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2447
Reduced Expression of Cyclin-Dependent Kinase
Inhibitor p27Kip1 Is an Indicator of Malignant Behavior
in Oral Squamous Cell Carcinoma
Yasusei Kudo, D.D.S.1
Takashi Takata, D.D.S., Ph.D.1
Wataru Yasui, M.D., Ph.D.2
Ikuko Ogawa, D.D.S., Ph.D.3
Mutsumi Miyauchi, D.D.S., Ph.D.1
Toshitsugu Takekoshi, D.D.S.1
Eiichi Tahara, M.D., Ph.D.2
Hiromasa Nikai, D.D.S., Ph.D.1
1
Department of Oral Pathology, Hiroshima University School of Dentistry, Hiroshima, Japan.
2
First Department of Pathology, Hiroshima University School of Medicine, Hiroshima, Japan.
3
Clinical Laboratory, Hiroshima University Dental
Hospital, Hiroshima, Japan.
Presented at the 39th Annual Meeting of the Japanese Association for Oral Biology, Kitakyushu,
Japan, October 1–2, 1997.
Supported in part by Grant No. 07457429 from the
Ministry of Education, Japan.
Address for reprints: Yasusei Kudo, D.D.S., Department of Oral Pathology, Hiroshima University
School of Dentistry, 1-2-3 Kasumi, Minami-ku,
Hiroshima 734, Japan.
Received February 23, 1998; revision received
April 27, 1998; accepted April 27, 1998.
© 1998 American Cancer Society
BACKGROUND.
Reduced expression of the cyclin-dependent kinase inhibitor
p27Kip1 has been reported to correlate with poor survival in cohorts of breast and
colorectal carcinoma patients. Posttranslational ubiquitin-mediated proteasomal
proteolysis is related to p27Kip1 protein levels. However, to the authors’ knowledge,
no previous study has examined the expression of p27Kip1 in oral squamous cell
carcinoma (OSCC).
METHODS. To examine the expression of p27Kip1 and its clinicopathologic roles in
OSCC, the authors studied the expression of p27Kip1 protein by immunohistochemistry in deparaffinized tissue sections of 20 normal oral mucosa specimens, 22
epithelial dysplasia specimens, and 70 OSCCs, and analyzed its correlation with
clinicopathologic parameters. They also studied the expression of p27Kip1 mRNA
and protein in six OSCC cell lines by Northern blot and Western blot analysis. To
examine the mechanism of reduced expression of p27Kip1, OSCC cell lines were
treated with the proteasome inhibitor LLnV.
RESULTS. All the normal oral mucosa specimens and 73% (16 of 22) of the oral
epithelial dysplasia specimens expressed p27Kip1 at high levels, whereas 87% of the
OSCCs (61 of 70) showed reduced expression of p27Kip1. Furthermore, the levels of
expression of this protein were significantly lower in carcinomas with metastasis
than those without metastasis. Although OSCC cell lines expressed p27Kip1 mRNA
at various levels, most of them expressed p27Kip1 protein at lower or undetectable
levels. LLnV induced the expression of p27Kip1 protein in HSC2 cells, in which
p27Kip1 protein was originally undetectable.
CONCLUSIONS. These findings suggest that 1) reduced expression of p27Kip1 may
correlate with the development and progression of OSCC and can be an indicator
of malignant behavior of this neoplasm, and 2) increased proteasome-mediated
degradation may play an important role in the reduction of p27Kip1 protein expression. Cancer 1998;83:2447–55. © 1998 American Cancer Society.
KEYWORDS: oral squamous cell carcinoma, p27Kip1, reduced expression, proteasome-mediated degradation, ubiquitin.
A
bnormalities in cell cycle regulators allow uncontrolled cell
growth and division, which may have a role in carcinogenesis.1,2
Multiple cyclins and cyclin-dependent kinases (CDKs) are positive
regulators of progress of the cell cycle. Cyclin/CDK complexes are
activated by phosphorylation by the CDK-activating kinase (CAK),
whereas cyclin/CDK complexes are negatively regulated by a number
of CDK inhibitors.3–11 CDK inhibitors belong to two large families
based on their structural and functional properties. The Ink4 family,
which includes p16Ink4a, p15Ink4b, p18Ink4c, and p19Ink4d, consists of
tandem repeats of an ankyrin-like sequence, whereas the Cip/Kip
2448
CANCER December 15, 1998 / Volume 83 / Number 12
TABLE 1
Age and Gender Distribution of the Patients
Age (yrs)
Normal oral mucosa
Epithelial dysplasia
OSCC
Gender
No. of cases
Mean
Range
Male
Female
20
22
70
55.85
59.91
58.67
31–80
29–83
24–88
12
14
36
8
8
34
OSCC: oral squamous cell carcinoma.
