Identification of overexpressed genes in hepatocellular carcinoma, with special reference to ubiquitin-conjugating enzyme E2C gene expression.
ABSTRACT This study consisted of 2 aims: (i) to determine genes associated with hepatocellular carcinoma (HCC) by microarray analysis; and (ii) to evaluate the clinicopathological significance of human ubiquitin-conjugating enzyme E2C (Ube2c) found to be overexpressed in HCC from microarray analysis. Laser microdissection and cDNA-microarray were performed to identify genes associated with HCC. We then focused on the Ube2c gene. Using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR), Ube2c expression status and clinicopathological significance were studied in 65 clinical HCC samples. A number of genes upregulated in HCC cells compared to noncancerous liver cells were identified, one of which was the Ube2c gene. Ube2c gene expression in the cancer tissue was higher than in the corresponding noncancerous tissue in 62 of the 65 cases (95.4%, p < 0.01). Tumors with high Ube2c expression showed higher frequencies of tumor invasion to capsular formation (fc-inf), invasion to portal vein (vp) and tumor de-differentiation (p < 0.05). Patients with high Ube2c expression also showed significantly worse disease-free survival rates than those with low Ube2c expression (p < 0.01). In addition, Ube2c expression was found to be an independent prognostic factor for disease-free survival rate in multivariate analysis. We identified differentially expressed genes between HCC and normal liver tissues. Of those, the Ube2c gene appeared to be associated with HCC progression, and may be useful as a prognostic indicator for HCC patients.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Cyclins are essential for cell proliferation, the cell cycle and tumorigenesis in all eukaryotes. UbcH10 regulates the degradation of cyclins in a ubiquitin-dependent manner. Here, we report that UbcH10 is likely involved in tumorigenesis. We found that cancer cells exposed to n-acetyl-leu-leu-norleucinal (ALLN) treatment and UbcH10 depletion exhibit a synergistic therapeutic effect. Abundant expression of UbcH10 drives resistance to ALLN-induced cell death, while cells deficient in UbcH10 were susceptible to ALLN-induced cell death. The depletion of UbcH10 hindered tumorigenesis both in vitro and in vivo, as assessed by colony formation, growth curve, soft agar and xenograft assays. These phenotypes were efficiently rescued through the introduction of recombinant UbcH10. In the UbcH10-deficient cells, alterations in the expression of cyclins led to cell cycle changes and subsequently decreases in tumorigenesis. The tumorigenesis of xenograft tumors from UbcH10-deficient cells treated with ALLN was decreased relative to wild-type cells treated with ALLN in nude mice. On the molecular level, we observed that UbcH10 deficiency enhances the activation of caspase 8 and caspase 3 but not caspase 9 to impair cell viability upon ALLN treatment. Collectively, our results suggest that, as an oncogene, UbcH10 is a potential drug target for the treatment of colorectal cancer.Scientific reports. 01/2014; 4:6910.
- [Show abstract] [Hide abstract]
ABSTRACT: Background:We previously conducted gene expression microarray analyses to identify novel indicators for colorectal cancer (CRC) metastasis and prognosis from which we identified PVT-1 as a candidate gene. PVT-1, which encodes a long noncoding RNA, mapped to chromosome 8q24 whose copy-number amplification is one of the most frequent events in a wide variety of malignant diseases. However, PVT-1 molecular mechanism of action remains unclear.Methods:We conducted cell proliferation and invasion assays using colorectal cancer cell lines transfected with PVT-1siRNA or negative control siRNA. Gene expression microarray analyses on these cell lines were also carried out to investigate the molecular function of PVT-1. Further, we investigated the impact of PVT-1 expression on the prognosis of 164 colorectal cancer patients by qRT-PCR.Results:CRC cells transfected with PVT-1 siRNA exhibited significant loss of their proliferation and invasion capabilities. In these cells, the TGF-β signalling pathway and apoptotic signals were significantly activated. In addition, univariate and multivariate analysis revealed that PVT-1 expression level was an independent risk factor for overall survival of colorectal cancer patients.Conclusion:PVT-1, which maps to 8q24, generates antiapoptotic activity in CRC, and abnormal expression of PVT-1 was a prognostic indicator for CRC patients.British Journal of Cancer advance online publication 5 November 2013; doi:10.1038/bjc.2013.698 www.bjcancer.com.British Journal of Cancer 11/2013; · 5.08 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Hepatocellular Carcinoma is a primary malignant tumor of the liver and gankyrin is an oncoprotein over-expressed in hepatocellular carcinoma. It has been found that Gankyrin protein reduces the level of p53 protein by increasing its ubiquitylation and deg-radation, following a MDM-2 mediated pathway. In-teraction of gankyrin with MDM2 enhances the ubi-quitylation of p53. Independent study of this protein molecule revealed that it is identical to the p28 sub-unit of the 26S proteasome, having seven similar al-pha helical ankyrin repeats. Gankyrin also binds to the Tumor Suppressor Protein (TSP) Retinoblastoma (RB), thereby accelerating its phosphorylation and proteasomal degradation. Blocking the expression of Gankyrin with MDM2 in cases of Hepatocellular Carcinoma (HCC) promoted apoptosis in cancer cells. Hence, Gankyrin can be used as a potential target for drug therapy against Hepatocellular Carcinoma.
