinoma

HCCs and classical HCCs; (2) presence of peripheral
areas of well differentiated HCC in both lesions or in
the smaller ones; and (3) multiple HCCs of obviously
different histology. All the studies using these humanmaterials had been approved by the Institutional Review Board.
RNA preparation and oligonucleotide array
Total RNA was extracted from bulk tissues using
Trizol reagent (Invitrogen Corp., Carlsbad, CA). Biotin-labeled cRNA was synthesized from 10 mg of total
RNA derived from each sample using a Super Script
Choice System (Gibco-BRL, Rockville, MD) and a
BioArray High Yield RNA Transcript Labeling Kit(Enzo Diagnostics, Farmingdale, NY), according to the
manufacturers’ instructions. The hybridization of eachcRNA to the probe array, HG-U95Av2 (Affymetrix,
Santa Clara, CA), and the detection of the signals were
performed as instructed by the manufacturer. A previously described method was used for the data analysis,16 and the hybridization intensity data were normalized to the nearest 1000 of the total signal intensity ineach array.
Real-time quantitative RT-PCR analysis
For the RT-PCR analysis, all RNA samples weretreated with DNase I (Promega Corp., Madison, WI)
to remove genomic DNA. The real-time quantitative
RT-PCR analysis was then performed using a previously reported method.20 The primer set 5 0 -ACTGGAGTCTCCTGTGGTGTGT-3 0 (forward) and 5 0 AGTGCTATTTCCCCTCCCTTC-3 0 (reverse) was
designed for the 3 0 untranslated region of HMGI. To
standardize the amount of RNA, the expression ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH)
in each sample was quantiﬁed using the primer set 5 0 GAAGGTGAAGGTCGGAGTC-3 0 (forward) and 5 0 CCCGAATCACATTCTCCAAGAA-3 0 (reverse). All
PCR reactions were performed using the SYBR GreenPCR Core Reagents kit (Perkin-Elmer Applied Biosystems, Foster City, CA) under the following conditions: 1 cycle at 50 C for 2 minutes; 1 cycle at 95 C for
10 minutes; and 40 cycles at 95 C for 15 seconds, 55 C
for 30 seconds, and 72 C for 1 minute. Real-time detection of the emission intensity of SYBR Green was
performed using an ABI prism 7700 Sequence Detector(Perkin-Elmer Applied Biosystems), using a previously
reported method.20 Quantitative RT-PCR was performed at least three times, including the use of a notemplate run as a negative control. Statistical analyses
were performed using unpaired t-tests.
Immunohistochemistry
Immunohistochemical staining was performed us-

ing formalin-ﬁxed, parafﬁn-embedded tissue sections
and previously described immunoperoxidase method.21
Brieﬂy, each section was deparafﬁnized, rehydrated,
and incubated with fresh 0.3% hydrogen peroxide inmethanol for 30 minutes at room temperature and thenwashed in phosphate-buffered saline. Normal swine serum (DAKO, Glostrup, Denmark) was applied for 30
minutes and removed. The sections were then incubated with goat polyclonal antibodies for HMGI (SC1564; Santa Cruz Biotechnology, Santa Cruz, CA) at adilution of 1 : 250 overnight at 4 C, washed three times
in phosphate-buffered saline, and incubated with secondary antibody for 30 minutes at room temperature. A
Vectastatin Elite ABC kit (Vector Laboratories, Burlingame, CA) was used to detect the antibody signals.
Staining evaluation
Staining was evaluated by two independent observers. An equal or more intense nuclear staining, compared with that in lymphocytes, was considered a positive result. Correlations between the overexpression of
HMGI protein and the clinicopathological features ofthe specimens were assessed, and the resulting data
were analyzed using the w 2 test and the Fisher’s exact
test. All statistical analyses were done using Stat View
(Version 5.0) software (Abacus Concepts, Berkeley,CA). A value of P < 0:05 was considered signiﬁcant.
Results
Identiﬁcation of geneswith different expression proﬁles in
HCCsFirst, we listed the upregulated and downregulated
genes in each tumor and selected the genes with thefollowing criteria: (1) presence (i.e., expressed in thesample), (2) more than 1,000 times the average difference (mean of the difference in signal intensities between the match and mismatch probe set), and (3) amore than 2-fold increase or decrease in the average
difference when compared to corresponding noncancerous liver tissues. Of the 142 genes that met the above
criteria, 61 genes were up-regulated in 50% or more of
the specimens and 81 genes were down-regulated in
50% or more of the specimens.
Two-way hierarchical clustering algorithm
To identify genes generally involved in intrahepatic
metastasis, we compared the expression proﬁles of the
20 HCCs with intrahepatic metastasis and the 10 HCCs
without intrahepatic metastasis using an oligonucleotide array and the 142 genes identiﬁed in the experiment described above. Next, we ﬁltered the 142 genes