family, which includes p21Cip1/Waf1, p27Kip1, and
p57Kip2, has a homologous amino-terminal domain
that contains contiguous cyclin and CDK binding regions. p27Kip1 is associated predominantly with cyclin
D/CDK4, but has the ability to inhibit various cyclin/
CDK complexes in vitro.7,12 p27Kip1 in combination
with newly formed cyclin E/CDK2 complexes blocked
cell cycle progression.7 The cyclin E/CDK2 complex
shows strong kinase activity shortly before cells enter
the S phase and leads to further phosphorylation of
the pRb protein.13–15 Cyclin E is overexpressed in certain types of human tumors.16 –18 p27Kip1 mediates G1
arrest induced by transforming growth factor-␤ (TGF␤), contact inhibition, or serum deprivation in epithelial cell lines.6 Although the role of p27Kip1 protein in
cancer cells has not been elucidated, reduced expression of p27Kip1 has recently been shown to correlate
with poor prognosis in breast and colorectal carcinomas.19 –22 Moreover, high levels of cyclin E and low
levels of p27Kip1 have been shown to be strongly predictive of increased mortality, both before and after
adjustment for other clinical and pathologic characteristics.19
Both in vivo and in vitro, p27Kip1 was found to be
degraded by the ubiquitin-proteasome pathway.23 In
various carcinomas, p27Kip1 mRNA levels are preserved, but protein levels are reduced.20,24 As carcinomas with low or absent p27Kip1 protein display enhanced proteolytic activity specific to p27Kip1, it has
been suggested that low p27Kip1 expression is brought
about by increased proteasome-mediated degradation
rather than altered gene expression.21
To our knowledge, however, there has been no
previous study of p27Kip1 expression in oral squamous
cell carcinoma (OSCC). Therefore, to study the value
of p27Kip1 expression in determining the prognoses of
OSCC patients, we examined the immunohistochemical expression of p27Kip1 in OSCC and its correlation
with clinicopathologic findings. We also investigated
whether a degradation pathway of p27Kip1 by a ubiquitin-proteasome complex is present in OSCC.
MATERIALS AND METHODS
Tissue Samples
Tissue samples of 20 normal oral mucosa specimens,
22 epithelial dysplasia specimens, and 70 OSCCs from
the years 1976 –1997 were retrieved from the Surgical
Pathology Registry of Hiroshima University Dental
Hospital. For the current analysis, only biopsied specimens from the tongue, taken before radiochemotherapy, were selected to avoid possible influences of
the lesional sites and treatment modalities on data.
The age and gender distributions of the patients are
summarized in Table 1. Of the 70 OSCC patients,
follow-up data were available for 51 (25 men and 26
women). At the time of diagnosis, their ages ranged
from 24 to 87 years (mean, 57.14 years). The mean
follow-up period for these OSCC patients was 79.96
months (range, 4 –185 months).
Tissues were fixed in 10% buffered formalin and
embedded in paraffin. The number of cases of each
degree of epithelial dysplasia and histologic grade of
OSCC is listed in Table 1. Histologic grade and stage of
tumor were classified according to the criteria of the
Japan Society for Head and Neck Cancer.25 Stage
grouping was based on the TNM classification as follows: Primary tumor (T): no evidence of primary tumor (T0); the greatest dimension of primary tumor
less than 2 cm (T1), 2– 4 cm (T2), or more than 4 cm
(T3); massive tumor more than 4 cm in greatest dimension with deep invasion to involve the antrum,
the pterygoid muscles, the root of the tongue, or the
skin of the neck (T4). Lymph node involvement (N):
No clinically positive lymph nodes (N0); a single clinically positive ipsilateral lymph node less than 3 cm in
greatest dimension (N1); a single clinically positive
ipsilateral lymph node 3– 6 cm in greatest dimension,
or multiple clinically positive ipsilateral lymph nodes,
none over 6 cm in greatest dimension (N2); massive
ipsilateral lymph nodes, bilateral lymph nodes, or
contralateral lymph nodes (N3). Distant metastasis
(M): No distant metastasis (M0), distant metastasis at
presentation (M1). Stage was grouped as follows: Stage
Reduced Expression of p27Kip1 in OSCC/Kudo et al.