Identification of overexpressed genes in hepatocellular carcinoma, with special
reference to ubiquitin-conjugating enzyme E2C gene expression
Keisuke Ieta1,2, Eiki Ojima1, Fumiaki Tanaka1, Yoshito Nakamura1, Naotsugu Haraguchi1, Koshi Mimori1, Hiroshi Inoue1,
Hiroyuki Kuwano2and Masaki Mori1*
1Department of Surgery and Molecular Oncology, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara,
Beppu 874-0838, Japan
2Department of General Surgical Science, Graduate School of Medicine, Gunma University, 3-39-22 Showa-machi,
Maebashi, Gunma, Japan
This study consisted of 2 aims: (i) to determine genes associated
with hepatocellular carcinoma (HCC) by microarray analysis;
and (ii) to evaluate the clinicopathological significance of human
ubiquitin-conjugating enzyme E2C (Ube2c) found to be overex-
pressed in HCC from microarray analysis. Laser microdissection
and cDNA-microarray were performed to identify genes associ-
ated with HCC. We then focused on the Ube2c gene. Using real-
time quantitative reverse transcription-polymerase chain reaction
(RT-PCR), Ube2c expression status and clinicopathological signifi-
cance were studied in 65 clinical HCC samples. A number of genes
upregulated in HCC cells compared to noncancerous liver cells
were identified, one of which was the Ube2c gene. Ube2c gene
expression in the cancer tissue was higher than in the correspond-
ing noncancerous tissue in 62 of the 65 cases (95.4%, p < 0.01).
Tumors with high Ube2c expression showed higher frequencies of
tumor invasion to capsular formation (fc-inf), invasion to portal
vein (vp) and tumor de-differentiation (p < 0.05). Patients with
high Ube2c expression also showed significantly worse disease-free
survival rates than those with low Ube2c expression (p < 0.01). In
addition, Ube2c expression was found to be an independent prog-
nostic factor for disease-free survival rate in multivariate analysis.
We identified differentially expressed genes between HCC and
normal liver tissues. Of those, the Ube2c gene appeared to be asso-
ciated with HCC progression, and may be useful as a prognostic
indicator for HCC patients.
' 2007 Wiley-Liss, Inc.
Key words: hepatocellular carcinoma; microarray; laser microdissection
(LMD); ubiquitin-conjugating enzyme E2C (Ube2c)
Hepatocellular carcinoma (HCC) is one of the most common
cancers in Japan, and its prevalence is increasing in America.
Most Japanese HCC cases develop from liver cirrhosis that is
almost entirely due to chronic hepatitis C or B.1–3Although recent
advances in molecular biology have elucidated the developmental
pathway of HCC from liver cirrhosis, few studies have determined
the differences in gene expression profiles between HCC and nor-
mal liver tissues. Therefore, we analyzed for differentially
expressed genes between cancerous tissues from HCC patients
and noncancerous liver tissues without liver cirrhosis from
patients with metastatic liver tumors using laser microdissection
(LMD) and cDNA-microarray. As a result, we detected 123 genes
that were overexpressed by more than 2-fold in HCC compared to
normal liver in at least 4 of the 6 samples examined. In the past,
our group has worked on the ubiquitination system in cancer cells.
For example, S-phase kinase-associated protein 2 (Skp2), a mem-
ber of the F-box family of substrate-recognition subunits of Skp1-
Cullin-F-box ubiquitin-protein ligase complexes, is necessary for
p27 ubiquitination and degradation. We reported that Skp2 gene
overexpression appeared to act as a prognostic factor for gastric
cancer and breast cancer.4–6Because of this interest, of the genes
observed to be upregulated in HCC, we decided to focus on the
Ube2c gene due to its involvement in the ubiquitination pathway.
The Ube2c gene, located on chromosome 20q13, belongs to the
E2 gene family and codes for a 19.6 kDa protein involved in ubiq-
uitin-dependent proteolysis. Rape and Kirschner showed that
cyclin A degradation was highly sensitive to the concentration of
Ube2c, and self-degradation of Ube2c is an autonomous sensor of
mitotic completion and provides the molecular switch that allows
cells to proceed from DNA segregation and cell division to a new
round of DNA duplication.7,8Ube2c has also been reported to be
highly expressed in various types of cancers.9–13However, the
relationship between Ube2c expression and clinicopathological
factors in HCC has not yet been investigated.
In the present study, we report the identification of overex-
pressed genes in HCC by LMD and cDNA-microarray analysis,
and then examine the clinicopathological significance of the
Ube2c gene, detected as an overexpressed gene in HCC patients.
Material and methods
Sixty-five patients with HCC were enrolled into this study. All
patients underwent resection of the primary tumor at the Kyushu
University Hospital at Beppu and affiliated hospitals between
2001 and 2003. Resected tumor and paired nontumor tissue speci-
mens were immediately cut from the resected liver and placed in
RNA Layter (TaKaRa, Japan) or embedded in Tissue Tek OCT
medium (Sakura, Tokyo, Japan), frozen in liquid nitrogen and
kept at 280?C until RNA extraction. Written informed consent
was obtained from all patients. The follow-up period ranged from
0.1 to 4.3 years with a median of 2.5 years.