Keio J Med 2004; 53 (2): 90–97

93

Fig. 1 A two-way hierarchical clustering algorithm. Cluster map and phylogenetic tree resulting from a two-way, pair-wise, average-linkage
cluster analysis. Each color patch in the resulting visual map represents the expression level of the associated gene in that tissue sample, with acontinuum of expression levels from blue (lowest) to bright red (highest). The two-way hierarchical clustering algorithm was able to successfully
distinguish between HCC with intrahepatic metastasis and HCC without intrahepatic metastasis. The scale bar reﬂects the fold increase (red) or
decrease (blue) for any given gene relative to the median level of expression across all samples.

using (4) a Mann-Whitney U-test with the signiﬁcanceset at P < 0:05 to identify genes expressed differently
between the two groups. Finally, 34 genes wereselected, and a two-way hierarchical clustering algorithm was used to successfully distinguished between
HCC with intrahepatic metastasis and HCC without
intrahepatic metastasis (Fig. 1). Nineteen of the 34
genes were up-regulated and 15 genes were down-

regulated in HCC with intrahepatic metastasis, compared to their levels in HCC without intrahepatic
metastasis.
HMG-I(Y) mRNA expression in HCC
Of the listed 19 genes that are up-regulated in HCC
with intrahepatic metastasis, we selected HMG-I(Y)94

Chuma M, et al: Expression proﬁling in hepatocellular carcinoma

Fig. 2 Real-time quantitative RT-PCR analysis. (A) The ratio of relative HMG-I(Y) mRNA expression levels in 30 HCCs. The expression
levels were normalized with GAPDH mRNA in each sample. The ratio of relative HMG-I(Y) mRNA expression (HMGI/GAPDH) in HCCs
(T) to that in corresponding noncancerous liver tissues (N) is indicated. (B) The average ratio in HCC with intrahepatic metastasis ( ) was
upregulated (2:37 G 0:17) versus that in HCC without intrahepatic metastasis ( ) (1:40 G 0:13), P < 0:01. Bars, SE.

and analyzed the relative expression level of HMGI(Y) mRNA (HMG-I(Y)/GAPDH) using real-timequantitative RT-PCR. In 12 of the 20 cases of HCC
with intrahepatic metastasis, the ratio of the relative
HMG-I(Y) mRNA expression in the HCC specimens
to the relative HMG-I(Y) mRNA expression in corresponding noncancerous liver tissues was more than twofold; the same was true in 2 out of the 10 cases of HCCs
without intrahepatic metastasis (Fig. 2A). The average
ratio in HCCs with intrahepatic metastasis was upregulated (2:37 G 0:17) versus that in HCCs without intrahepatic metastasis (1:40 G 0:13), P < 0:01 (Fig. 2B).

hepatic metastasis showed strong HMG-I(Y) immunoreactivity (Fig. 3B). To evaluate the relation between
HMG-I(Y) expression and prognostic factors, we divided these patients into two groups: an HMG-I(Y) positive expression (more than 20%) group and a low
HMG-I(Y) expression (lower than 20%) group. The
relations between HMG-I(Y) and seven prognostic
factors were then examined. Table 1 shows that tumor
histology grade and intrahepatic were signiﬁcantly
associated with HMG-I(Y) expression (P < 0:05).

Protein expression of HMG-I(Y)The recent development of cDNA microarray oroligonucleotide array technology, a high-throughput
method of monitoring gene expression, has made it
possible to analyze the expression of thousands ofgenes at once.22,23 Consequently, new classiﬁcations ofcancers can now be proposed based on the altered expression proﬁles of multiple genes in tumor tissues.10–16
For example, molecularly distinct subtypes have beenrecognized on the basis of differences in gene expression patterns in multistage hepatocarcinogenesis.16 To
investigate the detailed genetic mechanisms in cancer
metastasis, we compared the expression proﬁles of 20
HCCs with intrahepatic metastasis and 10 HCCs without intrahepatic metastasis using an oligonucleotide array. We showed that a two-way hierarchical clustering
algorithm successfully distinguished between HCC with

To determine whether HMG-I(Y) was also overexpressed at the protein level and if an increase in expression was associated with intrahepatic metastasis, we
performed an immunohistochemical study using an antibody against HMG-I(Y) (Fig. 3). Hepatocytes in noncancerous liver tissue with chronic hepatitis or cirrhosis
showed no immunostaining or only focal or faint staining in the nucleus. However, the lymphocytes always
stained strongly and thus served as an internal control
for positive staining (Fig. 3B). As shown in Fig. 3,
strong immunoreactivity was observed in the carcinoma
cells, localized mainly in the nucleus. Some HCC cases
without intrahepatic metastasis showed little HMGI(Y) immunoreactivity, whereas HCC cases with intra-