I, including T1, N0, M0; Stage II, including T2, N0, M0;
Stage III, including T3, N0, M0 or T1–T3, N1, M0; Stage
IV, including T4, N0 –1, M0 or any T, N2–3, M0 or any
T, any N, M1.
Cell Culture
Six OSCC cell lines (HSC2, HSC3, HSC4, Ca9-22, Ho1-U-1, and Ho-1-N-1) were examined. All the cell lines
were provided by the Japanese Cancer Research Resources Band (JCRB). They were routinely maintained
in RPMI-1640 (Kyokuto Pharmaceutical Industrial Co.,
Tokyo, Japan) and supplemented with 10% heat-inactivated fetal bovine serum (Boehringer Mannheim
K. K., Australia) and 100 U/mL penicillin-streptomycin
(Gibco BRL, Grand Land, NY) under conditions of 5%
CO2 in air at 37°C. For experiments, they were grown
to subconfluence in this medium.
Immunohistochemistry
Immunohistochemical detection of p27Kip1 was performed on 20 normal oral mucosa specimens, 22 epithelial dysplasia specimens, and 70 OSCCs of various
stages to analyze the relation with clinicopathologic
parameters of OSCC patients. We also studied immunoexpression of cyclin E to examine the correlation
between expression of p27Kip1 and cyclin E in 51 OSCC
cases with follow-up data. Immunostaining was performed using an antihuman p27Kip1 mouse monoclonal antibody (K25020, Transduction Laboratories, Lexington, KY), an anti– cyclin E monoclonal antibody
(14591A, Pharmingen, San Diego, CA), and a streptavidin-biotin kit (Nichirei, Tokyo, Japan). Deparaffinized tissue sections were immersed in methanol
containing 0.3% hydrogen peroxide for 30 minutes to
block endogenous peroxidase activity. Microwave pretreatment in citrate buffer was performed for 10 minutes 3 times to retrieve the antigenicity. The sections
were then incubated with normal rabbit serum for 30
minutes to block nonspecific antibody binding sites.
The sections were treated consecutively at 4°C with an
anti-p27Kip1 antibody (diluted 1:100) or an anti– cyclin
E antibody (diluted 1:200) overnight, biotinylated antimouse immunoglobulin G rabbit serum for 30 minutes, and a streptavidin-biotinylated horseradish peroxidase complex for 30 minutes. Peroxidase staining
was performed for 5–10 minutes using a solution of
3,3⬘-diaminobenzidine tetrahydrochloride in 50 mM
Tris-HCl (pH 7.5) containing 0.001% hydrogen peroxide. The sections were slightly counterstained with
Mayer’s hematoxylin.
Nuclear staining of p27Kip1 was scored on a semiquantitative scale (negative, medium, and high) by
evaluating the percentage of stained nuclei within representative areas of each tumor. For superficial carci-
2449
nomas, stained sections were observed throughout
the lesion. For advanced large tumors, at least 10
fields, including superficial, central, and deep invasive
areas, were observed, and the number of stained cells
and staining intensity were evaluated. In each field, we
counted at least more than 300 cells, using an eyepiece
graticule to prevent recounting. Although qualitative
differences in staining intensity were observed with
considerable intratumoral heterogeneity, all positive
cases showed obvious nuclear staining, at least focally.
The expression of p27Kip1 was graded as high (over
30% of tumor cells showed strong or diffuse immunopositivity), medium (5–30% of tumor cells showed
moderate or patchy immunopositivity), or negative
(less than 5% of tumor cells showed weak or focal
immunopositivity or no staining). The expression of
cyclin E was classified as strongly positive (over 30% of
tumor cells showed immunopositivity) or weakly positive/negative.