Identification of overexpressed genes in HCC
Samples. For the identification of overexpressed genes in
HCC, 6 randomly selected HCC cases were used (hepatitis B virus
(HBV) (1): 2 cases, hepatitis C virus (HCV) (1): 2 cases, HBV
(2) HCV (2): 2 cases). As a control, the samples of noncancerous
liver tissues were obtained from another 6 cases. These 6 were the
Grant sponsors: CREST, Japan Science and Technology Agency (JST);
Japan Society for the Promotion of Science (JSPS); Grant numbers:
18790964; Grant sponsor: The Ministry of Education, Culture, Sports, Sci-
ence and Technology (MEXT); Grant number: 18015039; Grant sponsor:
Third Term Comprehensive Ten-year Strategy for Cancer Control; Grant
*Correspondence to: Department of Surgery and Molecular Oncology,
Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara,
Beppu 874-0838, Japan. Fax: 181-977-27-1650/1651.
Received 11 November 2006; Accepted after revision 27 December
Published online 12 March 2007 in Wiley InterScience (www.interscience.
This article contains supplementary material available via the Internet at
Abbreviations: GAPDH, glyceraldehydes-3-phosphate dehydrogenase;
HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C
virus; LMD, laser microdissection; PCR, polymerase chain reaction; RT-
PCR, reverse transcription-polymerase chain reaction; Ube2c, ubiquitin-
conjugating enzyme E2C.
Int. J. Cancer: 121, 33–38 (2007)
' 2007 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
patients with metastatic liver tumor, and the liver showed normal
appearance without cirrhosis or infection with HCV or HBV.
Laser microdissection and RNA extraction. Cancer tissues
were microdissected using the LMD system (Leica Laser Micro-
dissection System, Leica Microsystems, Wetzlar, Germany) as
previously described.14Total RNA was extracted using an Rneasy
Mini Kit (Qiagen, Hilden, Germany) according to the manufac-
turer’s instructions. Total RNA from noncancerous samples was
obtained from 6 normal liver samples. Purity and concentration of
the RNA samples were determined with a Nano Drop (Nano Drop
Technologies, Wilmington) and Agilent 2100 Bioanalyzer (Agi-
lent Technologies, Palo Alto) as previously described.15
cDNA-microarray. T7-based RNA amplification was per-
formed using the Low RNA Input Fluorescent Linear Amplifica-
tion Kit (Agilent Technologies, USA). Total RNA (100 ng) was
reverse transcribed to cDNA using MMLV-RT and oligo dT pri-
mers, and used as a template for in vitro transcription reactions in
the presence of Cyanine labeled CTP (NEN Life Science, Boston,
MA) and T7 RNA polymerase. cRNA from noncancerous tissues
was labeled with Cyanine 3-CTP (Cy3), while cancer tissue cRNA
was labeled with Cyanine 5-CTP (Cy5). After purification using
an Rneasy Mini Kit, aliquots of the amplified cRNA were vali-
dated on an Agilent 2100 Bioanalyzer. The Cy3 and Cy5 labeled
cRNA were mixed and hybridized to a cDNA-microarray (Agilent
Human1:12814genes, Agilent Technologies, USA). A list of genes
on this cDNA microarray is available from http://www.agilent.-
com/chem/genelists. Scanning of the array slides was performed
using an Agilent dual laser DNA microarray scanner (Agilent
Data analysis. The intensity of each hybridization signal was
evaluated using Feature Extraction software (Agilent Technolo-
gies, USA). The common logarithm of the Cy5/Cy3 ratio for each
sample was calculated by averaging the spots. A cutoff value for
expression level was automatically calculated according to the
background fluctuation. Normalization of expression levels was
performed using LOWESS (locally weighted linear regression
curve fit) normalization.16Rosetta Laminator system version 2.0
(Rosetta Biosoftware, Kirkland, USA) was used to analyze gene
expression data. Candidate genes which fulfilled the following cri-
teria were selected: the fold changes were >2.0, and the p value
was <0.01. Moreover, within the selected genes that met these cri-
teria, genes that were upregulated in 4 or more samples (6 total
samples) were analyzed.
Evaluation of Ube2c gene expression
Clinical samples and cell lines. For evaluation of Ube2c gene
expression, all 65 tumors and nontumor samples, as described
above, were used. Human HCC cell lines (HuH-7, Hep-G2, Hep-
3B) were provided by the Cell Resource Center of Biomedical
Research, Institute of Development, Aging and Cancer (Tohoku
University, Sendai, Japan) and maintained in RPMI 1640 medium
or DMEM containing 10% fetal bovine serum (FBS) and antibiot-
ics at 37?C in a 5% humidified CO2atmosphere.
Oligonucleotide primers for Ube2c gene amplification by
RT-PCR. Total RNA was extracted from each clinical sample
and cDNA synthesized from 8.0 lg total RNA as previously
Ube2c-specific oligonucleotide primers were designed to give a
165 bp PCR product: sense primer 50-GGATTTCTGCCTTCCCT-
GAA-30; antisense primer 50-GATAGCAGGGCGTGAGGAAC-
30.12Primers were also designed for glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) (270 bp): sense primer 50-TTG-
GTATCGTGGAAGGACTCA-30; antisense primer 50-TGTCAT-
CATATTTGGCAGGTT-30. To avoid amplification of contami-
nating genomic DNA, the primers spanned more than 2 exons.
Amplification was performed for 29 cycles (22 cycles for
GAPDH) of 1 min at 95?C, 1 min at 60?C (56?C for GAPDH) and
1 min at 72?C. An 8.0 ll aliquot of each PCR-amplified DNA was
electrophoresed on 2% agarose gels containing ethidium bromide.