Discussion

Keio J Med 2004; 53 (2): 90–97

95

Fig. 3 Immunohistochemical localization of HMG-I(Y) in HCC. Representative histology of HCC with intrahepatic metastasis (A) (hematoxylin–eosin stain; original magniﬁcation, 20), and HMG-I(Y) immunostaining of the same section (B). Lymphocytes were used as a positive
control (arrows). HCC cases with intrahepatic metastasis showed strong HMG-I(Y) immunoreactivity, mainly in the nucleus.

intrahepatic metastasis and HCC without intrahepatic
metastasis. These results indicate that the molecularsignatures of HCC with intrahepatic metastasis and of
HCC without intrahepatic metastasis are distinctly different. We also identiﬁed 34 genes whose expression
levels were signiﬁcantly correlated with metastatic ability. We further showed that HMG-I(Y) was associatedwith intrahepatic metastasis and differentiation, although the reason why HMG-I(Y) expression increases
in HCC with intrahepatic metastasis and poorly differentiated HCC is not clear at present. The mammalian
HMG-I, HMG-Y, and HMGI-C proteins are membersof the HMG-I(Y) family of nonhistone chromatin proteins,24 with the former two proteins being derived
from the alternative processing of RNA transcripts
from a single functional gene.25 HMG-I(Y) has been
implicated in both positive and negative regulation ofgene transcription in vivo.26 Numerous studies havedemonstrated that HMG-I(Y) overexpression is frequently associated with both the neoplastic transformation of cells and metastatic tumor progression.27–29
Overexpression of HMG-I(Y) has been described inseveral cancers,29–33 and has been correlated with differentiation status.33 Moreover, signiﬁcant correlation
between elevated HMG-I(Y) expression and a poor
prognosis has been reported in patients with prostate33
and breast cancers.34 Recently, HMG-I(Y) has beenshown to be a c-myc target gene involved in neoplastictransformation.35 Several reports have concluded thatthe ampliﬁcation and overexpression of c-myc is signiﬁcantly correlated with the prognosis of HCC.36
Thus, we speculated that these interactions exist in
HCC and mediate key aspects of HCC progression.
In addition to HMG-I(Y), we were able to identify

several other candidate genes involved in intrahepatic metastasis. Some of the up-regulated genes
identiﬁed in the present analysis have been previously
correlated with tumor malignancy and metastasis inseveral types of cancers. For example, osteopontin is
known to be a secreted adhesive glycoprotein thatis associated with clinical outcome in prostate37 andbreast38 cancer and is also involved in intrahepatic metastasis in HCC.14 CD24 is a small heavily glycosylated
glycosylphosphatidylinositol-linked cell surface protein, which is expressed in a large variety of carcinomas,
including HCC.39 Aldose reductase, which efﬁciently
reduces aliphatic and aromatic aldehydes, is also overexpressed in HCC.40 RhoC, a ras-related GTP-binding
protein of member C in the rho subfamily, regulates thereorganization of the actin cytoskeleton and is speciﬁcally expressed in invasive breast carcinomas capableof metastasis.41 RhoC is also associated with tumor
progression in ovarian carcinoma42 and is involved invenous invasion of HCC.13 14-3-3 sigma, a member ofthe 14-3-3 family, plays a role in the G2 checkpoint response to DNA damage, and the epigenetic inactivationof 14-3-3 sigma caused by aberrant hypermethylation
has been reported in several cancers.43,44 However,
Christine recently reported the overexpression of 14-33 sigma in pancreatic cancer and the hypomethylationof the 14-3-3 sigma promoter.45 We also conﬁrmed
the overexpression of 14-3-3 sigma in HCC with intrahepatic metastasis using real-time quantitative RT-PCR
and immunohistochemistry (unpublished observations).
The overexpression of 14-3-3 sigma in HCC may be related to the anti-apoptotic role that has been described
for this gene. It would be necessary to analyze the expression and methylation status of the 14-3-3 sigma in96

Chuma M, et al: Expression proﬁling in hepatocellular carcinoma

larger HCC samples. Further analysis of these genes
should improve our understanding of intrahepatic metastasis.
In conclusion, the molecular signatures of HCC withintrahepatic metastasis and HCC without intrahepatic
metastasis were clearly different. HMG-I(Y) was associated with intrahepatic metastasis and may be a predictive marker for HCC with intrahepatic metastasis.

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13.
Acknowledgments: This study was supported by a Grant-Aid for theSecond Term Comprehensive 10-Year Strategy for Cancer Control
from the Ministry of Health, Labor and Welfare of Japan, for Scientiﬁc Research (B) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan, and by the Program for Promotionof Fundamental Studies in Health Sciences of the Organization for
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