Northern Blot Analysis
The expression of p27Kip1 mRNA was examined in
OSCC cell lines by Northern blot analysis. RNAs were
extracted by using the QuickPrep Micro mRNA Purification Kit (Pharmacia Biotech Inc., CA). Two ␮g of
poly(A)⫹-selected RNA were electrophoresed on 1.0%
agarose/formaldehyde gels and blotted onto nitrocellulose filter membranes. The filters were baked for 2
hours at 80°C under vacuum and hybridized with 32Plabeled probes using the random hexamer priming
method. After hybridization, the filters were washed
under stringent conditions and exposed for autoradiography to Fuji RX films with intensifying screens at
⫺80°C.26 A cDNA probe for p27Kip1 (a 0.69 kb Kpn I
and BamH I p27Kip1 fragment) was kindly provided by
Dr. J. Massague (Howard Hughes Medical Institute,
Memorial Sloan-Kettering Cancer Center, New York,
NY). A glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA probe was used as an internal control.
Western Blot Analysis
We examined the expression of p27Kip1 protein in OSCC
cell lines by Western blot analysis. The protein samples
were prepared, and Western blotting was carried out as
we described previously.27 Fifty ␮g of protein was subjected to 10% polyacrylamide gel electrophoresis followed by electroblotting onto a nitrocellulose filter. The
antibody was the same as for immunohistochemistry.
For detection of the immunocomplex, the ECL Western
blotting detection system (Amersham, Aylesbury, UK)
was used.
2450
CANCER December 15, 1998 / Volume 83 / Number 12
FIGURE 1. Immunostaining of p27Kip1 is shown in oral normal mucosa, epithelial dysplasia, and oral squamous cell carcinoma. (a) Oral normal mucosa, original
magnification ⫻150. (b) Severe dysplasia, original magnification ⫻100. Most epithelial cells showed high levels of p27Kip1 expression. (c) Moderately differentiated
carcinoma, original magnification ⫻150. Five percent to 30% of tumor cells showed immunopositivity. (d) Well differentiated carcinoma, original magnification
⫻100. Most tumor cells were negative for p27Kip1. Infiltrating lymphocytes were p27Kip1 positive.
Treatment with the Proteosomal Inhibitor NCarbobenzoxy-L-leucyl-L-leucyl-L-norvalinal (LLnV)
In human leukemic HL60 cells, proteasomal function
was blocked by LLnV and a clear accumulation of
p27Kip1 protein was caused by the addition of LLnV.28
Therefore, we used the specific proteasome inhibitor
LLnV to examine the proteasome-mediated degradation activity in OSCC cell lines. LLnV was obtained
from the Peptide Institute, Osaka. The compound was
dissolved in the amount of dimethyl sulfoxide required to establish a stock solution of 10 mM. LLnV
was added to the OSCC cells at a final concentration of
50 ␮M, and incubation was continued for 3, 6, 12, and
24 hours. The p27Kip1 levels were examined in lysates
of OSCC cells after incubation with LLnV by Western
blot analysis. The signal intensity was measured by
densitometric scanning, and relative expression levels
were presented using that of the control, with 0 h as a
standard (1.0).
Statistical Analysis
Patients survival data were used to determine whether
there was a correlation between p27Kip1 expression
levels and disease free survival time. Curves for survival were drawn according to the Kaplan–Meier
method, and differences between the survival rates of
three groups with different p27Kip1 protein and cyclin
E expression were examined. The statistical significance of these data was determined by the Mantel–
Cox test. A possible correlation between variables of
the analyzed tumor samples were tested for association by Fisher’s exact test. A P value of ⬍0.05 was
required for significance.
Reduced Expression of p27Kip1 in OSCC/Kudo et al.
2451
TABLE 2
Expression of p27 in Normal Oral Mucosa, Epithelial Dysplasia, and OSCC and Its Correlation with Clinicopathologic Parameters
Expression of p27b
Normal
Epithelial dysplasia
Mild or moderate
Severe
OSCC
Histologya
Well differentiated
Moderately differentiated
Poorly differentiated
Stagea
1
2
3
4
Metastasis
Negative
Positive
Cyclin E expression
Weakly positive/negative
Strongly positive
P valuec
No. of cases
High
Medium
Negative
20
20 (100%)
0 (0%)
0 (0%)
16
6
70
13 (81%)
3 (50%)
9 (13%)
3 (19%)
3 (50%)
33 (47%)
0 (0%)
0 (0%)
28 (40%)
30
33
7
5 (17%)
4 (12%)
0 (0%)
16 (53%)
15 (45.5%)
2 (29%)
9 (30%)
14 (42.5%)
5 (71%)
15
19
2
34
3 (20%)
3 (16%)
1 (50%)
2 (6%)
10 (67%)
11 (58%)
1 (50%)
11 (32%)
2 (13%)
5 (26%)
0 (0%)
21 (62%)
34
36
6 (17.5%)
3 (8%)
21 (62%)
12 (33%)
7 20.5%)
21 (58%)
0.0055
38
13
2 (5%)
3 (23%)
20 (53%)
3 (23%)
16 (42%)
7 (54%)
0.0704
⬍0.0001
0.3360
0.0149
OSCC: oral squamous cell carcinoma.