Real-time quantitative RT-PCR. PCR amplification for quanti-
fication of Ube2c and GAPDH mRNA in 65 clinical samples was
performed using the LightCycler system (Roche Applied Science,
IN) and the LightCycler-FastStart DNA Master SYBR Green I kit
(Roche Applied Science, IN) as previously described.17Amplifi-
cation conditions consisted of initial denaturation at 95?C for
10 min, followed by 40 cycles of denaturation at 95?C for 10 s,
annealing at 64?C (60?C for GAPDH) for 10 s and elongation at
72?C for 10 s. Melting curve analysis and electrophoresis on 2%
agarose gels were performed to ensure that the expected PCR
products were generated. To quantitate specific mRNA in the sam-
ples, a standard curve was produced for each run based on 3 points
from diluted HuH-7 cDNA. Relative Ube2c expression levels
were then obtained by normalizing the amount of Ube2c mRNA
divided by that of GAPDH mRNA as an endogenous control in
Western blot analysis. Total protein was extracted from 4 rep-
resentative pairs of samples and cell lines using protein extrac-
tion solution (PRO-PREP, iNtRON Biotecnology, Korea). Ali-
quots of total protein (60 lg for clinical samples or 20 lg for cell
lines) were electrophoresed in 12.5% concentrated READY
GELS J (Bio-Rad Laboratories, Japan) and then electroblotted
onto pure nitrocellulose membranes (Trans-Blot Transfer Me-
dium; Bio-Rad Laboratories, Japan) at 0.2 A for 120 min at 4?C.
Ube2c protein was detected using goat polyclonal antibody
(AB3935, Abcam, USA) diluted 1:200. Ube2c protein levels
were normalized to the level of b-actin protein (Cytoskelton,
Denver, CO) diluted 1:1,000. Blots were developed with horse-
radish peroxidase-linked anti-goat immunoglobulin (Promega,
Madison, WI) diluted 1:1,000. ECL Detection Reagents (Amer-
sham Biosciences, Piscataway, NJ) were used to detect antigen-
Immunohistochemistry. Immunohistochemical studies of Ube2c
were performed on surgical specimens from representative HCC
patients. Formalin-fixed, paraffin-embedded tissues were deparaffi-
nized, blocked, incubated with specific antibodies overnight at 4?C,
and detected using ENVISION reagents (ENVISION1 Dual Link/
HRP, Dako Cytomation, Denmark). All sections were counter-
stained with hematoxylin. Primary goat polyclonal anti-Ube2c anti-
body (AB3935, Abcam, USA) was used at a dilution of 1:200.
Ube2c RNA interference. Ube2c-specific siRNA (SilencerTM
Predesigned siRNA) and negative control siRNA (SilencerTM
Negative Control siRNA) were purchased from Ambion, USA.
Logarithmic growth-phase Hep3B cells were seeded at 1.5 3 105
or 2 3 103cells/well in a final volume of 2 ml or 100 ll, respec-
tively, in 6 or 96 well flat bottom microtitre plates, respectively,
and then cultured overnight to allow adherence. siRNA-Lipofecta-
mineTM2000 complexes were then added to each well as previ-
ously described,18and in vitro proliferation assay were performed
after 48 hr incubation from siRNA addition.
In vitro proliferation assay. Proliferation was determined by
(MTT) assay (Roche Diagnostics Corp., GmbH). After 48 hr incu-
bation from siRNA addition, cells were further cultured for 0–
96 hr. After 0–96 hr culture, spectrophotometrical absorbance of
the samples was measured as previously described.18Each inde-
pendent experiment was performed 3 times.
Statistical analysis. For continuous variables, data were
expressed as the means 6 SD. Differences between groups were
estimated using Student’s t test, v2 test, and repeated measures
ANOVA analysis. We applied the Student’s t-test for data in nor-
mal distribution and the nonparametric Wilcoxon/Kruskal–Wallis
tests for data without normal distribution. Overall survival curves
and disease-free survival curves were plotted according to the
Kaplan-Meier method, and measured from the day of surgery,
with the log-rank test applied for comparisons. Variables with a
value of p < 0.15 by univariate analysis were used in subsequent
multivariate analyses based on Cox’s proportional hazards model.
All differences were deemed significant at the level of p < 0.05.
IETA ET AL.
Statistical analyses were performed using the JMP 5 for Windows
software package (SAS Institute, Cary, NC).
Identification of overexpressed genes in HCC
Using a human cDNA-microarray, we determined differentially
expressed genes between HCC and noncancerous liver cells. We
identified 123 upregulated genes in the cancer cells compared to
the noncancerous cells (Supplementary 1). These genes were over-
expressed more than 2-fold in at least 4 of 6 cases, and included
genes previously reported to be associated with HCC, such as
PEG10.19–28Genes involved in cell cycle regulation or cell adhe-
sion, as well as growth factors and proteinases were also overex-
pressed and so could be considered as candidate cancer-related
genes. One of the overexpressed genes was Ube2c, associated
Evaluation of Ube2c gene expression
Expression of Ube2c mRNA in cell lines and clinical tissue
specimens. Ube2c mRNA expression in cell lines was examined
by reverse transcription-polymerase chain reaction (RT-PCR) and
revealed that all 3 cell lines tested, HuH-7, Hep-G2 and Hep-3B,
highly expressed Ube2c mRNA. Ube2c mRNA expression in can-
cerous and noncancerous tissues of HCC patients was examined
by RT-PCR and real-time quantitative PCR, with quantified values
used to calculate Ube2c/GAPDH expression ratios. Results indi-
cated that Ube2c mRNA expression levels were higher in cancer
tissues (0.074 6 0.066) than in noncancerous tissues (0.012 6
0.014) in 62 of the 65 cases (95.4%). This resulted in a significant
difference in mRNA expression level between cancer and normal
tissues (p < 0.01) (Figs. 1a and 1b). We classified the 65 HCC
cases into 2 groups according to median Ube2c mRNA expression
level in tumor tissues, as determined by quantitative RT-PCR, to
give high (n 5 33) and low (n 5 32) expression groups.
