Cancer Sci| November 2008|vol. 99| no. 11|2152–2159 doi: 10.1111/j.1349-7006.2008.00923.x
© 2008 Japanese Cancer Association
Blackwell Publishing Asia
p27Kip1 is overexpressed in very early stages
Shinichi Yachida,1,4 Michiie Sakamoto,2 Katsumi Imaida,3 Masanao Yokohira,3 Kousuke Saoo,3 Keiichi Okano,1
Hisao Wakabayashi,1 Hajime Maeta1 and Yasuyuki Suzuki1
Departments of 1Surgery and 3Pathology and Host-Defense, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793; 2Department of Pathology,
Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan
(Received May 11, 2008/Revised June 29, 2008/Accepted June 30, 2008/Online publication September 19, 2008)
Hepatocellular carcinoma (HCC) associated with chronic liver disease
evolves from precancerous lesions and early HCC to more malignant
forms. Despite the demonstrated importance of cell-cycle regulators
in tumor biology, there have been few studies of their role in multistep
hepatocarcinogenesis. Expression of p27Kip1 and a degradation
pathway associated protein, S-phase kinase-interacting protein 2
(Skp2), was therefore evaluated in surgically resected specimens of
eight adenomatous hyperplasias, 16 early HCC and 126 classical HCC.
Immunohistochemistry revealed no p27Kip1 expression in the majority
of hepatocytes from normal and cirrhotic liver, whereas positive
staining for p27Kip1 protein was found in 75.0% and 93.8% of
adenomatous hyperplasias and early HCC, respectively. The average
p27Kip1 labeling indices (LI) for adenomatous hyperplasias, early
HCC, well differentiated HCC, moderately differentiated HCC and
poorly differentiated HCC were 36.99, 43.59, 47.73, 49.24, and 30.21,
respectively. Real-time quantitative reverse transcription–polymerase
chain reaction (RT-PCR) analyses confirmed the increases. Skp2 LI were
also significantly elevated in accordance with stepwise progression
of hepatocarcinogenesis. Increased expression of Skp2 mRNA was
observed most frequently in less differentiated tumors and Kaplan–
Meier survival analysis showed a significantly association with a
poor prognosis (P = 0.0496). In conclusion, a high level of p27Kip1
expression is evident from early stages of hepatocarcinogenesis,
indicating that this parameter could be a useful diagnostic marker
for precancerous lesions and early HCC. In addition, Skp2 expression
correlates with tumor dedifferentiation and may contribute to
biological aggression in HCC. (Cancer Sci 2008; 99: 2152–2159)
death in Japan. As with other cancers, HCC are characterized by
an obvious multistage process of tumor progression.(1–3) It has
been shown that small, nodular hypercellular lesions known as
adenomatous hyperplasias (AH) appear in damaged liver
infected with hepatitis virus B or C. These lesions develop into
early HCC, which correspond to carcinoma in situ or microinvasive
carcinoma, in which the portal tracts within nodules are preserved,
and then into progressed HCC (classical HCC) through the
transition stage of nodule-in-nodule-type HCC (progressed HCC
within early HCC).(4,5)
The proliferation of cancer cells has been found to be closely
related to abnormalities in expression of cell-cycle regulators.
p27Kip1 a member of the CIP/KIP family, causes G1 arrest by
inhibiting the activities of G1 cyclin–cyclin-dependent kinases.
As a negative regulator of the cell cycle, p27Kip1 is a new class of
tumor suppressor controlling cell-cycle progression.(6–8) Recent
findings indicate that decreased expression of p27Kip1 protein is
associated with high aggressiveness and a poor prognosis in a
large variety of cancers, including classical HCC.(9–11) There are a
number of key molecules involved in post-translational regulation
of p27Kip1. One is S-phase kinase-interacting protein 2 (Skp2),
he hepatocellular carcinoma (HCC) is one of the most common
malignant tumors worldwide and a leading cause of cancer
which has been shown to be required for ubiquitin-mediated
degradation of p27Kip1.(12,13) Overexpression of Skp2 has been
observed to be reciprocally correlated with p27Kip1 expression in
Despite the demonstrated importance of cell-cycle regulators
in tumor biology, to our knowledge there have been no studies
of cell-cycle regulators in liver precancerous lesions and early
HCC. In the present study, we therefore investigated the possible
role of cell-cycle regulators, especially p27Kip1 and the degradation
related Skp2, in surgically resected specimens of AH, early
HCC and classical HCC.
Materials and Methods
Patients. The present study involved 122 patients with precan-
cerous lesions and/or HCC (150 lesions) undergoing hepatic
resection between 1990 and 2006 at Keio University Hospital,
Tokyo, and Kagawa University Hospital, Kagawa, Japan. The
median age of the patients was 65.1 years (range 36–82 years),
and the male : female ratio was 6.6 : 1. Of these patients, 28
were hepatitis B surface antigen (HBs-Ag) positive, 63 were
hepatitis C antibody (HCV-Ab) positive, 2 were positive for
both HBs-Ag and HCV-Ab, 15 were negative for both HBs-Ag
and HCV-Ab in the presence of chronic liver disease, and the
remaining 3 without chronic liver disease were negative for both
HBs-Ag and HCV-Ab (unknown, 11 patients). With regard to
background liver disease, 67 and 42 patients had chronic hepatitis
and liver cirrhosis, respectively, and 13 patients had a normal
liver. Informed consent for the use of resected tissues was
obtained from every patient before the study was started and the
study protocol confirms to the ethical guidelines of the Declaration
of Helsinki in 1995.
Tissue samples histological evaluation. The tumors were classified
in accordance with the World Health Organization (WHO)
classification,(17) and that proposed by the Liver Cancer Study
Group of Japan.(18) Each tumor was re-evaluated for macroscopic
type, microscopic vascular invasion, intrahepatic metastasis and
degree of histological differentiation. In total, eight AH samples,
16 early HCC samples and 126 classical HCC samples were
examined. When progressed HCC areas within early HCC are
growing expansively, the nodules often have a ‘nodule-in nodule’
appearance.(5,17,18) Among the classical HCC, six were of the
nodule-in-nodule type and 13 had early HCC-like areas in the
AH, also known as dysplastic nodules, are characterized by a
moderate increase in cell density with a slightly irregular trabecular
pattern.(17) There are many portal tracts within the nodules, but
Current address: Shinichi Yachida, Gastrointestinal/Liver Division, Department of
Pathology, Johns Hopkins Medical Institutions, 1550 Orleans Street, CRBII Rm 316,
Baltimore MD 21231, USA
4To whom correspondence should be addressed.
E-mail: email@example.com; firstname.lastname@example.org
Yachida et al.
Cancer Sci| November 2008| vol. 99| no. 11| 2159
© 2008 Japanese Cancer Association
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