a
According to the criteria of the Japanese Classification of Head and Neck Cancer.25 Stage grouping as follows: Stage I includes T1, N0, M0; Stage II includes T2, N0, M0; Stage III includes T3, N0, M0 or T1–T3,
N1, M0; Stage IV includes T4, N0–1, M0, any T, N2–3, M0, or any T, any N, M1.
b
Grades of p27 positive cells were classified as high (⬎30%), medium (5–30%), or negative (⬍5%).
c
Correlation was analyzed by Fisher’s exact test, and P values are shown. P values less than 0.05 were regarded as statistically significant.
RESULTS
Immunohistochemical Analysis of p27Kip1 in OSCC
In the normal oral epithelium, cells in the prickle cell
and granular cell layers showed strongly positive
staining for p27Kip1 in their nuclei, but cells in the
basal layer did not (Fig. 1a). p27Kip1 protein was also
consistently positive in inflammatory cells, fibroblasts,
muscle cells, and endothelial cells. p27Kip1 staining in
these components could be regarded as an internal
control for the immunohistochemistry. The incidence
of p27Kip1 expression in normal oral mucosa, epithelial
dysplasia, and OSCC is summarized in Table 2.
In epithelial dysplasia, 81% of mild-to-moderate
dysplasia specimens (13 of 16) and 50% of severe
dysplasia specimens (3 of 6) expressed p27Kip1 at high
levels (Table 2, Fig. 1b). Three cases (19%) of mild-tomoderate dysplasia and half of severe dysplasia cases
showed decreased p27Kip1 immunoexpression. However, none of the dysplasia cases were negative.
In contrast to epithelial dysplasia, 40% of OSCCs (28
of 70) were negative and only 9 (13%) showed p27Kip1
staining at high levels. High p27Kip1 expression was frequently found in early stage OSCCs without metastasis.
Reduced expression of p27Kip1 was associated with ad-
vanced stage and metastasis, and there was a significant
correlation between them (P ⬍ 0.05) (Table 2, Fig. 1c,d).
In comparison with well-differentiated carcinomas,
poorly differentiated carcinomas tended to show reduced expression of p27Kip1, but there was no statistically significant correlation. Though we also examined
the influence of age, gender, and smoking habits of
OSCC patients on the expression of p27Kip1, there were
no correlations among them. The expression of p27Kip1
in histologic sections was heterogenous in some cases,
and there was a tendency to express less p27Kip1 in the
invasive front. Metastatic lesions also expressed less
p27Kip1 (data not shown).
As we reported previously, for gastric carcinoma
there was an inverse correlation between the expression of p27Kip1 and that of cyclin E.24 Therefore, we
also studied the expression of cyclin E in OSCC (Table
3). Strong expression of cyclin E was found in 13 (25%)
of 51 OSCCs. Expression of cyclin E was not found in
most normal oral mucosa specimens (data not
shown). Compared with poorly differentiated carcinomas, well-differentiated carcinomas tended to show
strong expression of cyclin E, but the expression of
cyclin E was not associated with advanced stage and
2452
CANCER December 15, 1998 / Volume 83 / Number 12
TABLE 3
Expression of Cyclin E in OSCC and Its Correlation with
Clinicopathologic Parameters
Expression of cyclin Eb
Histologya
Well differentiated
Moderately differentiated
Poorly differntiated
Stagea
I
II
III
IV
Metastasis
Negative
Positive
No. of cases
Strongly
positive
Weakly positive/
negative
22
25
4
7 (32%)
6 (24%)
0 (0%)
15 (68%)
19 (76%)
4 (100%)
9
15
1
26
3 (33%)
2 (20%)
0 (0%)
7 (27%)
6 (67%)
12 (80%)
1 (100%)
19 (73%)
24
27
6 (25%)
7 (26%)
18 (75%)
20 (74%)
OSCC: oral squamous cell carcinoma.
a
According to the criteria of the Japanese Classification of Head and Neck Cancer.25
b
Grades of cyclin E expression were classified as strongly positive (⬎30%).
metastasis (Table 3). There was no inverse correlation
between p27Kip1 and cyclin E in OSCC (Table 2).