The clinicopathological significance of Ube2c mRNA expres-
sion in HCC. Clinicopathological features were analyzed in rela-
tion to Ube2c expression status (Table I). The incidence of tumor
invasion to capsular formation (fc-inf) was significantly higher (p
< 0.05) in the high expression group (23 of 33 fc-inf positive,
69.7%) than in the low expression group (13 of 32 fc-inf positive,
40.6%). Likewise, the incidence of invasion to portal vein (vp)
was higher (p < 0.05) in the high expression group (23 of 33 vp
positive, 69.7%) than in the low expression group (13 of 32 vp
positive, 40.6%), and poorly-differentiated HCC showed higher
Ube2c expression levels than well-differentiated HCC (p < 0.05).
No other significant differences were observed with respect to age,
gender, tumor size, capsular formation (fc), invasion to hepatic
vein (vv), invasion to bile duct (b) and number of tumors.
Analysis of disease-free survival curves showed that patients in
the high expression group had a significantly poorer prognosis
than those in the low expression group (p < 0.01) (Fig. 2). How-
ever, overall survival rates between the 2 groups were not statisti-
cally different (data not shown).
Univariate analysis identified Ube2c expression (low or high
expression), tumor size (? or >3 cm) and number of tumors (soli-
tary or multiple) as adverse prognostic factors for disease-free sur-
vival after hepatic resection. Variables with a p value of less than
0.15 by univariate analysis were selected for multivariate analysis
using Cox’s proportional hazards model. Ube2c expression (rela-
tive risk (RR): 1.51, confidence interval (CI): 1.06–2.22, p 5
0.02) was found to be a significant factor affecting disease-free
survival rate following hepatic resection (Table II).
Ube2c protein expression in clinical tissue specimens. Ube2c
protein expression in tumor and normal tissues from representative
FIGURE 1 – (a) Expression of Ube2c mRNA as assessed by RT-PCR in representative HCC cases. (T, cancer tissue; N, noncancerous tissue;
n, negative control; p, positive control; m, marker) (b) Ube2c mRNA expression in cancer and noncancerous tissues from HCC patients as
assessed by real-time quantitative PCR (n 5 65). Horizontal lines indicate mean value of each group. (T, cancer tissue; N, noncancerous tissue)
(c) Expression of Ube2c protein by western blot in representative HCC patient tissues. Ube2c protein was detected as a band of ?20 kDa. (T,
cancer tissue; N, noncancerous tissue; p, positive control; m, marker) (d) Immunohistochemistry with Ube2c antibody on HCC patient samples.
The majority of staining occurred in cancer cells. (a): cancer tissue, original magnification 3200, Ube2c stain, (b): noncancerous tissue, original
magnification 3200, Ube2c stain.
OVEREXPRESSED GENES IN HCC; A SPECIAL REFERENCE TO Ube2c
HCC patients was examined by western blot. Strong Ube2c pro-
tein expression was observed in the cancer tissues (Fig. 1c).
Immunohistochemical staining. Ube2c staining was stronger in
cancer tissues than in corresponding noncancerous liver tissues.
Ube2c expression was localized to cell nuclei (Fig. 1d). Expres-
sion of Ube2c in poorly differentiated HCC was detected in nuclei
Effect of Ube2c gene silencing on an HCC cell line. As
described earlier, Hep-3B cells showed high Ube2c expression
levels. Suppression of Ube2c mRNA was confirmed by real-time
quantitative PCR (50% suppression) (Fig. 3a). Protein expression
was suppressed by Ube2c-specific siRNA in western blots
(Fig. 3b). As shown in Figure 3c, suppression of Ube2c inhibited
the proliferation rate of Hep3B HCC cells (96 hr Ube2c siRNA:
1.92 6 0.40, negative siRNA: 2.97 6 0.36, control: 3.29 6 0.29)
(p < 0.01).
This study identified differentially expressed genes between
HCC and normal liver tissues (Supplementary 1). Diubiquitin
(FAT10) was the most upregulated gene and belongs to the ubiqui-
tin-like modifier (UBL) family. It has been reported that upregula-
tion of FAT10 expression in tumors was observed in 90% of HCC
patients.19Another highly upregulated gene was glypican-3
(GPC3), a member of the glypican family of glycosyl-phosphati-
dylinositol-anchored cell-surface heparin sulfate proteoglycans.20–
22GPC3 is also reported to be expressed in most HCC cells, but
not in normal hepatocytes and benign liver lesions,23,24and serum
GPC3 protein levels are elevated in a large proportion of HCC
patients.25Midkine (MDK) is a member of the heparin-binding
growth factor family and increased MDK expression has been
reported in various human cancers including HCC, and signifi-
cantly elevated serum MDK levels are observed in cancer patients.