The correlation between p27Kip1 protein expression and the survival rates of 51 OSCC patients with
follow-up data was also examined. Figure 2 shows the
Kaplan–Meier survival curves of the patients grouped
by the immunoreactivity of p27Kip1 in their tumors.
The cumulative survival rate for patients who were
negative for expression of p27Kip1 was lower than that
for patients with medium-to-high expression of
p27Kip1, and there was a statistical significance between p27Kip1 expression and the survival rate. High
levels of cyclin E and low levels of p27Kip1 were
strongly predictive of increased mortality among patients with breast carcinoma.19 However, in the current study, the survival rate for patients who had
strongly positive expression of cyclin E and who were
negative for expression of p27Kip1 was higher than the
overall rate for patients who were negative for expression of p27Kip1.
Expression of p27Kip1 in OSCC Cell Lines
Expression of p27Kip1 mRNA and protein in 6 OSCC
cell lines is shown in Figure 3a. The same filter was
reprobed with a GAPDH cDNA probe. Most of these
cell lines expressed p27Kip1 mRNA at high levels. We
examined expression of p27Kip1 protein in the 6 cell
lines by Western blot analysis (Fig. 3b). Ca9-22 and
HSC4 cells expressed p27Kip1 protein at higher levels,
and HSC3 and Ho-1-U-1 cells expressed p27Kip1 protein at lower levels. p27Kip1 protein was not found in
HSC2 or Ho-1-N-1. The expression levels of p27Kip1
protein did not correspond with the results of Northern blot analysis.
We also searched for macroscopic alterations of
the p27Kip1 genes in these cell lines by Southern blot
analysis. Neither gene amplification nor rearrangement of the p27Kip1 genes was found in any of these
cell lines (data not shown).
Accumulation of p27Kip1 after Treatment with the
Proteosomal Inhibitor LLnV in HSC2 and Ca9-22
We examined the p27Kip1 levels in HSC2 and Ca9-22
cells after incubation with LLnV by Western blot analysis. As shown in Figure 3, HSC2 cells lacked p27Kip1
protein and Ca9-22 cells preserved p27Kip1 protein,
whereas both expressed p27Kip1 mRNA at high levels.
LLnV induced the expression of p27Kip1 protein in
HSC2 cells, whereas it did not do so in Ca9-22 cells
(Fig. 4). In HSC2 cells, the signal intensity of p27Kip1
protein became larger with time after the treatment
with LLnV, and at 24 hours was about 5 times as much
as that at 0 h.
DISCUSSION
The proliferation and progression of cancer cells may
be caused by abnormalities of various positive and
negative cell cycle regulators.1,2 Reduced expression of
p27Kip1 was recently found in various cancers,19 –22,24
and so we focused on the expression of p27Kip1 in
OSCC. In this study, we demonstrated the reduced
expression of p27Kip1 in 87% of OSCCs as described in
breast, gastric, and colorectal carcinomas.19 –22,24 The
reduced expression of p27Kip1 was observed in 56% of
breast carcinomas, 70% of colorectal carcinomas, and
74% of gastric carcinomas.20,21,24 The reduced expression of p27Kip1 may be a common event in various
carcinomas.
Normal oral mucosa specimens and 73% of epithelial dysplasia specimens expressed p27Kip1 at high
levels, but only 13% of OSCCs expressed this protein
immunohistochemically. Moreover, 81% of mild-tomoderate dysplasia specimens showed high p27Kip1
expression in comparison with 50% of severe dysplasia
specimens. However, negative expression was not
found in epithelial dysplasia. These findings suggest
that the reduction of p27Kip1 protein expression may
begin at an early stage in the development of OSCC,
but it is necessary to confirm this in a large number of
epithelial dysplasia cases. Epithelial dysplasia with reduced expression of p27Kip1 may indicate the possibility of progression to cancer.
The most interesting finding of the current study
is that the immunoexpression of p27Kip1 protein was
closely associated with tumor stage and metastasis.
Reduced Expression of p27Kip1 in OSCC/Kudo et al.
2453
FIGURE 2. The relation of p27Kip1 and cyclin E expression to survival is shown. Kaplan–Meier plots for disease
free survival show the association of survival and expression of cyclin E and p27Kip1 in patients with oral squamous
cell carcinoma. The prognoses for patients who were
negative for expression of p27Kip1 (n ⫽ 23; median survival ⫽ 57 months) was poorer than the prognoses for
patients with medium-to-high expression of p27Kip1 (n ⫽
28; median survival ⫽ 110 months) or those with high
expression of cyclin E who were negative for expression of
p27Kip1 (n ⫽ 7; median survival ⫽ 79 months). The
statistical significance of these data was measured by the
Mantel–Cox test.
FIGURE 3. Expression of p27Kip1 mRNA and protein in oral squamous cell
carcinoma cell lines is shown. (a) Northern blot analysis: Two ␮g of poly
(A)⫹-selected RNA was subjected to Northern blot analysis as described in
“Materials and Methods.” A Glyceraldehyde-3-phosphate dehydrogenase cDNA
probe was applied as an internal control. Most of these cell lines expressed
p27Kip1 mRNA at various levels. (b) Fifty ␮g of protein was subjected to Western
blot analysis as described in “Materials and Methods.” Ca9-22 and HSC4 cells
expressed p27Kip1 at higher levels, but the rest showed reduced expression of
p27Kip1 protein.
The degree of reduction is increased in line with tumor progression. The incidence of p27Kip1 positive
tumors was significantly lower in Stage IV cases and
cases with metastasis.
Furthermore, most metastatic tumors also showed
the same or reduced immunoreactivity as that of primary tumors (data not shown). It has been reported that
antisense oligonucleotide–mediated down-regulation of
p27Kip1 in EMT-6 mammary tumor cell spheroids reduced intercellular adhesion and increased cell proliferation.29 We suppose that reduced expression of p27Kip1
may be related to the loss of intercellular adhesion and
result in metastasis. Interaction between expression of
p27Kip1 and adhesion molecules was suggested, but the
mechanism of p27Kip1-mediated metastasis is unknown.30
Figure 2 shows that the prognoses of patients with
negative expression of p27Kip1 were poorer than those
of patients with medium-to-high expression of
p27Kip1. The prognoses of OSCC patients with high
expression of cyclin E and negative expression of
p27Kip1 were not poor, and this result was different
from the result for breast carcinoma.19 In breast carcinoma, most of high grade tumors were found to
show cyclin E overexpression, and overexpression of
cyclin E correlated with increasing tumor stage and
grade.18 The overexpression of cyclin E did not correlate with various parameters for malignancy of OSCC
(Table 3), and it seemed to be a major reason for this
result. The correlation between p27Kip1 and cyclin E in
OSCC cells should be examined in a large number of
cases.
The reduced expression of p27Kip1 protein was
detected in OSCC cell lines, whereas most of them
preserved the expression of p27Kip1 mRNA and none of
them had macroscopic alterations of the p27Kip1 gene
(Fig. 3). This was consistent with the findings reported
for gastric, breast, and colorectal carcinomas.19 –22,24
As reduced expression of p27Kip1 protein was not
brought about by reduced expression of p27Kip1 mRNA
or gene alteration, we examined proteasome-mediated degradation of p27Kip1 protein. We showed that
accumulation of p27Kip1 protein was originally found
after treatment with proteasome inhibitor LLnV in
2454
CANCER December 15, 1998 / Volume 83 / Number 12
8.
9.
10.
11.
12.
13.
FIGURE 4.
Treatment of HSC2 and Ca9-22 cells with the proteasome
inhibitor LLnV is shown. Fifty ␮g of protein was obtained from the cells after
treatment with 50 ␮M LLnV for the indicated periods and analyzed as in Figure
3. LLnV induced the expression of p27Kip1 protein in HSC2 cells, whereas it did
not do so in Ca9-22 cells.
14.
HSC2 cells without p27Kip1 protein expression (Fig. 4).
Our results suggest that the discrepancy between the
expression of mRNA and protein is associated with
increased proteasome-mediated degradation at posttranslational levels. We should examine whether the
reduced expression of p27Kip1 is really caused by proteasome-mediated degradation in OSCC tissues.
Reduced expression of p27Kip1 could predict malignant behavior in OSCC. As it is difficult to predict
the survival of patients with OSCC, we should examine
the significance of p27Kip1 as a prognostic factor. In
OSCC, inhibition of proteasome-mediated degradation of p27Kip1 protein may be used as a possible target
of novel cancer therapy.
17.
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