It is thought that MDK acts as an antiapoptotic factor in HepG2
cells through the down-regulation of caspase-3 activity. Other
genes reported to be associated with HCC (SPINK1, ROBO1,
PLA2G2A, PEG10 and so on) were also included in the list of
FIGURE 2 – Kaplan-Meier disease-free survival curves for HCC
patients according to the level of Ube2c mRNA expression. The recur-
rence rate for patients in the high expression group was significantly
higher than that for patients in the low expression group (p < 0.01).
High expression group (n 5 33): Ube2c/GAPDH ? median value,
Low expression group: Ube2c/GAPDH < median value (n 5 32).
TABLE I – Ube2c GENE EXPRESSION AND CLINICOPATHOLOGICAL
FEATURES FOR 65 HCC PATIENTS
Clinicopathologic variableHigh expression
group (n 5 33)
group (n 5 32)
Capsular formation (fc)
Invasion to capsular formation (fc-inf)
Invasion to portal vein (vv)
Invasion to hepatic vein (vp)
Invasion to bile duct (b)
Number of tumors
65.3 6 11.266.7 6 9.1 0.59
4.2 6 3.83.2 6 1.90.21
High expression group (Ube2c/GAPDH ? median value), Low
expression group (Ube2c/GAPDH < median value). Well, well differ-
entiated; poor, poorly differentiated; moderate, moderately differenti-
ated; HBV, hepatitis B virus; HCV, hepatitis C virus; LC, liver cirrho-
sis; fc-inf, invasion to capsule or outside of capsule. Cases with no
capsule formation were included in fc-inf (2).
TABLE II – RESULTS OF UNIVARIATE AND MULTIVARIATE ANALYSES OF
CLINICOPATHOLOGICAL FACTORS AFFECTING DISEASE FREE-SURVIVAL
RATE FOLLOWING SURGERY
Number of tumors
n, number of patient; CI, confidence interval; fc, capsular formation;
fc-inf, tumor invasion to capsular formation; vp, invasion to portal vein;
vv, invasion to hepatic vein; b, invasion to bile duct; Ube2c, Ube2c
expression; high, high expression group (Ube2c/GAPDH ? median
value); low, low expression group (Ube2c/GAPDH < median value).
IETA ET AL.
Of the overexpressed genes we identified, we further studied the
Ube2c gene that was expressed 3 times higher in HCC cells than
in normal liver cells. Ube2c is highly expressed in various human
cancers, including ovarian carcinoma, metastatic prostate carci-
noma and thyroid anaplastic carcinoma.9–13Okamoto et al.
showed that Ube2c expression was low in many normal tissues
but was prominent in the majority of cancer cell lines, and that
high Ube2c expression levels were observed in primary tumors
derived from lung, stomach, uterus and bladder when compared
with corresponding normal tissues.11Ube2c has also been reported
to be overexpressed in primary colorectal cancers and in subse-
quent liver metastases and was identified as an overexpressed and
myc-interacting gene in human glioma.29However, none of the
previous studies investigated the clinicopathological significance
of increased Ube2c expression in HCC.30In the present study, we
compared various clinicopathological factors with respect to
Ube2c expression status in HCC. Our findings indicated that
Ube2c expression was higher in HCC than in noncancerous liver
tissues, and that Ube2c overexpression was significantly associ-
ated with fc-inf, vp, tumor grade (p < 0.05) and poor prognosis in
terms of disease free survival (p < 0.01) in HCC patients. In addi-
tion, multivariate analysis showed that Ube2c expression was an
independent prognostic factor associated with the disease-free sur-
vival rate. These findings suggested that enhanced expression of
Ube2c may play an important role in various pathological proc-
esses of HCC. Our finding of high expression levels detected in
poorly-differentiated HCC agreed with reports that showed that
Ube2c expression was associated with poor tumor differentiation
in ovarian, breast, lung, bladder and brain cancers.11,12
The function of the Ube2c gene product is closely linked to cell
cycle progression and the destruction of mitotic cyclins. Rape and
Kirschner showed that the decision between cyclinA degradation
and APC inactivation is determined by Ube2c availability.7,8Our
study demonstrated that siRNA-mediated suppression of Ube2c
expression inhibited the growth rate of an HCC cell line, which
was consistent with reports that NIH3T3 cells stably transfected
with Ube2c exhibited a more malignant phenotype than the paren-
tal NIH3T3 cells,11such that Ube2c gene silencing by siRNA
inhibited cell proliferation without inducing cell death, with cell
cycle analysis by FACS following Ube2c siRNA treatment show-
ing arrest at the G2/M phase.12,31,32Furthermore, when combined
with agonists for the DR5/TNF-related apoptosis inducing ligand
(TRAIL) receptor, inhibition of Ube2c by siRNA enhanced tumor
cell killing.12Therefore, in HCC patients, high Ube2c expression
may lead to increased malignant potential of the tumor, such that
the Ube2c gene may have some utility as a therapeutic target.
The Ube2c gene was found to be overexpressed in gastro-
esophageal cancer, with chromosomal amplification at the Ube2c
locus, 20q13.1, shown.12,3320q amplification is common among
various carcinomas,34–37including HCC,38so it is likely that this
amplification would induce Ube2c overexpression, thereby
increasing the malignant potential of HCC cells.
In conclusion, our study identified upregulated genes in HCC
cells compared to normal liver cells, and also showed that one of
the upregulated genes, Ube2c, may play an important role in
HCC, and may prove useful as a novel prognostic marker for
patients with HCC.
We thank Ms. T. Shimooka, Ms. K. Ogata, Ms. M. Oda,
Ms. M. Kasagi and Ms. Y. Nakagawa for their technical assistance.
1. Ferlay J, Bray F, Pisani P, Parkin DM. GLOBOCAN 2000: cancer
incidence, mortality and prevalence worldwide, version 1.0. IARC
Cancer Base No. 5., Lyon: IARC, 2001.
Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet
Bosch FX, Ribes J, Diaz M, Cleries R. Primary liver cancer: world-
wide incidence and trends. Gastroenterology 2004;127:S5–16.
4.Sonoda H, Inoue H, Ogawa K, Utsunomiya T, Masuda TA, Mori M.
Significance of skp2 expression in primary breast cancer. Clin Cancer
Masuda TA, Inoue H, Nishida K, Sonoda H, Yoshikawa Y, Kakeji Y,
Utsunomiya T, Mori M. Cyclin-dependent kinase 1 gene expression is
associated with poor prognosis in gastric carcinoma. Clin Cancer Res
FIGURE 3 – (a,b). Ube2c expres-
sion suppressed by Ube2c specific-
siRNA in Hep-3B cells. At 48 h af-
ter siRNA addition, Ube2c expres-
sion was measured by real-time
quantitative PCR (a) and western
blot (b) (m, marker; u, Ube2c
siRNA; n, negative siRNA; c, con-
trol; p, positive control) (c) Effect
of Ube2c suppression on Hep-3B
cell proliferation as assessed by
MTT assay. Ube2c-suppressed cells
were less proliferative than control
cells (p < 0.01). Data represent the
OVEREXPRESSED GENES IN HCC; A SPECIAL REFERENCE TO Ube2c
6. Masuda TA, Inoue H, Sonoda H, Mine S, Yoshikawa Y, Nakayama
K, Nakayama K, Mori M. Clinical and biological significance of S-
phase kinase-associated protein 2 (Skp2) gene expression in gastric
carcinoma: modulation of malignant phenotype by Skp2 overexpres-
sion, possibly via p27 proteolysis. Cancer Res 2002;62:3819–25.
Rape M, Kirschner MW. Autonomous regulation of the anaphase-pro-
moting complex couples mitosis to S-phase entry. Nature 2004;432:
Lukas J, Bartek J. Cell division: the heart of the cycle. Nature 2004;
Welsh JB, Zarrinkar PP, Sapinoso LM, Kern SG, Behling CA, Monk
BJ, Lockhart DJ, Burger RA, Hampton GM. Analysis of gene expres-
sion profiles in normal and neoplastic ovarian tissue samples identifies
candidate molecular markers of epithelial ovarian cancer. Proc Natl
Acad Sci USA 2001;98:1176–81.
10. LaTulippe E, Satagopan J, Smith A, Scher H, Scardino P, Reuter V,
Gerald WL. Comprehensive gene expression analysis of prostate can-
cer reveals distinct transcriptional programs associated with meta-
static disease. Cancer Res 2002;62:4499–506.
11. Okamoto Y, Ozaki T, Miyazaki K, Aoyama M, Miyazaki M, Nakaga-
wara A. UbcH10 is the cancer-related E2 ubiquitin-conjugating
enzyme. Cancer Res 2003;63:4167–73.
12. Wagner KW, Sapinoso LM, El-Rifai W, Frierson HF, Butz N, Mestan
J, Hofmann F, Deveraux QL, Hampton GM. Overexpression, genomic
amplification and therapeutic potential of inhibiting the UbcH10 ubiq-
uitin conjugase in human carcinomas of diverse anatomic origin.
13. Pallante P, Berlingieri MT, Troncone G, Kruhoffer M, Orntoft TF,
Viglietto G, Caleo A, Migliaccio I, Decaussin-Petrucci M, Santoro M,
Palombini L, Fusco A. UbcH10 overexpression may represent a
marker of anaplastic thyroid carcinomas. Br J Cancer 2005;93:464–
14. Mori M, Mimori K, Yoshikawa Y, Shibuta K, Utsunomiya T, Sada-
naga N, Tanaka F, Matsuyama A, Inoue H, Sugimachi K. Analysis of
the gene-expression profile regarding the progression of human gas-
tric carcinoma. Surgery 2002;131:S39–47.
15. Nishida K, Mine S, Utsunomiya T, Inoue H, Okamoto M, Udagawa
H, Hanai T, Mori M. Global analysis of altered gene expressions dur-
ing the process of esophageal squamous cell carcinogenesis in the rat:
a study combined with a laser microdissection and a cDNA microar-
ray. Cancer Res 2005;65:401–9.
16. Quackenbush J. Microarray data normalization and transformation.
Nat Genet 2002;32:496–501.
17. Ogawa K, Utsunomiya T, Mimori K, Tanaka F, Inoue H, Nagahara H,
Murayama S, Mori M. Clinical significance of human kallikrein gene
6 messenger RNA expression in colorectal cancer. Clin Cancer Res
18. Ieta K, Tanaka F, Utsunomiya T, Kuwano H, Mori M. CEACAM6
gene expression in intrahepatic cholangiocarcinoma. Br J Cancer 2006;
19. Lee CG, Ren J, Cheong IS, Ban KH, Ooi LL, Yong Tan S, Kan A,
Nuchprayoon I, Jin R, Lee KH, Choti M, Lee LA. Expression of the
FAT10 gene is highly upregulated in hepatocellular carcinoma and
other gastrointestinal and gynecological cancers. Oncogene 2003;22:
20. Filmus J, Selleck SB. Glypicans: proteoglycans with a surprise. J Clin
21. Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, Lincecum
J, Zako M. Functions of cell surface heparan sulfate proteoglycans.
Annu Rev Biochem 1999;68:729–77.
22. Filmus J, Church JG, Buick RN. Isolation of a cDNA corresponding
to a developmentally regulated transcript in rat intestine. Mol Cell
23. Hsu HC, Cheng W, Lai PL. Cloning and expression of a developmen-
tally regulated transcript MXR7 in hepatocellular carcinoma: biologi-
cal significance and temporospatial distribution. Cancer Res 1997;57:
24. Zhu ZW, Friess H, Wang L, Abou-Shady M, Zimmermann A, Lander
AD, Korc M, Kleeff J, Buchler MW. Enhanced glypican-3 expression
differentiates the majority of hepatocellular carcinomas from benign
hepatic disorders. Gut 2001;48:558–64.
25. Capurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E,
Filmus J. Glypican-3: a novel serum and histochemical marker for he-
patocellular carcinoma. Gastroenterology 2003;125:89–97.
26. Koide N, Hada H, Shinji T, Ujike K, Hirasaki S, Yumoto Y, Hanafusa
T, Kadomatsu K, Muramatsu H, Muramatsu T, Tsuji T. Expression of
the midkine gene in human hepatocellular carcinomas. Hepatogas-
27. Ikematsu S, Yano A, Aridome K, Kikuchi M, Kumai H, Nagano H,
Okamoto K, Oda M, Sakuma S, Aikou T, Muramatsu H, Kadomatsu
K, et al. Serum midkine levels are increased in patients with various
types of carcinomas. Br J Cancer 2000;83:701–6.
28. Ohuchida T, Okamoto K, Akahane K, Higure A, Todoroki H, Abe Y,
Kikuchi M, Ikematsu S, Muramatsu T, Itoh H. Midkine protects hepa-
tocellular carcinoma cells against TRAIL-mediated apoptosis through
down-regulation of caspase-3 activity. Cancer 2004;100:2430–6.
29. Bredel M, Bredel C, Juric D, Harsh GR, Vogel H, Recht LD, Sikic
BI. Functional network analysis reveals extended gliomagenesis path-
way maps and three novel MYC-interacting genes in human gliomas.
Cancer Res 2005;65:8679–89.
30. Wilting SM, Snijders PJ, Meijer GA, Ylstra B, van den Ijssel PR,
Snijders AM, Albertson DG, Coffa J, Schouten JP, van de Wiel MA,
Meijer CJ, Steenbergen RD. Increased gene copy numbers at chromo-
some 20q are frequent in both squamous cell carcinomas and adeno-
carcinomas of the cervix. J Pathol 2006;209:220–30.
31. Townsley FM, Aristarkhov A, Beck S Hershko A, Ruderman JV.
Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/
UbcH10 blocks cells in metaphase. Proc Natl Acad Sci USA
32. Salvesen GS, Dixit VM. Caspases: intracellular signaling by proteoly-
sis. Cell 1997;91:443–6.
33. Takahashi Y, Ishii Y, Nishida Y, Ikarashi M, Nagata T, Nakamura T,
Yamamori S, Asai S. Detection of aberrations of ubiquitin-conjugat-
ing enzyme E2C gene (UBE2C) in advanced colon cancer with liver
metastases by DNA microarray and two-color FISH. Cancer Genet
34. El-Rifai W, Frierson HF, Jr, Moskaluk CA, Harper JC, Petroni GR,
Bissonette EA, Jones DR, Knuutila S, Powell SM. Genetic differences
between adenocarcinomas arising in Barrett’s esophagus and gastric
mucosa. Gastroenterology 2001;121:592–8.
35. Tsafrir D, Bacolod M, Selvanayagam Z, Tsafrir I, Shia J, Zeng Z, Liu
H, Krier C, Stengel RF, Barany F, Gerald WL, Paty PB et al. Rela-
tionship of gene expression and chromosomal abnormalities in color-
ectal cancer. Cancer Res 2006;66:2129–37.
36. Fujita Y, Sakakura C, Shimomura K, Nakanishi M, Yasuoka R, Ara-
gane H, Hagiwara A, Abe T, Inazawa J, Yamagishi H. Chromosome
arm 20q gains and other genomic alterations in esophageal squamous
cell carcinoma, as analyzed by comparative genomic hybridization
and fluorescence in situ hybridization. Hepatogastroenterology 2003;
37. Fukushige S, Waldman FM, Kimura M, Abe T, Furukawa T, Suna-
mura M, Kobari M, Horii A. Frequent gain of copy number on the
long arm of chromosome 20 in human pancreatic adenocarcinoma.
Genes Chromosomes Cancer 1997;19:161–9.
38. Wong N, Lai P, Lee SW, Fan S, Pang E, Liew CT, Sheng Z, Lau JW,
Johnson PJ. Assessment of genetic changes in hepatocellular carci-
noma by comparative genomic hybridization analysis: relationship
to disease stage, tumor size, and cirrhosis. Am J Pathol 1999;154:
IETA ET AL.